bluuebunny/crossref_metadata_2025 huggingface dataset:
Excitation of rat colonic afferent fibres by 5‐HT3 receptors
Authors: Hicks Gareth A. , Coldwell Jonathan R. , Schindler Marcus , Ward Philip A. Bland , Jenkins David , Lynn Penny A. , Humphrey Patrick P. A. , Blackshaw L. Ashley
Published: November 2002

The gastrointestinal tract contains most of the body's 5‐hydroxytryptamine (5‐HT) and releases large amounts after meals or exposure to toxins. Increased 5‐HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5‐HT levels correlate with pain episodes. 5‐HT3 receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored. Here we investigated effects of 5‐HT on sensory afferents from the colon and the expression of 5‐HT3 receptors on their cell bodies in the dorsal root ganglia (DRG). Distal colon, inferior mesenteric ganglion and the lumbar splanchnic nerve bundle (LSN) were placed in a specialized organ bath. Eighty‐six single fibres were recorded from the LSN. Three classes of primary afferents were found: 70 high‐threshold serosal afferents, four low‐threshold muscular afferents and 12 mucosal afferents. Afferent cell bodies were retrogradely labelled from the distal colon to the lumbar DRG, where they were processed for 5‐HT3 receptor‐like immunoreactivity. Fifty‐six percent of colonic afferents responded to 5‐HT (between 10−6 and 10−3 M) and 30 % responded to the selective 5‐HT3 agonist, 2‐methyl‐5‐HT (between 10−6 and 10−2 M). Responses to 2‐methyl‐5‐HT were blocked by the 5‐HT3 receptor antagonist alosetron (2 × 10−7 M), whereas responses to 5‐HT were only partly inhibited. Twenty‐six percent of L1 DRG cell bodies retrogradely labelled from the colon displayed 5‐HT3 receptor‐like immunoreactivity. We conclude that colonic sensory neurones expressing 5‐HT3 receptors also functionally express the receptors at their peripheral endings. Our data reveal actions of 5‐HT on colonic afferent endings via both 5‐HT3 and non‐5‐HT3 receptors.
Oestrogen augments the fetal ovine hypothalamus–pituitary–adrenal axis in response to hypotension
Authors: Purinton Scott C. , Wood Charles E.
Published: November 2002

In the fetal sheep, parturition is triggered by an increase in the activity of the fetal hypothalamus‐ pituitary‐adrenal (HPA) axis which, in turn, augments the biosynthesis of oestrogen by the placenta. Parturition can be prevented or delayed by destruction of the paraventricular nucleus (PVN), pituitary or adrenal, or stimulated by infusions of adrenocorticotropin (ACTH) or glucocorticoids. We have previously reported that physiological increases in fetal plasma concentrations of oestradiol have a neuroendocrine effect to increase both basal and hypotension‐stimulated ACTH secretion. The present study was performed to test the effect of oestradiol on the central baroreceptor and chemoreceptor reflex pathways. We used immunohistological techniques to identify various neuroanatomical regions which are activated by hypotension and, subsequently, those areas modified by oestrogen's action and baroreceptor and chemoreceptor denervation. We assessed cellular activation in these brain regions by immunostaining for Fos, the protein product of c‐fos, an immediate early response gene. We found that oestradiol increased Fos abundance in nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVLM), and PVN, and augmented the increase in Fos in these regions in response to a 10 min period of brachiocephalic arterial occlusion (BCO). Carotid sinus denervation blocked the Fos response to BCO, but not to oestrogen alone, in these regions. In contrast, the hippocampus responded to BCO with increase Fos in intact fetuses, but did not respond to oestrogen treatment. None of the treatments altered Fos expression in cerebral cortex or in cerebellum. We conclude that oestradiol augments the activity of the central baroreceptor and chemoreceptor reflex pathways, and that it may influence fetal ACTH secretion via this site of action.
Antioxidants prevent depression of the acute hypoxic ventilatory response by subanaesthetic halothane in men
Authors: Teppema Luc J. , Nieuwenhuijs Diederik , Sarton Elise , Romberg Raymonda , Olievier Cees N. , Ward Denham S. , Dahan Albert
Published: November 2002

