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9,178,577
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Anatomy of the ductus venosus in neonatal dogs (Canis familiaris).
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Anatomical features of the ductus venosus in 84 neonatal dogs are described. The ductus venosus was a straight conduit 1-3 mm wide and 4-12 mm long in pups with a crown-rump length of 80-200 mm. It arose from the left main portal vein branch opposite the umbilical vein, passed between the left lateral liver lobe and the papillary process of the caudate lobe, and terminated in the dorsal aspect of the proximal part of the left hepatic vein. The left hepatic vein was dilated at this point. There was no variation in the location of the ductus venosus in the animals studied.
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9,178,578
|
A quantitative study of ganglion cells in the goat retina.
|
As in a number of mammals, the most prominent feature of the ganglion-cell layer in the retina of the murciano-granadina goat is an increase in the density of ganglion cells in the central area, as well as a concentration along a ridge extending horizontally across the retina, below the optic disc, and in the upper temporal retina. Thus, there is an area of maximum density and two streaks that are known as the 'horizontal' and 'vertical' streak. The isodensity lines of ganglion-cell distribution is toughly concentric, with their values varying from 304 cells/mm2 in the periphery to 3592 cells/mm2 in the central area, with the cells densely packed. There were some individual differences amongst the animal studied, although all of them were purebred animals.
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9,178,579
|
Transcranial cerebral oximetry--is it clinically useless at this moment to interpret absolute values obtained by the INVOS 3100 cerebral oximeter?
|
Dynamic changes of regional cerebral oxygenation (rSO2) were measured simultaneously to laser Doppler flowmetry in a 35-year-old female during cerebral aneurysm surgery. In addition, to demonstrate the uselessness of the absolute values obtained by the INVOS 3100, cerebral oximeter readings from a pumpkin ("Styrian Cucurbita") are demonstrated. The results of the present study and of other reports discussed in this paper demonstrate that the INVOS 3100 cerebral oximeter should not be used at this moment as a measure reflecting absolute rSO2 values. However, it seems to be relevant that the INVOS 3100 system can monitor the dynamic changes in rSO2, although limitations have to be taken into consideration.
|
9,178,586
|
Management of infected total knee arthroplasty.
|
Deep wound infection after total knee arthroplasty is an infrequent but very serious complication. Treatment is difficult and challenging. Antibiotic therapy alone can not replace surgery in the management of infected total knee arthroplasty.
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9,178,588
|
Hyperbaric oxygen in the treatment of diabetic foot infection.
|
Although hyperbaric oxygen therapy to treat diabetic foot lesions has been approved for insurance reimbursement in Taiwan, its clinical application has not yet been well accepted. This study evaluated multiple healing predictive factors in patients with diabetic foot infections to determine the usefulness of adjunctive hyperbaric oxygen in the treatment of such patients.
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9,178,580
|
Reorganization of contractile systems and protein tyrosine phosphorylation in platelet aggregation.
|
Platelet aggregation is accompanied with reorganization of contractile systems. This event involves tyrosine phosphorylation of various proteins. When tyrosine phosphorylation is induced by an intracellular trigger as vanadate, a phosphorylated cytosolic 60 Kd protein acts as a nucleating center on which new actin filaments grow. When aggregation is induced by an extracellular signal as ristocetin, actin filaments of the membrane-associated skeleton and the 60 Kd protein bound to this structure migrate toward the cytosol and only in this soluble state the 60 Kd protein undergoes tyrosine phosphorylation and consequently triggers reorganization of contractile systems.
|
9,178,589
|
Diagnosis of prostate cancer: comparison of serum prostate specific antigen, digital rectal examination and transrectal ultrasonography.
|
While prostate specific antigen (PSA) is useful as a tumor marker for monitoring patients with prostate cancer after definitive therapy, limitations have been noted when it is used for early detection of prostate cancer.
|
9,178,581
|
The carotenoid pigments of a marine Bacillus firmus strain.
|
As carotenoids have important biological functions, it is important to discover new natural sources of these pigments. The bacterial strains isolated from a sea water rock pool were cultivated on marine agar containing yeast extract and identified by conventional methods. The bacterial pigments were extracted with methanol and analyzed by reversed-phase HPLC with diode array detection. The major pigment of a Bacillus firmus strain was identified as astaxanthin; the results obtained suggest potential use of this bacterium in aquaculture and in pharmaceutical field.
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9,178,587
|
Comparison of the clinical diagnostic value between pleural needle biopsy and analysis of pleural effusion.
|
Many diseases are manifested by pleural effusion. Chest echo-guided thoracentesis and pleural biopsy are the two major procedures in diagnosing pleural effusion, but the validity is still under debate. To compare the diagnostic value of echo-guided pleural biopsy with pleural effusion analysis, we designed this retrospective study.
|
9,178,590
|
Necrotizing enterocolitis in newborn: nine years' experience.
|
Necrotizing enterocolitis (NEC) is the most significant acquired gastrointestinal (GI) emergency in the neonatal intensive care unit.
|
9,178,591
|
Ketoacidosis with hyperglycemia in heavy drinkers: a report of 12 cases.
|
Heavy alcohol intake (> 45 g daily) might be a cause of diabetes. The short-term risks of heavy alcohol intake include ketoacidosis, glucose intolerance and pancreatitis. Alcoholic ketoacidosis (AKA) in combination with hyperglycemia mimics diabetic ketoacidosis (DKA). We described the characteristics of heavy drinkers with ketoacidosis and hyperglycemia but without a prior history of diabetes.
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9,178,592
|
Endoscopic sinus surgery for the treatment of frontoethmoidal mucocele complicated with orbital abscess: a case report.
|
Mucoceles may develop after several decades following sinus surgery. They can progressively expand over many years and destroy the surrounding bones, which may result in severe complications such as orbital infection or intracranial lesions. A 68-year-old woman was hospitalized with the chief complaints of exacerbated right orbital swelling and progressive visual loss for one week in May 1995. Under the impression of the right frontoethmoidal mucocele complicated with orbital abscess, she underwent endoscopic sinus surgery for marsupialization and wide drainage of the mucocele. The symptoms subsided one week post-operatively without ophthalmological sequelae. There has been no recurrence one-year post-operatively. Endoscopic sinus surgery is an effective treatment for frontoethmoidal mucocele.
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9,178,593
|
Obstetrical hysterectomy and placenta previa/accreta: three bladder injury case reports.
|
Pregnancies complicated with placenta previa and a history of cesarean section are well known to be at increased risk for placenta accreta. Bladder injury is occasionally encountered in these patients during operation. From January 1992 to December 1995, 25 obstetrical hysterectomy were performed at Chang Gung Memorial Hospital, Linkou Medical Center. Of these 25 patients three had bladder injury. Ultrasonography is a unique way to screen and detect prenatally the abnormal placentation and intraplacental lacunae. Color Doppler ultrasonic scanning further discloses that the lacunae are mainly venous spaces. Elevated mid-trimester maternal serum x-fetoprotein (MSAFP) frequently leads to a suspicion of the abnormal placentation, and magnetic resonance imaging (MRI) can clearly identify the placenta accreta/increta. Uncontrollable bleeding frequently occurs in these patients during cesarean section, warranting emergency hysterectomy. Emergency obstetrical hysterectomy should be decided upon and performed by an experienced obstetrician. Massive hemorrhage and bladder injury are the major complications encountered in such operations. We review the literature and propose a protocol of management. As the incidence of cesarean section continues to rise world-wide, the problem of placenta previa/accreta is likely to become more common. Obstetricians should be ready to face the late sequelae of today's decision for cesarean section.
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9,178,583
|
Assay of total protein kinase activity in mouse brain cortex.
|
In previous studies we demonstrated that the adrenergic system is impaired in old animals and that the main alterations were observed at the level of receptor density and adenylate-cyclase activity. The decreased ability to produce cAMP could influence the activity of the cAMP dependent protein kinase (PKA), one of the enzymes responsible for the phosphorylation of protein substrates. Since protein phosphorylation is one of the most common and important mechanisms through which a cell regulates its activity, the characteristics of the phosphorus incorporation reaction were studied. Kinase activity was measured in homogenate of young mouse brain cortex prepared avoiding gross manipulations in order to maintain conditions as close to those present in the living animal as possible. Results show that phosphate incorporation is proportional to protein content and strictly dependent on ATP availability. Increasing the ATP concentration from 10 to 500 mumol/l, the length of incorporation phase increases, suggesting that the limiting point of the reaction is better represented by energy availability than by enzyme or protein substrate concentrations.
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9,178,585
|
Changes in plasminogen activator inhibitor-1 levels in non-small cell lung cancer.
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Increased urokinase plasminogen activator (uPA) levels are increased in a number of malignancies and have been correlated with decreased disease-free interval and decreased overall survival. We have, therefore, examined components of this plasminogen activating system in patients with Non-Small Cell Lung Cancer (NSCLC). Levels of uPA, urokinase-plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) were measured semiquantitatively in paraffin sections of tumours from 147 patients with NSCLC. Immunohistochemically stained sections of tumour were allocated a score for stain intensity and results correlated to: survival; tumour stage(T); nodal stage(N); stage grouping (I to IIIb), survival status and sex. Increased levels of PAI-1 were associated with a decreased survival in squamous cell carcinoma (SCC) X2 = 5.72, p = 0.017 (n = 74). There was a significant positive relationship between PAI-1 levels and N-stage (p = < 0.05), presence of nodal metastases (p = < 0.05), stage grouping (p = < 0.01) and extent of disease (p = < 0.05) in the total group and the SCC subgroup, but not adenocarcinoma. There was a significant positive relationship between PAI-1 levels and T-stage (p = < 0.05) in the total group, and survival status (p = < 0.05) in the SCC subgroup alone. uPA and uPAR levels were not significantly associated with tumour staging or survival. We conclude that increased PAI-1 antigen levels may be associated with a decreased survival in patients with SCC.
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9,178,594
|
Glucocorticoid remediable aldosteronism: a case report.
|
Glucocorticoid remediable aldosteronism (GRA) is a hereditary cause of mineralocorticoid hypertension. The most common presentation is asymptomatic hypertension. Hypokalemia, hyperaldosteronism and suppressed plasma renin activity are other forms of primary hyperaldosteronism. However, the aldosterone secretion in these patients is regulated by adrenocortico-tropic hormone (ACTH) rather than the reninangiotension system. Here, we report a patient with a 12-year history of hypertension without response to any treatment until dexamethasone was administered. The diagnosis of GRA was confirmed by elevated plasma level of 18-oxocortisol, which is a unique steroid biochemical abnormality of this disease. In GRA, hybrid steroids (18-oxocortisol and 18-hydroxycortisol) are synthesized at the C-18 carbon of cortisol in a similar way as when corticosterone is converted to aldosterone. The gene duplication defect is on chromosome 8 codes for a chimerical 11 beta-hydroxylase/aldosterone synthase enzyme, causing ectopic expression of aldosterone synthase in zona fasiculata. Because this hypertension is remediable by exogenous glucocorticoid, this case was reported to raise attention about treatable aldosteronism.
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9,178,595
|
Color Doppler energy in prenatal diagnosis of meconium peritonitis: a case report.
|
Meconium peritonitis is an uncommon chemical peritonitis of a fetus resulting from antenatal bowel perforation. We reported a case of meconium peritonitis with pseudocystic formation diagnosed by color Doppler energy (CDE) at 34 gestational weeks. An echogenic substance inside a fetal abdominal mass was detected using ultrasound. By conventional color Doppler, there was minimal blood flow in the cystic wall or septums of the mass. Using CDE, bowel hyperperistalsis was observed in multiple small bowel loops and the region of intestinal loops into the mass was easily detected. Therefore, the angle independent nature of CDE will play a significant role in the early and accurate diagnosis of meconium peritonitis before birth.
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9,178,596
|
Retained vaginal gauze with unusual complication: a case report.
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We report a case of a postpartum retained vaginal gauze which migrated to the bladder and presented as a bladder stone. The patient had received numerous clinical evaluations for her chronic abdominal pain, of all which failed to detect or indicate the presence of retained gauze. Retained surgical gauze is a preventable problem but continues to occur periodically. Prevention remains the key to this problem. The gauze packed within the vagina either after transvaginal surgery or delivery should be cared for as in other parts of the body. Though extremely rare, retained surgical gauze should be considered in the differential diagnosis in postpartum patients with chronic abdominal pain, irritable bladder symptoms or pelvic abscess.
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9,178,598
|
Attacking the attacker: gay Christians talk back.
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This paper analyses the accounts constructed by 60 gay male Christians in partnership as stigma management strategies at the level of cognition and rhetoric. Four strategies are identified: (i) attacking the stigma; (ii) attacking the stigmatizer; (iii) use of positive personal experience; and (iv) use of the ontogeneric argument. These strategies are interchangeably and collectively used to dismiss the credibility of the institutionalized Church and the validity of its unfavourable official position on the issue of homosexuality. The effective use of these strategies demonstrates the positive personal identity these gay Christians have developed in this advanced stage of their moral career.
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9,178,597
|
Vulvovaginal tuberculosis: a case report.
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A rare case of vulvovaginal tuberculosis is reported. A 76-year-old woman presented with a painful ulcer at the posterior fourchette and lower vagina for 6 weeks. Direct biopsy for histologic examination revealed mycobacterial infection. Anti-tuberculosis treatment was effective for this patient. We suggest prompt biopsies for suspicious vulvar or vaginal lesions.
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9,178,582
|
Changes of glycosaminoglycan composition in aging chicken brain. A preliminary investigation.
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The qualitative and quantitative pattern of GAGs was examined by electrophoresis in aging chicken brain in four different stages starting from day 1 to 30 months. GAG content referred to defatted dry tissue exhibits constant decrease. Four main GAGs have been identified with a mobility corresponding to hyaluronate, condroitin sulfate, heparan sulfate and dermatan sulfate controls. Hyaluronate appears the main GAG represented while dermatan sulfate the minor one. Our data show that in chicken brain GAG percentage undergoes age-related changes.
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9,178,584
|
Choline feeding depresses the phospholipase C activity in the regenerating liver of female rats.
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The administration of an excess of choline for 3 weeks is able to delay the proliferative response to partial hepatectomy (PH) in female rats. Choline feeding can affect the phospholipid composition of cell membranes and, as a consequence, the transduction of the mitogenic signals. On these bases, we studied the turnover of phosphatidylinositol-4,5-biphosphate (PIP2) in the regenerating liver of female rats. The hydrolysis of PIP2 is catalysed by a specific phospholipase C (PL-C) and it generates the second messenger molecules, namely diacylglycerol and inositol-1,4,5-triphosphate (IP3). Our results showed that the administration of an excess of choline to females was able to reduce the PL-C activity and the membrane IP3 content in the quiescent liver. Both parameters remained lower than controls during liver regeneration, even if they were higher 1 and 2 h after PH in comparison with the quiescent liver, in choline-fed females. These data suggest that the delay in the liver regeneration by choline is due, at least in part, to the alteration in the pathway of PIP2 turnover for the transduction of mitogenic signals.
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9,178,600
|
The eight myths of Operation 'Desert Storm' and Gulf War syndrome.
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Several conventional claims regarding Gulf War Syndrome are criticized: that Gulf War veterans are no sicker than the civilian population as a whole; that Gulf War Syndrome is a myth invented by the press; that GWS cannot be defined as a legitimate medical syndrome; that since its cause cannot be determined, it is not a problem associated with Operation 'Desert Storm'; that the US and UK governments are doing all they can to investigate and treat illness in veterans or deny existence of over 100,000 cases in veterans and their families; that GWS will settle without treatment; that the armed forces were well prepared for integrated conflict involving chemical and biological warfare in the Middle East, increasing the risk of this in the future.
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9,178,599
|
The vision within our grasp.
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This article takes stock of recent progress in human development and poverty reduction, drawing on the 1996 Human Development Report. It presents a vision of human development and targets for the twenty-first century, to provide a frame within which we can all work in the years ahead, and identifies three specific actions for IPPNW to support.
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9,178,609
|
Enhanced closed-state inactivation in a mutant Shaker K+ channel.
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Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed "multiple-state inactivation" model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels.
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9,178,610
|
Regulation of cation-selective channels in liver cells.
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In liver cells, cation-selective channels are permeable to Ca2+ and have been postulated to represent a pathway for receptor-mediated Ca2+ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca2+], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl-], a physiological response to Ca2+-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na+ and Ca2+ influx.
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9,178,611
|
Ethanol activates maxi Ca2+-activated K+ channels of clonal pituitary (GH3) cells.
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The effect of ethanol on maxi Ca2+-activated K+ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity. 30 mm (1.4 per thousand) ethanol significantly increased mean channel open time and channel open probability by 26.3 +/- 9% and 78.8 +/- 10%, respectively; single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19-31) pseudosubstrate inhibitor as well as by AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122. Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity and hence may influence action potentials duration and hormone secretion.
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9,178,612
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Dihydropyridine receptor-ryanodine receptor uncoupling in aged skeletal muscle.
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The mechanisms underlying skeletal muscle functional impairment and structural changes with advanced age are only partially understood. In the present study, we support and expand our theory about alterations in sarcolemmal excitation-sarcoplasmic reticulum Ca2+ release-contraction uncoupling as a primary skeletal muscle alteration and major determinant of weakness and fatigue in mammalian species including humans. To test the hypothesis that the number of RYR1 (ryanodine receptor) uncoupled to DHPR (dihydropyridine receptor) increases with age, we performed high-affinity ligand binding studies in soleus, extensor digitorum longus (EDL) and in a pool of several skeletal muscles consisting of a mixture of fast- and slow-twitch muscle fibers in middle-aged (14-month) and old (28-months) Fisher 344 Brown Norway F1 hybrids rats. The number of DHPR, RYR1, the coupling between both receptors expressed as the DHPR/RYR1 maximum binding capacity, and their dissociation constant for high-affinity ligands were measured. The DHPR/RYR1 ratio was significantly reduced in the three groups of muscles (pool: 1.03 +/- 0.15 and 0.80 +/- 0.11, soleus: 0.44 +/- 0. 12 and 0.26 +/- 0.10, and EDL: 0.95 +/- 0.14 and 0.68 +/- 0.10, for middle-aged and old muscles, respectively). These data support the concept that DHPR-RYR1 uncoupling results in alterations in the voltage-gated sarcoplasmic reticulum Ca2+ release mechanism, decreases in myoplasmic Ca2+ elevation in response to sarcolemmal depolarization, reduced Ca2+ supply to contractile proteins and reduced contraction force with aging.
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9,178,614
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Regulation of metabolite flux through voltage-gating of VDAC channels.
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The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, "closed" state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5-11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically.
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9,178,613
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Identification of anion-selective channels in the basolateral membrane of mitochondria-rich epithelial cells.
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Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl- channels. The two major populations constituted Ohmic Cl- channels with limiting conductance (gamma125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (+/-0.1) x 10(-14), 5.7 (+/-0.7) x 10(-14), and 32 (+/-2) x 10(-14) cm3/sec, for the 10, 30 and 150 pS Cl- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl- channel was studied in patches generating 'noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near ECl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl- channels are organized in clusters.
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9,178,615
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On the role of calcium in the regulatory volume decrease (RVD) response in Ehrlich mouse ascites tumor cells.