We studied the effect of the antioxidants (AOX) ascorbic acid (2 g, I.V.) and α‐tocopherol (200 mg, P.O.) on the depressant effect of subanaesthetic doses of halothane (0.11 % end‐tidal concentration) on the acute isocapnic hypoxic ventilatory response (AHR), i.e. the ventilatory response upon inhalation of a hypoxic gas mixture for 3 min (leading to a haemoglobin saturation of 82 ± 1.8 %) in healthy male volunteers. In the first set of protocols, two groups of eight subjects each underwent a control hypoxic study, a halothane hypoxic study and finally a halothane hypoxic study after pretreatment with AOX (study 1) or placebo (study 2). Halothane reduced the AHR by more than 50 %, from 0.79 ± 0.31 to 0.36 ± 0.14 l min−1 %−1 in study 1 and from 0.79 ± 0.40 to 0.36 ± 0.19 l min−1 %−1 in study 2, P < 0.01 for both. Pretreatment with AOX prevented this depressant effect of halothane in the subjects of study 1 (AHR returning to 0.77 ± 0.32 l min−1 %−1, n.s. from control), whereas placebo (study 2) had no effect (AHR remaining depressed at 0.36 ± 0.27 l min−1 %−1, P < 0.01 from control). In a second set of protocols, two separate groups of eight subjects each underwent a control hypoxic study, a sham halothane hypoxic study and finally a sham halothane hypoxic study after pretreatment with AOX (study 3) or placebo (study 4). In studies 3 and 4, sham halothane did not modify the control hypoxic response, nor did AOX (study 3) or placebo (study 4). The 95 % confidence intervals for the ratio of hypoxic sensitivities, (AOX + halothane) : halothane in study 1 and (AOX ‐ sham halothane) : sham halothane in study 3, were [1.7, 2.6] and [1.0, 1.2], respectively. Because the antioxidants prevented the reduction of the acute hypoxic response by halothane, we suggest that this depressant effect may be caused by reactive species produced by a reductive metabolism of halothane during hypoxia or that a change in redox state of carotid body cells by the antioxidants prevented or changed the binding of halothane to its effect site. Our findings may also suggest that reactive species have an inhibiting effect on the acute hypoxic ventilatory response.
Recruitment of Ca2+ release channels by calcium‐induced Ca2+ release does not appear to occur in isolated Ca2+ release sites in frog skeletal muscle
Authors: Fénelon Karine , Pape Paul C.
Published: November 2002

Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle in response to small depolarisations (e.g. to ‐60 mV) should be the sum of release from many isolated Ca2+ release sites. Each site has one SR Ca2+ release channel activated by its associated T‐tubular voltage sensor. The aim of this study was to evaluate whether it also includes neighbouring Ca2+ release channels activated by Ca‐induced Ca2+ release (CICR). Ca2+ release in frog cut muscle fibres was estimated with the EGTA/phenol red method. The fraction of SR Ca content ([CaSR]) released by a 400 ms pulse to ‐60 mV (denoted fCa) provided a measure of the average Ca2+ permeability of the SR associated with the pulse. In control experiments, fCa was approximately constant when [CaSR] was 1500‐3000 μm (plateau region) and then increased as [CaSR] decreased, reaching a peak when [CaSR] was 300‐500 μm that was 4.8 times larger on average than the plateau value. With 8 mm of the fast Ca2+ buffer BAPTA in the internal solution, fCa was 5.0‐5.3 times larger on average than the plateau value obtained before adding BAPTA when [CaSR] was 300‐500 μm. In support of earlier results, 8 mm BAPTA did not affect Ca2+ release in the plateau region. At intermediate values of [CaSR], BAPTA resulted in a small, if any, increase in fCa, presumably by decreasing Ca inactivation of Ca2+ release. Since BAPTA never decreased fCa, the results indicate that neighbouring channels are not activated by CICR with small depolarisations when [CaSR] is 300‐3000 μm.
Carbohydrate ingestion reduces skeletal muscle acetylcarnitine availability but has no effect on substrate phosphorylation at the onset of exercise in man
Authors: Watt Matthew J. , Heigenhauser G. J. F. , Stellingwerff Trent , Hargreaves Mark , Spriet Lawrence L.
Published: November 2002