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The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mM Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mM Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 microM), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.
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9,178,616
|
Ionic permeability on isolated mouse liver nuclei: influence of ATP and Ca2+.
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Patch-clamp experiments on isolated nuclei revealed the existence of ionic channels on the nuclear envelope, but their exact localization and function are still unknown. Recent studies have demonstrated that ATP and calcium ions play an important role in nucleocytoplasmic protein traffic. ATP is essential to allow big molecules in and out of the nucleus. However, a cytoplasmic rise of calcium ions above 300 nm decreases both ATP-dependent transport and passive diffusion through the nuclear envelope. The use of isolated nuclei placed in a saline solution provides the possibility for testing only the compounds added in the bath or in the recording pipette. In the present study, we show that ATP is responsible for an increase of nuclear ionic permeability on isolated nuclei. This result not only confirms data previously reported in in situ nuclei, but also suggests that ATP is directly involved in the modulation of passive ionic permeability. In these particular experimental conditions, calcium ions decrease the channel current starting from a concentration of 1 microM. The parallelism in the modulation action of ATP and Ca++ between nuclear pores and ionic channels present on the nuclear envelope contributes to the support of the idea that an ionic pathway is associated with the pore complex.
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9,178,617
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Separate determination of the electrical properties of the tonoplast and the plasmalemma of the giant-celled alga Valonia utricularis: vacuolar perfusion of turgescent cells with nystatin and other agents.
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In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 microM of the H+-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to -22 mV and in an increase of the area-specific membrane resistance from 6.8 x 10(-2) to 40.6 x 10(-2) Omegam2. The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81-94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 microM of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively form the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 x 10(-2) compared to 0.77 x 10(-2) Fm(-2) of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system.
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9,178,618
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Electrophysiological actions of quinine on voltage-dependent currents in dissociated rat taste cells.
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How taste receptor cells participate in encoding disparate compounds into distinct taste qualities represents a fundamental problem in the study of gustatory transduction mechanisms. Quinine is the most common stimulus employed to represent bitterness yet its electrophysiological consequences on voltage-dependent ion channels in the taste receptor cell have not been elucidated in detail. This study examines such effects on taste receptor cells dissociated from the foliate and circumvallate papillae of the rat. Outward potassium currents, which include transient, sustained and calcium-activated components, were reversibly inhibited by bath application of quinine, with an IC50 of 5.1x10(-6)M. The time course of the current traces, along with voltage shifts in normalized conductance and inactivation curves, suggests that multiple mechanisms of inhibition may be occurring. Inwardly rectifying potassium currents were unaffected. Sodium currents, to somewhat higher concentrations of quinine (IC50 = 6.4x10(-5)M), were also reduced in magnitude without noticeable effects on activation or reversal potential but with a shift in inactivation. Calcium currents, visualized with barium as a charge carrier, were enhanced in magnitude by the presence of low concentrations of quinine (10(-5)M) but were suppressed by higher concentrations (10(-4)M). Quinine broadened the waveform of the gustatory action potential and increased the input resistance. These data serve as genesis to future investigations of the signal transduction mechanism of quinine on voltage-dependent currents.
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9,178,620
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Interference of a short-chain phospholipid with ion transport pathways in frog skin.
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The effects of mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC; with two saturated 10-carbon acyl chains) on active Na+ transport and transepithelial conductance (G) in the frog skin (Rana temporaria) were investigated. Active Na+ transport was measured as the amiloride-sensitive short-circuit current (ISC) and G was determined from transepithelial voltage-clamp pulses under short-circuit conditions. DDPC dose-dependently inhibited ISC with an ID50 of about 0.05% (w/v) and a maximal effect ( approximately 55%) at >/= 1% DDPC. G increased to steady-state values above control level. Simultaneously, equal increases in unidirectional sucrose permeabilities (PSu; measured from [14C]sucrose fluxes) were observed, and a positive correlation was demonstrated between DDPC-induced changes in PSu and G. Since amiloride did not prevent the increase in G by DDPC, these results suggest that the DDPC-induced increase in G represents an increase in the paracellular shunt conductance. The effects of mucosal DDPC were almost fully reversible within 8 h. The results indicate that DDPC inhibits amiloride-sensitive Na+ channels in the apical membrane of the frog skin epithelium and opens a paracellular tight junction pathway. Both effects may be caused by incorporation of DDPC in the apical cell membrane.
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9,178,619
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Regulation of Cl- transport by IBMX in renal A6 epithelium.
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We studied regulation of Cl- transport by cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 microM, a Ca2+ channel blocker) or 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 microM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 microM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl- release is regulated by an increased cytosolic Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl- channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl- release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration.
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9,178,621
|
Effects of nitric oxide on force-generating proteins of skeletal muscle.
|
Nitric oxide (NO) has recently been identified as a physiologically important intracellular messenger modulating the contractile activity of skeletal muscle [Kobzik L, Reid MB, Bredt DS, Stamler JS (1994) Nature 372: 546-548]. However, the mechanism of action of NO is not yet known. We used skinned (demembranated) muscle fibres to investigate the mechanism of NO function in muscle contraction. Maximally Ca2+-activated single fibres of rat skeletal muscle were exposed to physiologically relevant NO concentrations by adding NO donor molecules into the bath solution. Donor application caused a decline both in the contractile properties and in the myofibrillar adenosine triphosphatase (ATPase) activity. These results reveal a novel molecular mechanism of NO action: a direct inhibition of the force-generating proteins in skeletal muscle.
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9,178,623
|
Imaging excised apical plasma membrane patches of MDCK cells in physiological conditions with atomic force microscopy.
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We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i. e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per micron(2), with heights between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer - allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.
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9,178,622
|
Decline in isokinetic force with age: muscle cross-sectional area and specific force.
|
Humans produce less muscle force (F) as they age. However, the relationship between decreased force and muscle cross-sectional area (CSA) in older humans is not well documented. We examined changes in F and CSA to determine the relative contributions of muscle atrophy and specific force (F/CSA) to declining force production in aging humans. The proportions of myosin heavy chain (MHC) isoforms were characterized to assess whether this was related to changes in specific force with age. We measured the peak force of isokinetic knee extension in 57 males and females aged 23-80 years, and used magnetic resonance imaging to determine the contractile area of the quadriceps muscle. Analysis of MHC isoforms taken from biopsies of the vastus lateralis muscle showed no relation to specific force. F, CSA, and F/CSA decreased with age. Smaller CSA accounted for only about half of the 39% drop in force that occurred between ages 65-80 years. Specific force dropped about 1.5% per year in this age range, for a total decrease of 21%. Thus, quantitative changes in muscle (atrophy) are not sufficient to explain the strength loss associated with aging.
|
9,178,624
|
Annexins from Ehrlich ascites cells inhibit the calcium-activated chloride current in Xenopus laevis oocytes.
|
The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.
|
9,178,625
|
The outer hair cell motor in membrane patches.
|
We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 Momega pipettes with tip diameters of 3 micron the measured maximum voltage-dependent capacitance was 56 +/- 6 fF at -36 mV when the resting membrane potential was -20 mV. The calculated total charge movement was 5.6 +/- 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/micron2. Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings.
|
9,178,626
|
Sustained GABA-induced regulation of the L-type Ca2+ conductance in crustacean muscle fibers.
|
The sustained effects of gamma-aminobutyric acid (GABA) on voltage-gated conductances, and excitatory and inhibitory postsynaptic currents (EPSC and IPSC, respectively) in crayfish opener muscle fibers were analyzed using the two-electrode voltage-clamp technique. GABA (1.0 mM) was applied for 1-2 min and measurements were performed 30 min after restoring control Ringer solution. The L-type Ca2+ current (ICa) was reduced by > 33%. The ICa conductance (gCa) was reduced and the activation and inactivation were slowed down by GABA. The ICa regulation outlasted GABA superfusion (150 min). A small decrease (< 19%) of the Ca2+-activated K+ current (IKCa), due to the ICa reduction, was also recorded. The leak (IL), the delayed-rectifier (IK) and the hyperpolarization-activated (IAB) currents were not affected. Picrotoxin (0.5 mM) and bicuculline (0.2 mM) blocked the ICa reduction. Neither the GABAB antagonist saclofen (1.0 mM) nor the agonist baclofen (1.0 mM) had any effect. Therefore, the ICa regulation was probably mediated through GABAA receptors. EPSCs, but not IPSCs, were reduced (30%) for prolonged periods (> 100 min.) after GABA application. We describe a new, potentially functional, role for GABA receptors in the mediation of a sustained reduction of presynaptic and postsynaptic excitability in crustacean muscle.
|
9,178,627
|
Heart rate, blood pressure, and running speed responses to mesencephalic locomotor region stimulation in anesthetized rats.
|
The decerebrate rat locomotor preparation described in a previous study requires extensive brain surgery with the possibility of significant blood loss. The purpose of this study was to improve on the previous model by using lightly anesthetized instead of decerebrated rats. After initial surgery consisting of boring a small hole through the parietal bone, the animals were maintained on low levels of halothane anesthetic. The mesencephalic locomotor region was then located by physiological criteria using stereotaxic coordinates from the previous study. Locomotor speed, blood pressure and heart rate responses were then measured over a wide range of stimulation currents that elicited a maximal running speed. Stimulation currents ranged from 36 microA for walking to 82 microA for fast galloping. Locomotor speeds ranged from 20 m/min for walking to 64 m/min for fast galloping. Some animals easily achieved galloping speeds beyond 100 m/min. Blood pressure and heart rate increased with increasing stimulation currents. Blood pressure also increased during stimulation after muscular paralysis. This was not due to current spread, suggesting that the mesencephalic locomotor region might be involved in central command mechanisms. Heart rate did not increase after paralysis. This supports other multi-joint dynamic studies suggesting that movement per se may be necessary to induce heart rate changes, presumably via joint mechanoreceptors. The range of locomotor patterns and cardiovascular responses were obtained under self-supported conditions. By defining the mesencephalic locomotor region via physiological criteria, and by grouping blood pressure and heart rate measurements by gait rather than by stimulation currents, the potential use of the intact model for cardiovascular control studies was demonstrated. The animals were able to run and gallop at high speeds considering they were anesthetized. The simplified preparation will be useful for more complex cardiovascular experiments requiring intact and self-supported conditions.
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9,178,628
|
The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells.
|
The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.
|
9,178,629
|
The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones.
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Induction of heat shock proteins (HSPs) following cell injury contributes to the protection of vital cell functions. It was, therefore, of interest to study the effects of transient renal ischaemia on the abundance and distribution of two HSPs, HSP25 and HSP72, in renal tissue using Western-blot techniques. Analyses were performed on the supernatant (HSP25, HSP72) and pellet (HSP25) of homogenates obtained from cortex (CX) and outer (OM) and inner (IM) medulla of the rat kidney immediately after 60 min of ischaemia followed by varying periods of reperfusion. Ischaemia of the left kidney caused HSP25 contents to decrease in CX, OM and IM by 73, 89 and 54% respectively, compared with the corresponding zones of the contralateral control kidney. This initial decrease in supernatant HSP25 was accompanied by an increased abundance of HSP25 in the pellet. Following reperfusion, HSP25 contents in the supernatant gradually increased in CX and OM, reaching, after 24 h, values that were 5.4- and 2.5-fold higher, respectively, than those in the control kidneys. After 7 or 14 days of reperfusion, HSP25 contents had not completely normalised in CX, but had reached control levels in OM. In IM, the HSP25 content remained below control throughout the entire reperfusion period. HSP72 (supernatant) was below the detection limit in the CX of the control kidney. Similar to the level of HSP25, that of HSP72 was also markedly lower in OM and IM immediately after ischaemia. The intrarenal distribution of HSP72 and the sequence of zonal changes in HSP72 contents were similar to those observed for HSP25. These results are compatible with the view that, during ischaemia and the initial reperfusion period, HSP25 migrates from the cytoplasmic compartment (supernatant) into the nucleus and/or associates with cytoskeletal structures. The observation that both HSP25 and HSP72 are transiently induced in CX and OM, but not in IM, may be explained by the fact that, while all kidney cells are exposed to ischaemic stress, only inner medullary cells experience a major postischaemic attenuation of osmotic stress.
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9,178,630
|
Paracellular calcium transport across Caco-2 and HT29 cell monolayers.
|
Intestinal calcium absorption has been shown to include two processes, a saturable transcellular movement and a non-saturable paracellular pathway. The potential utility of cell monolayers for studying transepithelial intestinal calcium transport has already been demonstrated; however, simultaneous evaluation of the contribution of the saturable transcellular and of the non-saturable paracellular processes to the total transepithelial transport has not yet been attempted. The aim of this study was to investigate the contribution both of transcellular and paracellular transport processes to the total transepithelial calcium transport in two cell culture monolayers. Caco-2 cells and a clone derived from HT-29 cells (HT29-Cl.19A), two cell lines derived from colon adenocarcinomas which are known to be able to exhibit typical enterocytic differentiation, were used. Cell monolayers were grown on a permeable support and used after 15 days of culture when these cells express enterocytic differentiation and high transepithelial resistance. Isotopic transport rate measurements were performed in the absence of a chemical gradient. The paracellular route was evaluated using [3H]mannitol. Calcium and [3H]mannitol transport rates across cell monolayers were not significantly different. Augmentation of calcium uptake by 200 mM sorbitol did not significantly increase calcium or mannitol transepithelial transport; however, calcium accumulation in the cells was increased by about 200%. Modulation of the monolayer permeability by addition of 10 nM vasoactive intestinal polypeptide (VIP) or 0.5 mM carbachol treatment, which respectively increased and decreased the transepithelial resistance, consequently modified calcium and mannitol transport in a parallel manner. Our results show that Caco-2 and HT29-Cl.19A cell monolayers are good models for studying the calcium paracellular transport pathway.
|
9,178,631
|
NH4+ conductance in Xenopus laevis oocytes. I. Basic observations.
|
Current-clamp and voltage-clamp techniques were used to study the effects of NH4+ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH4Cl resulted in a depolarization of the oocyte's cell membrane potential and, at a clamp potential of -70 mV, in an inward current. The magnitude of the inward current was proportional to the NH4Cl concentration in the extracellular solution and on membrane potential. The reversal potential, Erev , was -35.5 +/- 11.6 mV under control conditions and -3.1 +/- 11.0 mV (n = 19) in the presence of NH4Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca2+-free solution affected the NH4Cl-evoked response only marginally. Replacement of extracellular Na+ by N-methyl-D-glucamine+ markedly reduced, but did not eliminate, the NH4Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH4Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K+ conductance, Ba2+ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH4Cl for Na+ and NH4+. The mechanism by which NH4Cl activates the conductance remains unknown.
|
9,178,632
|
Modulation of ligand-gated dopamine channels in Helix neurones.
|
Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.
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9,178,633
|
Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia.
|
The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase). In order to contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved. Epithelia were treated for 1 h with 10(-6 )M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied. Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase alpha1 and beta1 subunit mRNAs were slightly increased (1.2- to 1.4-fold). In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR). After 1 h of aldosterone treatment, approximately 0.5% of these appeared to be regulated. Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned and for two of them cDNAs containing entire coding sequences were isolated by library screening. ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D. The K1/2 values were similar or slightly below those reported for stimulation of Na transport. These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.
|
9,178,634
|
Force and myosin content variation in isolated intact single muscle fibres from Rana temporaria.
|
We studied the relation between force normalized by dry mass per unit length and the myosin fraction of muscle dry mass. The two tibialis anterior muscles were dissected from 12 frogs (Rana temporaria). Then, from one muscle, two single fast-twitch fibres were isolated. Each fibre was mounted isometrically in Ringer's solution, and electrically stimulated using a standardized protocol. Peak force production, normalized by the fibre's dry mass per unit length, varied by a factor of 1.4. Little variation in normalized force was measured between fibres from the same animal, whereas between animals a significant difference was found (P<0.05). The contralateral muscle was used to determine the myosin fraction of the dry mass. The relationship between the fraction myosin of the dry mass and force normalized by dry mass per unit length showed a high correlation (r = 0.81; n = 12). From this we conclude that variation in normalized tetanic force is determined greatly (65%) by variations in myosin content.
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9,178,635
|
Whole-cell currents from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell CCL39.
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A conventional patch-clamp technique was used to record the whole-cell current from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell. Ca2+ was extracellularly applied to the Na+-loaded cell to activate the outward current by operating the reverse mode of NCX1. No measurable outward current was ever elicited from the nontransfected cell. Na+/Ca2+ exchange blocker 5 mM Ni2+ or 3 microM KB-R7943 that was applied extracellularly abolished the outward current. With 140 mM external Li+ (replacing Na+), the outward current was transient during the Ca2+ application. In contrast, with 140 mM external Na+, the outward current was maintained without any inactivation during the Ca2+ application. I-V relations predicted from the whole-cell clamp protocols used were obtained both before and during the Ca2+ application. The exchanger whole-cell currents are thus successfully detectable from NCX1 which is overexpressed in this stable transfectant system.
|
9,178,636
|
Augmentation of lipopolysaccharide-induced thymocyte apoptosis by interferon-gamma.
|
The role of interferon (IFN)-gamma on thymocyte apoptosis in response to lipopolysaccharide (LPS) was investigated. The administration of LPS into mice induced marked apoptosis of thymocytes in vivo, but the simultaneous injection of anti-IFN-gamma antibody with LPS completely prevented thymocyte apoptosis. Pretreatment of mice with IFN-gamma markedly enhanced LPS-induced thymocyte apoptosis. Thymocyte apoptosis augmented by IFN-gamma occurred in the thymic cortex, and target cells undergoing apoptosis were CD4+8+ immature thymocytes. IFN-gamma itself did not induce thymocyte apoptosis in vivo and in vitro. IFN-gamma exhibited no synergistic action with effector molecules, such as tumor necrosis factor (TNF)-alpha and glucocorticoids. Further, it was shown that IFN-gamma did not enhance the susceptibility of thymocytes to apoptosis. Pretreatment of mice with IFN-gamma significantly augmented the serum TNF-alpha level and the serum cortisol level in response to LPS. Therefore, we suggest that IFN-gamma might augment LPS-induced thymocyte apoptosis through elevating serum TNF-alpha and cortisol levels.
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9,178,637
|
An analysis of alternatively spliced CD45 mRNA transcripts during T cell maturation in humans.
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CD45 is a transmembrane protein tyrosine phosphatase found on nucleated hematopoietic cells. In humans, multiple protein isoforms of CD45 are produced by alternative mRNA splicing of exons 4, 5, and 6 coding for the extracellular portion. We measured all eight possible CD45 mRNA transcripts using RT-PCR in human thymocytes and T cell lines. We report that only six mRNA transcripts are present in T cells. The high mw CD45 mRNA transcripts containing exon 4 correlated with the stage of T cell maturation: abundant high mw transcripts (30.7% of all CD45 mRNA transcripts) were present in immature, CD3-4-8 triple-negative thymocytes which decreased (7.7%) in intermediate, CD4+8+ double-positive (DP) thymocytes and then increased (13.8% or 16.8%) in mature, CD4+8- or CD4-8+ single-positive thymocytes. In addition, there was a complex variation in the spliced mRNA transcripts coding for CD45R(O), CD45R(B), CD45R(BC), CD45R(AB), and CD45R(ABC) protein isoforms. High mw CD45 mRNA transcripts accumulated immediately prior to an important physiologic event such as thymocyte expansion. In addition, we identified linkage between RNA splicing of exons 5 and 6, and splicing of exon 5 only and exons 4, 5, and 6 in FACS-purified CD4+ and CD8+ thymocytes. These data support the developmental regulation of alternatively spliced CD45 mRNA transcripts and suggest that specific CD45 isoforms may play an important role at critical stages of T cell development.
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9,178,638
|
CD40-triggered protein tyrosine phosphorylation on Vav and on phosphatidylinositol 3-kinase correlates with survival of the Ramos-Burkitt lymphoma B cell line.
|
Signals transduced through CD40 rescue cells of the Ramos-Burkitt lymphoma (Ramos-BL) B cell line from surface immunoglobulin M (sIgM)-triggered growth arrest and apoptosis. This study investigates whether protein tyrosine kinase (PTK) activity and tyrosine phosphorylation on p95(vav) and on the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase) play a role in the regulation of Ramos-BL B cell survival. The PTK inhibitor herbimycin A (HA) triggers significant growth arrest prior to apoptosis from the G1-phase of the cell cycle, indicating that tyrosine phosphorylation of key proteins is critical for Ramos-BL cell cycle progression and survival. Indeed, signals transduced through CD40 fail to rescue Ramos-BL B cells from HA-triggered growth arrest and apoptosis. Since Vav and PI3 kinase are intimately involved in the regulation of cellular growth, their tyrosine phosphorylation status was determined in unstimulated and anti-IgM- and anti-CD40-treated Ramos-BL B cells: Vav and p85 are devoid of tyrosine-phosphorylated epitopes in control cells whereas p85, but not Vav, is significantly phosphorylated following ligation of sIgM and anti-CD40 triggers tyrosine phosphorylation on both proteins. Thus, tyrosine-phosphorylated Vav may be a critical effector of CD40-mediated survival. As tyrosine-phosphorylated PI3 kinase is common to both sIgM-triggered death and CD40-triggered survival pathways, its lipid kinase activity was correlated with tyrosine phosphorylation on p85: Ramos-BL B cells exhibit high basal levels of PI3 kinase activity, determined by immunoprecipitation with anti-p85 and 32P incorporation into phosphatidylinositol, which is not significantly affected by stimulation with anti-IgM but which is elevated by 36 +/- 2.9% following ligation of CD40. Thus, tyrosine phosphorylation on p85 correlates with the CD40-triggered increase in PI3 kinase activity but not with basal levels nor with sIgM-triggered levels of enzymatic activity: these data suggest the presence of different PI3 kinase isoforms or the existence of multiple regulatory pathways for the same PI3 kinase isotype in Ramos-BL B cells.
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9,178,639
|
Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in humans in vitro.
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We studied the effects of antioxidants from chocolate, cacao liquor polyphenol (CLP), on human immune functions in vitro. CLP is an enriched polyphenol fraction purified from cacao liquor that is a major component of chocolate. It has been shown that polyphenols have antioxidant activity, and reactive oxygen species (ROS) are involved in immune responses. CLP inhibited both hydrogen peroxide and superoxide anion, typical ROS, production by phorbol myristate acetate-activated granulocytes. CLP also inhibited menadione-induced production of both hydrogen peroxide and superoxide anion in normal human peripheral blood lymphocytes (PBL). CLP treatment of normal PBL in vitro inhibited mitogen-induced proliferation of T cells and polyclonal Ig production by B cells in a dose-dependent manner. CLP treatment inhibited both IL-2 mRNA expression of and IL-2 secretion by T cells. These results suggest that antioxidant CLP has immunoregulatory effects.
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9,178,640
|
HLA class I-restricted and tumor-specific cytotoxic T lymphocytes from metastatic lymph nodes of esophageal cancers.
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This paper investigates the presence of HLA class I-restricted and tumor-specific cytotoxic T lymphocytes (CTL) in tumor sites of esophageal cancers. Five CTL lines were established from the metastatic lymph nodes or pleural effusion by incubation with interleukin-2 of tumor-infiltrating lymphocytes: cases 1 and 5, HLA-A26- and HLA-A33-restricted and squamous cell carcinoma (SCC)-specific CTL; case 2, HLA-Cw0102-restricted and esophageal SCC-specific CTL; case 3, HLA-A24- and HLA-A26-restricted CTL recognizing histologically different tumor cells; and case 4, HLA-A26-restricted and esophageal SCC-specific CTL. These results suggest the existence of HLA class I-restricted and tumor-specific CTL in metastatic esophageal SCC.
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9,178,641
|
Expansion of an unusual population of Gr-1+CD3int cells in the lymph nodes and other peripheral organs of mice carrying the lpr gene.
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Granulocytes and extrathymic T cells are often activated simultaneously, but they are absolutely separate populations in normal mice. However, some abnormal extrathymic T cells (i.e., CD3int cells) seen in mice carrying the lpr gene were found to express a granulocyte marker, Gr-1. Such mice include MRL-lpr/lpr mice and SCG mice. In parallel with an age-associated increase of IL-2Rbeta(low)CD3int cells which contained double-negative CD4-8- and B220+CD2- cells, Gr-1+CD3int cells increased in number in the lymph nodes and other peripheral organs. In addition to a major population of IL-2Rbeta(low)CD3int cells, there is a small population of IL-2Rbeta(high)CD3int cells which produce normal Fas mRNA and Fas molecule from the lpr gene. It was found that both IL-2Rbeta(low)CD3int and IL-2Rbeta(high)CD3int cell populations contained Gr-1+ cells. IL-2Rbeta(high)CD3int cells tended to contain a higher proportion of Gr-1+ cells than did IL-2Rbeta(low)CD3int cells. More interestingly, Gr-1+CD3int cells expressed a considerable level of mRNA of the mG-CSF receptor, similar to granulocytes. The present study thus yielded further information on an unusual property of abnormally expanding CD3int cells in mice carrying the lpr gene.
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9,178,642
|
Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures.
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In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.
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9,178,643
|
Long-term effect of primary immunization on subsequent immune responsiveness.
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Specific antigen/adjuvant combinations preferentially induce type 1 or type 2 cytokine responses. For example, BALB/c mice primed with TNP-ovalbumin in complete Freund's adjuvant (TNP-OVA/CFA) produce a type 2-dominated response characterized by the activation of IL-4-secreting cells and the production of IgG1 and IgE anti-TNP antibodies. In contrast, mice primed with TNP conjugated to Brucella abortus (TNP-BA) produce a type 1 response dominated by the secretion of IFN-gamma and IgG2a anti-TNP antibodies. We examined whether treating young mice with these antigen/adjuvant combinations altered the cytokine profile of their subsequent immune responses. Mice immunized with TNP-BA and boosted several months later with TNP-OVA/CFA developed a cytokine and antibody profile similar to the priming rather than boosting antigen. This was also observed in mice immunized with TNP-OVA/CFA and boosted with TNP-BA. Both the ratio of IL-4:IFN-gamma-secreting cells and the isotype of antibodies produced by these mice were altered by primary immunization. Analysis of Con A-responsive cells from these animals showed that long-lived changes in the frequency of T lymphocytes available to secrete type 1 versus type 2 cytokines were induced by strong primary immunogens.
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9,178,645
|
CD4+ hepatic cancer-specific cytotoxic T lymphocytes in patients with hepatocellular carcinoma.
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We investigated T cell immunity against hepatocellular carcinoma (HCC), and showed that both peripheral blood mononuclear cells and tumor-infiltrating lymphocytes incubated with interleukin-2 alone displayed HLA-nonrestricted but hepatic cancer-specific cytotoxicity in a majority of patients with HCC. Namely, they lysed both HCC and cholangiocellular carcinomas in an HLA-nonrestricted manner, but they did not lyse any tumors with the other histological types tested, normal hepatocytes, or the cells transfected with hepatitis C virus or MUC1 gene. These CTL lines and clones were phenotypically CD3+CD4+CD8-. These unique CTL could play important roles in T cell immunity against HCC.
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9,178,644
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Th1 CD4+ cells adoptively transfer experimental hypersensitivity pneumonitis.
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Cultured cells from Micropolyspora faeni-sensitized donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). To determine whether the CD4+ cells responsible for transfer have characteristics of Th1 or Th2 cells, we established cell lines from lung-associated lymph nodes of M. faeni-sensitized C3H/HeJ mice by culturing with antigen and either IFN-gamma, IL2, and anti-IL4, or IL4. Cell lines were stimulated regularly with antigen, fresh antigen-presenting cells, and the cytokine/anti-cytokine antibody cocktail. At various times after initiation of culture, cells were injected intravenously into recipients, which were then challenged intratracheally with M. faeni and sacrificed and the extent of pulmonary inflammatory response was determined. IFN-gamma, IL4, and IL10 levels were determined in supernatants of cell cultures stimulated with M. faeni to characterize the cell lines as Th1 (IFN-gamma, but low IL4 and IL10 secretion) or Th2 (IL4 and IL10, but low IFN-gamma secretion). Cell lines were differentiated into either Th1 (IFN-gamma = 310 +/- 45 U/ml, IL4 = 0.10 +/- 0.1 U/ml, IL10 = 1750 +/- 75 pg/ ml, >99% CD4+) cell lines by Day 16 of culture or Th2 cell lines (IFN-gamma = 1.8 +/- 1.0 U/ml, IL4 = 830 +/- 388 U/ml, IL10 = 51,700 +/- 10,900 pg/ml, >96% CD4+) by Day 30. Th1 cell lines were able to adoptively transfer EHP whereas Th2 cell lines were unable to adoptively transfer EHP. The ability to transfer EHP was directly related to the amount of IFN-gamma and inversely to the amount of IL4 secreted by antigen-stimulated cells. We conclude that it is possible to produce CD4+ cell lines with either Th1 or Th2 characteristics from lung-associated lymph nodes of mice exposed to M. faeni and that only Th1 CD4+ cell lines can adoptively transfer EHP.
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9,178,646
|
Characterization of a new human macrophage cell line 2MAC. 1. Expression of functional macrophage CD16 (Fc gammaRIIIA/gamma) and tissue factor induction on ligation of HLA-DR.
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A human macrophage-like line, designated 2MAC, has been established from peripheral blood. 2MAC expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules. In particular, 2MAC expresses CD16/Fc gammaRIII, the low-affinity Fc receptor for IgG, as well as high levels of HLA class I and class II. Consistent with this macrophage assignment, we present evidence that 2MAC expresses the macrophage form of CD16, namely, Fc gammaRIIIA/gamma. By several criteria also applicable to signal transducing NK CD16 and T cell CD3/TCR complexes, including modulation from the cell surface and Ca2+ mobilization in response to ligation by specific monoclonal antibody, CD16 expressed by 2MAC is functional. Ligation of 2MAC HLA class II, but not HLA class I, by specific mAb induces an increase in free cytoplasmic Ca2+ concentration ([Ca2+]i). This Ca2+ flux appears to be physiologically relevant, as ligation of HLA-DR, but not HLA class I, by mAb results in the efficient, Ca2+ mobilization-dependent induction of tissue factor by 2MAC. 2MAC, therefore, should prove useful for studying signal transduction through macrophage CD16 and HLA class II.
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9,178,647
|
The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity.
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Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF), which inhibits NK activity and displays an antiabortive effect in mice. Our previous data provide indirect evidence for the importance of PIBF in the maintenance of normal gestation. This study was aimed at investigating whether neutralization of endogenous PIBF production influences pregnancy outcome and if so, what are the mechanisms that participate in this process. Syngeneically pregnant Balb/c mice on Day 8.5 of pregnancy were injected ip with 0.3 mg/kg of RU 486 or with 0.5 mg of rabbit anti-PIBF IgG alone, or together with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. On Day 10.5 the ratio of living and resorbed embryos and NK activity of the spleen cells were determined. In mice treated with anti-PIBF the ratio of resorbed fetuses was significantly higher than that in untreated controls. In RU 486-treated mice we also observed significantly increased resorption rate, which was associated with the inability of spleen cells to produce PIBF. Both anti-PIBF treatment and that with progesterone receptor blocker resulted in increased splenic NK activity. There was a positive relationship between NK activity and the rate of resorptions. All the above effects were corrected by simultaneous treatment with anti-NK or anti-NC (natural cytotoxic) antibodies. These data allow the conclusion that PIBF contributes to normal gestation in mice and that the effect of PIBF is manifested via blocking NK and/or NC activity.
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9,178,648
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Dopamine receptor antagonists block nerve growth factor-induced hyperactivity.
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The role of dopamine receptors in mediating nerve growth factor (NGF)-induced locomotor stimulation was investigated by examining the effects of selective dopamine D1 and D2 receptor antagonists on the motor hyperactivity induced by NGF. A single intracerebroventricular administration of NGF (5.1 microg) increased locomotor activity immediately after injection in normal adult rats. This hyperactivity was partly blocked by the dopamine D1 receptor antagonist SCH23390 (R-(+)-7-chloro-2,3,4,5-tetrahydro-3-methyl-1-phenyl-1H-3-benzazepine-8- ol) and by the dopamine D2 antagonist raclopride ((S)-3,5-dichloro-N-((1-ethyl-2-pyrrolidinyl)methyl)-2-hydroxy-6-methoxy benzamide). Effective doses of raclopride did not alter spontaneous levels of activity in control rats. These results suggest that stimulation of both subtypes of dopamine receptors is necessary for eliciting NGF-induced hyperactivity in the rat. The role of the dopamine D2 receptor in mediating the behavioral actions of NGF appears to be more important than that of the dopamine D1 receptor.
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9,178,649
|
The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures.
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The influence of 2-chloroadenosine, a non-metabolizable adenosine A1 receptor agonist, was tested on the development of electrically kindled amygdala and on the seizure responses of fully kindled rats. Focal intra-amygdaloid injection of 2-chloroadenosine (1-10 nmol/0.5 microl) 20 min before applying the daily kindling stimulus prevented the development of the kindling process. The behavioural seizure score and the afterdischarge duration were reduced below their initial values. The antiepileptogenic effects of 1 and 10 nmol of 2-chloroadenosine were reversible 8-10 days after withdrawal of the drug. When 2-chloroadenosine was tested on fully developed stage 5 amygdala-kindled seizures, it increased the generalised seizure threshold in a dose-dependent manner. A maximum efficiency of 125% (P < 0.001) was achieved with 5 nmol and the median effective dose was 0.55 nmol. Higher doses resulted in the reduced anticonvulsant effect (P < 0.05). With the same daily stimulation, 2-chloroadenosine 5 nmol in 0.5 microl vehicle, significantly reduced the maximum seizure score by 90%, the afterdischarge duration by 88% and completely blocked the generalised seizure duration. The antiseizure activity of the drug lasted for 3 days. In conclusion, 2-chloroadenosine not only acts as an anticonvulsant against electrically induced kindled seizures as described here, and against audiogenic seizures, electroshock and a variety of chemical convulsants as described by others, it prevents the development of the epileptic state by kindling-stimulation, i.e., it is antiepileptogenic. We theorise here that this is due to its blockade of presynaptic glutamate release.
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9,178,650
|
Duration of catalepsy correlates with increased intrastriatal sulpiride.
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To investigate the mechanism underlying sulpiride-induced catalepsy, we simultaneously examined cataleptic behavior and the kinetics of the dopamine receptor antagonist, sulpiride of dopamine, and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), using in vivo voltammetry. After intrastriatal administration of sulpiride to freely moving rats, the levels increased, peaked at 20 min, and remained elevated for more than 3 h. Sulpiride-induced cataleptic behavior also continued for 3 h. Levels of DOPAC peaked 180 min after the injection and did not return to baseline within the experimental period. Thus, the time-course of cataleptic behavior correlated better with elevated extracellular levels of sulpiride than with that of DOPAC. These findings suggest that sulpiride induces catalepsy via a direct action.
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9,178,651
|
The role of 5-HT(1B/1D) receptors in the modulation of 5-hydroxytryptamine levels in the frontal cortex of the conscious guinea pig.
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The role of 5-HT(1B/1D) receptors in modulating extracellular 5-hydroxytryptamine (5-HT) levels in the guinea pig was investigated with the non-selective 5-HT(1B/1D) receptor inverse agonist, methiothepin, and the selective 5-HT(1B/1D) receptor partial agonists, GR 127935 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazole-3-yl)[1,1'-biphenyl]-4-carboxamide) and GR 125743 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-3-methyl-4-(4-pyri dinyl)benzamide). Extracellular 5-HT levels were measured using the technique of brain microdialysis, in the frontal cortex of the freely moving guinea-pig. Extracellular 5-HT was tetrodotoxin sensitive and calcium dependent, and increased when perfused with a high concentration of K+. In addition, extracellular 5-HT levels were lowered by the 5-HT(1B/1D) receptor agonist, sumatriptan, and the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin, while perfusion of the selective serotonin re-uptake inhibitor, paroxetine, increased 5-HT in a concentration-dependent manner. Perfusion of methiothepin, GR 127935 and GR 125743 into the frontal cortex caused significant but transient increases of extracellular 5-HT. However, systemic administration of methiothepin, GR 127935 and GR 125743, at 0.3 mg/kg i.p., produced significant decreases in extracellular 5-HT, to minima of 27 +/- 3%, 31 +/- 12% and 27 +/- 13% of basal, respectively. The increase of extracellular 5-HT, following 5-HT(1B/1D) receptor inverse and partial agonist perfusion into the frontal cortex, was probably a consequence of attenuation of an endogenous 5-HT tone at terminal 5-HT autoreceptors. The unexpected decrease in 5-HT levels following systemic administration may be a result of additional attenuation of endogenous 5-HT tone at cell body autoreceptors in the raphe. Such an increase in local 5-HT levels could then stimulate 5-HT1A receptors to inhibit cell firing and hence decrease 5-HT levels in the terminal regions. This was confirmed when co-administration of the 5-HT1A receptor antagonist, WAY 100635, significantly attenuated the GR 127935 decrease in 5-HT.
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9,178,652
|
The role of spinal delta1-opioid receptors in inhibiting the formalin-induced nociceptive response in diabetic mice.
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Injection of formalin into the hindpaw of mice produced a biphasic nociceptive response consisting of immediate (first-phase) and tonic (second-phase) components. In diabetic mice, the flinching response of the first phase was increased while that in the second phase was decreased in diabetic mice relative to the results in non-diabetic mice. To examine the role of supraspinal and/or spinal endogenous delta1-opioid receptors in inhibiting the formalin-induced nociceptive response in diabetic mice, we assessed the effect of 7-benzylidenenaltrexone, a selective delta1-opioid receptor antagonist, and naltriben, a selective delta2-opioid receptor antagonist, administered either i.c.v. or i.t., on the formalin-induced flinching response. The second-phase response appeared when diabetic mice were pretreated with 7-benzylidenenaltrexone (0.1 and 0.3 mg/kg, s.c.), but not with naltriben (0.3 and 1 mg/kg, s.c.). On the other hand, while 7-benzylidenenaltrexone (0.1, 0.3 and 1 microg/mouse) administered i.t. had no significant effect on the first phase, it significantly and dose-dependently increased the second phase of the formalin-induced flinching response in diabetic mice. 7-Benzylidenenaltrexone (1 and 3 microg/mouse) administered i.c.v. had no significant effect on either the first- or the second-phase response in both non-diabetic and diabetic mice. These results suggest that a spinal delta1-opioid receptor-mediated endogenous antinociceptive system may inhibit the formalin-induced second phase of the nociceptive response in diabetic mice.
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9,178,653
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Cardiac effects of isoliquiritigenin.
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The effects of isoliquiritigenin on force of contraction (Fc), L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat ventricular heart muscle. Isoliquiritigenin increased Fc and I(Ca) and, after longer exposure times, resting tension and [Ca2+]i. The effect of isoliquiritigenin (100 microM) on I(Ca) was diminished by Rp-cAMPS (30 microM). 1H-[1,2,4]oxa- diazolo[4,3-a]quinoxalin-1-one (50 microM) did not influence the effects of isoliquiritigenin on Fc and I(Ca). The positive inotropic effects of isoprenaline and forskolin, but not of 3-isobutyl-1-methylxanthine, were potentiated by isoliquiritigenin (100 microM). In the presence of milrinone (10 microM), no further effects of isoliquiritigenin (100 microM) on Fc and I(Ca) were observed. It is suggested that the increase in Fc and I(Ca) by isoliquiritigenin is due to an accumulation of cyclic AMP. These effects are probably unrelated to an effect of the drug on soluble guanylyl cyclase, as reported for smooth muscle, but rather due to a direct inhibition of phosphodiesterase III activity.
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9,178,654
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Effect of an aldose reductase inhibitor on abnormalities of electroretinogram and vascular factors in diabetic rats.
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The effect of an aldose reductase inhibitor, [5-(3-thienyl) tetrazol-1-yl] acetic acid (TAT), on the electroretinogram was determined in rats with streptozotocin-induced diabetes. Laboratory chow containing 0.05% TAT was given to rats for 2 months, while other diabetic rats were untreated. Groups of TAT-treated and untreated normal rats were also studied. Treatment with TAT produced significant improvement of the electroretinogram. TAT shortened the peak latencies of the b-wave oscillatory potentials, which were significantly prolonged in untreated diabetic rats (P < 0.0001 vs. untreated normal rats). This was accompanied by a significant decrease in the retinal sorbitol and fructose concentrations (by 46.5% and 25.7%, respectively). TAT treatment of diabetic rats also markedly reduced ADP-induced platelet aggregation and significantly increased the red blood cell 2,3-diphosphoglycerate level, accompanied by a marked reduction in sorbitol and fructose concentrations of platelet and red blood cells. There were significant correlations between the summed b-wave peak latencies and platelet aggregation or the 2,3-diphosphoglycerate level in diabetic rats. These findings suggest that an aldose reductase inhibitor, TAT, has therapeutic value for diabetic retinopathy.
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9,178,655
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Endogenous nitric oxide inhibits leukotriene B4 release from rat alveolar macrophages.
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Effects of endogenous nitric oxide (NO) on the release of mediators of the lipoxygenase and cyclo-oxygenase pathway from rat alveolar macrophages were studied. Alveolar macrophages, freshly isolated or after 18-h culture, were incubated in (amino acid-free) Krebs medium and labelled with [3H]arachidonic acid. The release of [3H]leukotriene B4 and [3H]prostanoids (separated by high performance liquid chromatography) was determined. A 23187 was used as stimulus, as rising intracellular Ca2+ activates directly the phospholipase A2 and lipoxygenase pathway. A 23187 (10 microM) enhanced [3H]leukotriene B4 release from freshly prepared alveolar macrophages about 65-fold, but only 5- to 6-fold from cultured alveolar macrophages. Evoked [3H]leukotriene B4 release and spontaneous [3H]prostanoid release were inhibited when L-arginine (300 microM) was added to the Krebs incubation medium of alveolar macrophages, in which marked NO synthase had been induced by culture with lipopolysaccharides (10 microg/ml). Inhibitory effects of L-arginine were prevented by N(G)-monomethyl-L-arginine (L-NMMA, 100 microM). Inhibition of NO synthase during the culture period by L-NMMA (culture medium, in contrast to Krebs medium, already contains the substrate of NO synthase, L-arginine), resulted in attenuation of the 'culture-dependent' decline of the evoked release of [3H]leukotriene B4 and allowed lipopolysaccharides to cause an increase in spontaneous [3H]prostanoid release (i.e., to induce cyclo-oxygenase activity). In conclusion, in rat alveolar macrophages, endogenous NO appears to inhibit the release of mediators of the cyclo-oxygenase and lipoxygenase pathway through multiple sites of action.
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9,178,657
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Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons.
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Pyrroloquinoline quinone may act as a free radical scavenger and also as a modulator of the NMDA receptor associated redox modulatory site. Using the oxidation sensitive dye dihydroethidium, we examined the effects of pyrroloquinoline quinone on free radical production in cultured forebrain neurons following glutamate receptor activation. Both glutamate (100 microM) and hydrogen peroxide (30 mM) produced a rapid increase in dihydroethidium fluorescence indicating dye oxidation. Pyrroloquinoline quinone (5-200 microM) effectively inhibited dihydroethidium fluorescence induced by glutamate but not by hydrogen peroxide. Glutamate-induced dihydroethidium fluorescence was inhibited by the thiol oxidant 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). Pyrroloquinoline quinone (50 microM) inhibited glutamate responses in control and in dithiothreitol treated neurons. However, pyrroloquinoline quinone did not further decrease the response to glutamate in DTNB treated neurons. These results suggest that pyrroloquinoline quinone inhibits the free radical-generating response to glutamate by oxidizing the NMDA receptor redox site and not by scavenging reactive oxygen species.
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9,178,656
|
Bradykinin-induced knee joint incapacitation involves bradykinin B2 receptor mediated hyperalgesia and bradykinin B1 receptor-mediated nociception.
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The participation of B1 and B2 types of bradykinin receptors was studied in the rat knee-joint incapacitation test. Five intra-articular successive hourly administrations of bradykinin produced progressive incapacitation, thus indicating that bradykinin induced sensitization to its own nociceptive effect. Four co-injections of bradykinin with the bradykinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin were without nociceptive effect. However, a 5th injection of bradykinin alone produced intense incapacitation. The bradykinin B2 receptor antagonist HOE-140 ([D-Arg)[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), or indomethacin, prevented the bradykinin-induced incapacitation. However, successive co-injections of bradykinin with prostaglandin E2, in contrast to bradykinin alone, did induce incapacitation in animals pretreated with indomethacin or HOE-140. The injection of the bradykinin B1 receptor agonist des-Arg9-bradykinin into prostaglandin E2-treated joints did induce incapacitation, although administration of the bradykinin B1 receptor agonist or prostaglandin E2 alone did not induce incapacitation. In conclusion, in ongoing articular inflammation, it is suggested that the bradykinin B1 receptor is particularly involved with nociceptor activation, while the bradykinin B2 receptor is related to nociceptor sensitization.
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9,178,658
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Influence of receptor density on the patterns of beta2-adrenocepter desensitization.
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Sustained stimulation of the beta2-adrenoceptor leads to a desensitization of the receptor-mediated adenylyl cyclase stimulation. While desensitization promoted by nanomolar concentrations of isoproterenol involves the phosphorylation of the beta2-adrenoceptor by protein kinase A alone, both protein kinase A- and beta-adrenoceptor kinase-mediated phosphorylation leading to the binding of beta-arrestin contribute to the desensitization evoked by micromolar concentrations of agonist. In the present study, we assessed the influence of receptor density on the patterns of desensitization induced by these two different levels of stimulation. Murine L cells were transfected with a cDNA encoding the human beta2-adrenoceptor and clonal cell lines expressing various levels of beta2-adrenoceptor were used for the study. In cell lines expressing the highest number of receptor, approx. 150000 sites/cell (approx. 3000 fmol/mg of membrane proteins), pretreatment with micromolar concentrations of isoproterenol causes a desensitization pattern characterized by a reduction in both the potency and the efficacy of isoproterenol to further stimulate the adenylyl cyclase activity. In contrast, desensitization induced by 10 nM isoproterenol resulted only in a decrease in the potency of isoproterenol. This distinct pattern of desensitization is not seen in cells expressing 12000 receptors/cell (approx. 200 fmol/mg of membrane proteins) and, in that case, pretreatment with 10 nM isoproterenol leads to a reduction in both the sensitivity and the maximal response. Similar effects on the beta-adrenoceptor-stimulated adenylyl cyclase were observed in these cells following treatment with dibutyryl cAMP. Receptor density therefore dramatically influences the pattern of desensitization evoked by low level of stimulation. The results also demonstrate that although different molecular events are involved in the desensitization evoked by different levels of stimulation, its phenotypic expression can be qualitatively identical in cells expressing a relatively small number of receptors. Hence, protein kinase A-mediated desensitization cannot be qualitatively distinguished from the beta-adrenoceptor kinase-mediated process in these cells.
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9,178,659
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Characterization of metabotropic glutamate receptors in rat C6 glioma cells.
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Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[3H]glutamate and [3H(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ([3H](+/-)-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a Kd = 1250 +/- 101 nM and Bmax = 12.1 +/- 1.8 pmol/mg protein when the assays were performed with L-[3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When [3H](+/-)-trans-ACPD was used as radioligand, the kinetic parameters obtained were Kd = 2605 +/- 1042 nM and Bmax = 13.66 +/- 5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[3H]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity L-glutamate > L-quisqualic acid > (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) > ibotenic acid >>> (2S, 'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[3H]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the L-glutamate dose-response curve, increasing the IC50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.
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9,178,660
|
Role of glutathione metabolism in the glutamate-induced programmed cell death of neuronal-like PC12 cells.
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In addition to its well-known interaction with ionotropic and metabotropic receptors, glutamate may, at high concentrations, interfere with a cystine-glutamate antiport designated as Xc- and lead to a significant decrease in cystine uptake and intracellular glutathione level. These effects, in turn, may induce death in various cellular bodies including astrocytes, rat glioma cells and cortical neurons in culture. In the present paper we demonstrate that the toxicity evoked by glutamate in a neuronal-like model is indeed related to the metabolism of glutathione since glutamate toxicity is preceded by a significant depletion of intracellular glutathione and is abolished in the presence of precursors of glutathione synthesis such as cystine and N-acetylcysteine. It also appears that prolonged incubation in cystine-free medium leads to cell detachment and death, a phenomenon which is progressively abolished in the presence of increasing concentrations of cystine. In addition, buthionine sulfoximine, a known inhibitor of glutathione synthesis, also induces cell lysis with a time-course very similar to that of glutamate. However, depletion of glutathione is probably not sufficient to trigger the death signal since cycloheximide, which inhibits the toxic effect of both glutamate and buthionine sulfoximine, does not block the decrease in cellular glutathione content induced by these drugs. Our results therefore confirm that oxidative stress and intracellular glutathione depletion are able to trigger programmed cell death in neuronal-like cells, although the exact nature of the death mechanisms remains largely unknown.
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9,178,661
|
Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor.
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The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.
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9,178,668
|
Surveillance of patients with Barrett's esophagus for dysplasia and cancer with balloon cytology.
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A less costly cancer surveillance method for Barrett's esophagus is desirable. The aim of this study was to compare nonendoscopic balloon cytology with biopsy and brush cytology for detecting dysplasia and carcinoma in patients with Barrett's esophagus.
|
9,178,669
|
Speed of healing and symptom relief in grade II to IV gastroesophageal reflux disease: a meta-analysis.
|
Esophagitis healing proportions are often incorrectly called the healing rate. The aim of this study was to compare different drug classes by expressing the speed of healing and symptom relief through a new approach.
|
9,178,670
|
Large paraesophageal varices on endosonography predict recurrence of esophageal varices and rebleeding.
|
Recurrence of varices and rebleeding after endoscopic therapy is very common. Data on the prediction of recurrent varices after initial obliteration by endoscopic therapy are few. The aim of this study was to correlate the presence and the size of paraesophageal varices (PEVs) in patients after endoscopic variceal ligation with recurrent varices and rebleeding.
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9,178,671
|
Famotidine for healing and maintenance in nonsteroidal anti-inflammatory drug-associated gastroduodenal ulceration.
|
Nonsteroidal anti-inflammatory drugs (NSAIDs) are strongly associated with gastroduodenal ulceration. How to manage patients with NSAID-associated ulcers is a common clinical dilemma. High-dose famotidine in the healing and maintenance of NSAID-associated gastroduodenal ulceration was therefore evaluated.
|
9,178,672
|
Thalidomide: a novel therapy for microsporidiosis.
|
Microsporidiosis is a common cause of chronic diarrhea in human immunodeficiency virus (HIV)-seropositive individuals and often does not respond to treatment. Fecal tumor necrosis factor alpha (TNF-alpha) is elevated in microsporidiosis; therefore, thalidomide, an anti-TNF-alpha agent, was used as therapy.
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9,178,673
|
The prevalence and causes of chronic diarrhea in patients with celiac sprue treated with a gluten-free diet.
|
The majority of patients with celiac sprue experience diarrhea before diagnosis. There have been no studies of the prevalence or causes of chronic diarrhea in these patients after treatment with a gluten-free diet.
|
9,178,674
|
Role of pancreatic impairment in growth recovery during gluten-free diet in childhood celiac disease.
|
Clinical significance and duration of insufficient release of pancreatic enzymes in childhood celiac disease have not been clarified. The aim of this study was to evaluate the role that pancreatic impairment plays in growth recovery and the duration of this impairment.
|
9,178,675
|
Genetic markers may predict disease behavior in patients with ulcerative colitis.
|
Recent studies have suggested that HLA DRB1*0103 and allele 2 of the interleukin 1 receptor antagonist (IL-1RA) gene predict severe and extensive ulcerative colitis, respectively. The aim of this study was to test these hypotheses in patients undergoing surgery for their colitis.
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9,178,676
|
Quantification of the placebo response in ulcerative colitis.
|
There is consistently a measurable benefit noted among placebo users in treatment trials of ulcerative colitis (UC). The aim of this study was to define the placebo response in active UC and identify study features that influence the placebo response.
|
9,178,677
|
The effect of gastroenterology training on the efficiency and cost of care provided to patients with diverticulitis.
|
National trends emphasize the need for cost-efficient medical care with no diminution in quality. The most appropriate role for various physician groups has yet to be determined. The aim of this study was to investigate the efficiency of medical care provided by family practitioners (FPs), internists (IMs), and gastroenterologists (GIs) for acute diverticulitis.
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9,178,678
|
Virtual colonoscopy with magnetic resonance imaging: in vitro evaluation of a new concept.
|
Screening for colonic polyps is desirable. A new concept based on cross-sectional and endoscopic analysis of a magnetic resonance (MR) data set is presented.
|
9,178,679
|
Deletion analysis of the p16 tumor suppressor gene in gastrointestinal mucosa-associated lymphoid tissue lymphomas.
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The molecular mechanisms responsible for initiation and progression of gastrointestinal mucosa-associated lymphoid tissue (MALT) lymphomas are largely unknown. The aim of this study was to analyze the p16 tumor suppressor gene in MALT lymphomas of the stomach and colon.
|
9,178,680
|
CD4+ T-cell population mediates development of inflammatory bowel disease in T-cell receptor alpha chain-deficient mice.
|
Increase of T cells expressing CD4 and T-cell receptor (TCR) alpha- beta+ (beta[dim]) was observed in the mucosal and peripheral lymphoid tissues of TCR alpha-/- mice with inflammatory bowel disease (IBD). The aim of this study was to characterize the CD4+ TCR alpha-beta+ T cells.
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9,178,681
|
Early functional effects of Clostridium difficile toxin A on human colonocytes.
|
Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells.
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9,178,682
|
Enhanced leukocyte binding by intestinal microvascular endothelial cells in inflammatory bowel disease.
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Microvascular endothelial cells mediate leukocyte homing, angiogenesis, and inflammation and healing and show tissue-specific adhesion molecules and functions. The activation of human intestinal mucosal microvascular endothelial cells (HIMECs) was studied in vitro to uncover possible abnormalities associated with inflammatory bowel disease.
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9,178,683
|
Pattern of adhesion molecule expression on vascular endothelium in Helicobacter pylori-associated antral gastritis.
|
The inflammatory response in Helicobacter pylori-associated gastritis (HAG) is characterized by an intense infiltrate of granulocytes and lymphocytes. The emigration of white blood cells into sites of inflammation is mediated by receptors on endothelial cells and on blood leukocytes. The aim of this study was to characterize endothelial adhesion molecule expression in HAG leading to leukocyte infiltration.
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9,178,684
|
Overexpressed nitric oxide synthase in portal-hypertensive stomach of rat: a key to increased susceptibility to damage?
|
Portal hypertension predisposes gastric mucosa to increased injury. The aim of this study was to determine whether overexpression of constitutive nitric oxide synthase (cNOS) is responsible for increased susceptibility of portal-hypertensive (PHT) gastric mucosa to damage.
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9,178,685
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Deranged hydrophobic barrier of the rat gastroduodenal mucosa after parenteral nonsteroidal anti-inflammatory drugs.
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Parenteral administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may cause gastrointestinal mucosal lesions. The aim of this study was to investigate whether parenteral NSAIDs alter surface hydrophobicity of the gastroduodenal mucosa.
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9,178,686
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The effect of gastrin-releasing peptide on gastrin and somatostatin messenger RNAs in humans infected with Helicobacter pylori.
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Gastrin-releasing peptide stimulates gastrin secretion but also inhibits its release via somatostatin. Exogenous gastrin-releasing peptide stimulates a greater increase in plasma gastrin concentrations in patients infected with Helicobacter pylori than in uninfected controls. Because this infection suppressed gastric mucosal somatostatin, we studied whether the increased gastrin response was a result of an abnormal response of the somatostatin cell.
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9,178,687
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Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract.
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The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH).
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9,178,688
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Oxyntomodulin stimulates intestinal glucose uptake in rats.
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Enteroglucagon peptides have long been proposed as mediators of intestinal adaptation, including mucosal growth and nutrient absorptive capacity. The hypothesis that infusions of oxyntomodulin, a bioactive form of enteroglucagon, would stimulate glucose and amino acid uptake was tested and its effects were compared with those of glucagon.
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9,178,690
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Gum arabic promotes rat jejunal sodium and water absorption from oral rehydration solutions in two models of diarrhea.
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We have shown that addition of gum arabic (GA) to a 90 mmol/L sodium-111 mmol/L glucose oral rehydration solution (ORS) enhances its effectiveness for water and electrolyte absorption in normal rats. The present study extends these observations on GA in ORS to two rat models of diarrheal disease.
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9,178,689
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Effects of intestinal stasis on intercellular adhesion molecule 1 expression in the rat: role of enteric bacteria.
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The mechanisms underlying the inflammatory changes associated with intestinal stasis are poorly understood. The objective of this study was to assess whether endothelial expression of intercellular adhesion molecule 1 (ICAM-1) and leukocyte recruitment are altered after intestinal stasis.
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