This study investigated the effect of reduced acetylcarnitine availability on oxidative metabolism during the transition from rest to steady‐state exercise. Eight male subjects completed two randomised exercise trials at 68 % of the peak rate of O2 uptake (V̇O2,peak). On one occasion subjects ingested 1 g (kg body mass)−1 glucose 75 min prior to exercise (CHO), whereas the other trial acted as a control (CON). Muscle samples were obtained pre‐ and 75 min post‐ingestion, and following 1 and 10 min of exercise. Plasma glucose and insulin were elevated (P < 0.05), and plasma free fatty acids (FFA) were lower at the onset of exercise in CHO. Acetylcarnitine (CON, 4.8 ± 1.8; CHO, 1.5 ± 0.9 mmol (kg dry mass (d.m.))−1, P < 0.05) and acetyl CoA (CON, 13.2 ± 2.3; CHO, 6.3 ± 0.6 μmol (kg d.m.)−1, P < 0.05) were lower at rest, whereas pyruvate dehydrogenase activation (PDHa) was greater in CHO compared with CON (CON, 0.78 ± 0.07; CHO, 1.44 ± 0.19 mmol min−1 (kg wet mass (w.m.))−1). Respiratory exchange ratio (RER) was significantly elevated during exercise in CHO. The acetyl groups increased at similar rates at the onset of exercise (1 min) and there was no difference in substrate phosphorylation as determined from lactate accumulation and phosphocreatine degradation between trials. Subsequently, oxidative metabolism during the transition from rest to steady‐state exercise was not affected by prior carbohydrate ingestion. Although exercise resulted in the rapid activation of PDH in both trials, PDHa was greater at 1 min in CHO (CON, 2.36 ± 0.22; CHO, 2.91 ± 0.18 mmol min−1 (kg w.m.)−1). No differences in muscle metabolite levels and PDHa were observed after 10 min of moderate exercise between trials. In summary, at rest, carbohydrate ingestion induced multiple metabolic changes which included decreased acetylcarnitine availability and small increases in PDHa. The prior changes in PDHa and acetylcarnitine availability had no effect on substrate phosphorylation and oxidative metabolism at the onset of exercise. These data suggest that acetylcarnitine availability is unlikely to be the site of metabolic inertia during the transition from rest to steady‐state moderate intensity exercise.
Decreased shortening velocity and altered myosin isoforms in guinea‐pig hypertrophic intestinal smooth muscle
Authors: Löfgren Mia , Fagher Katarina , Wede Oskar Karlsson , Arner Anders
Published: November 2002

The aims of this study were to investigate whether hypertrophy of the small intestinal smooth muscle leads to alterations of myosin isoform composition and shortening velocity and whether possible changes correlate with a change in the sensitivity to ADP of shortening velocity in this tissue. A partial occlusion was introduced in the distal part of the ileum of guinea‐pigs. After 2 weeks, the part of the small intestine just proximal of the stenosis was hypertrophied (indicated by a significantly increased cross‐sectional area). The most proximal part of the small intestine was used as control, thus enabling comparisons between hypertrophic and normal tissue from the same animal. The outer longitudinal layer of the intestinal wall was gently peeled off and used for biochemistry, RT‐PCR and mechanical experiments. The desmin/actin ratio was significantly increased following hypertrophy, although myosin and actin expression were similar in control and hypertrophic tissue. In hypertrophic tissue, the myosin heavy chain mRNA with a 21 base pair insert decreased significantly. The composition of the mRNA encoding the myosin essential light chains changed towards more of the basic type (LC17b). No change in the expression of non‐muscle myosin heavy chains A and B was detected. The maximal shortening velocity (Vmax) of maximally activated skinned preparations was significantly lower in the hypertrophic tissue (≈50 % of control). The sensitivity of Vmax to ADP was increased in the hypertrophic smooth muscle tissue. We conclude that myosin expression is altered following intestinal hypertrophy and that these alterations affect reactions in the cross‐bridge interaction, leading to a slower and more economical contractile function.
Lactate and glucose interactions during rest and exercise in men: effect of exogenous lactate infusion
Authors: Miller Benjamin F. , Fattor Jill A. , Jacobs Kevin A. , Horning Michael A. , Navazio Franco , Lindinger Michael I. , Brooks George A.
Published: November 2002

To test the hypothesis that lactate plays a central role in the distribution of carbohydrate (CHO) potential energy for oxidation and glucose production (GP), we performed a lactate clamp (LC) procedure during rest and moderate intensity exercise. Blood [lactate] was clamped at ≈4 mm by exogenous lactate infusion. Subjects performed 90 min exercise trials at 65 % of the peak rate of oxygen consumption (V̇O2,peak; 65 %), 55 % V̇O2,peak (55 %) and 55 % V̇O2,peak with lactate clamped to the blood [lactate] that was measured at 65 % V̇O2,peak (55 %‐LC). Lactate and glucose rates of appearance (Ra), disappearance (Rd) and oxidation (Rox) were measured with a combination of [3‐13C]lactate, H13CO3−, and [6,6‐2H2]glucose tracers. During rest and exercise, lactate Ra and Rd were increased at 55 %‐LC compared to 55 %. Glucose Ra and Rd were decreased during 55 %‐LC compared to 55 %. Lactate Rox was increased by LC during exercise (55 %: 6.52 ± 0.65 and 55 %‐LC: 10.01 ± 0.68 mg kg−1 min−1) which was concurrent with a decrease in glucose oxidation (55 %: 7.64 ± 0.4 and 55 %‐LC: 4.35 ± 0.31 mg kg−1 min−1). With LC, incorporation of 13C from tracer lactate into blood glucose (L → GNG) increased while both GP and calculated hepatic glycogenolysis (GLY) decreased. Therefore, increased blood [lactate] during moderate intensity exercise increased lactate oxidation, spared blood glucose and decreased glucose production. Further, exogenous lactate infusion did not affect rating of perceived exertion (RPE) during exercise. These results demonstrate that lactate is a useful carbohydrate in times of increased energy demand.
Differential exocytosis from human endothelial cells evoked by high intracellular Ca2+ concentration
Authors: Zupančič G. , Ogden D. , Magnus C. J. , Wheeler‐Jones C. , Carter T. D.
Published: November 2002

Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high‐resolution membrane capacitance (Cm) measurements. The total whole‐cell Cm and the amplitudes and times of discrete femtoFarad (fF)‐sized Cm steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca2+]i was elevated by intracellular photolysis of calcium‐DM‐nitrophen to evoke secretion and monitored with the low‐affinity Ca2+ indicator furaptra. Sustained elevation of [Ca2+]i to > 20 μm evoked large, slow increases in Cm of up to 5 pF in 1‐2 min. Exocytotic and endocytotic steps of amplitude 0.5‐110 fF were resolved, and accounted on average for ≈33 % of the total Cm change. A prominent component of Cm steps of 2.5‐9.0 fF was seen and could be attributed to exocytosis of von‐Willebrand‐factor‐containing Weibel‐Palade bodies (WPb), based on the near‐identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5‐9.0 fF Cm steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca2+]i elevation, whereas total Cm increased immediately due to the secretion of small, non‐WPb granules. The results show that following a large increase of [Ca2+]i, corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9‐110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.
Increased incidence of gap junctional coupling between spinal motoneurones following transient blockade of NMDA receptors in neonatal rats
Authors: Mentis George Z. , Díaz Eugenia , Moran Linda B. , Navarrete Roberto
Published: November 2002

Neonatal rat motoneurones are electrically coupled via gap junctions and the incidence of this coupling declines during postnatal development. The mechanisms involved in this developmental regulation of gap junctional communication are largely unknown. Here we have studied the role of NMDA receptor‐mediated glutamatergic synaptic activity in the regulation of motoneurone coupling. Gap junctional coupling was demonstrated by the presence of graded, short latency depolarising potentials following ventral root stimulation, and by the transfer of the low molecular weight tracer Neurobiotin to neighbouring motoneurones. Sites of close apposition between the somata and/or dendrites of the dye‐coupled motoneurones were identified as potential sites of gap junctional coupling. Early postnatal blockade of the NMDA subtype of glutamate receptors using the non‐competitive antagonist dizocilpine maleate (MK801) arrested the developmental decrease in electrotonic and dye coupling during the first postnatal week. These results suggest that the postnatal increase in glutamatergic synaptic activity associated with the onset of locomotion promote the loss of gap junctional connections between developing motoneurones.
Fast synaptic transmission mediated by α‐bungarotoxin‐sensitive nicotinic acetylcholine receptors in lamina X neurones of neonatal rat spinal cord
Authors: Bradaïa A. , Trouslard J.
Published: November 2002

Using patch clamp recordings on neonatal rat spinal cord slices, we have looked for the presence of α‐bungarotoxin‐sensitive nicotinic ACh receptors (nAChRs) on sympathetic preganglionic neurones (SPNs) surrounding the central canal of the spinal cord (lamina X) and examined whether they were implicated in a fast cholinergic synaptic transmission. SPNs were identified either by their morphology using biocytin in the recording electrode and/or by antidromic stimulation of the ventral rootlets. The selective α7‐containing nAChR (α7*nAChR) agonist choline (10 mm) induced a fast, rapidly desensitizing inward current, which was fully blocked by α‐bungarotoxin (α‐BgT; 50 nm) and strychnine (1 μm), two antagonists of α7*nAChRs. TheI‐Vrelationship of the choline‐induced current showed a strong inward‐going rectification. Electrically evoked excitatory postsynaptic currents (eEPSCs) could be recorded. At ‐60 mV, eEPSCs peaked at ‐26.2 pA and decayed monoexponentially with a mean time constant of 8.5 ms. The current‐voltage relationship for eEPSCs exhibited a strong inward rectification and a reversal potential close to 0 mV, compatible with a non‐selective cationic current. The appearance of eEPSCs was entirely suppressed by the application of 100 μmACh or nicotine. Choline (10 mm) and 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP; 100 μm) both reduced the amplitude of eEPSCs, whereas cytisine (100 μm) had no effect. Strychnine (1 μm) and α‐BgT (50 nm) both suppressed the eEPSCs. Blocking the P2X purinergic and 5‐HT3receptors had no effect on eEPSCs. DMPP induced four types of current, which differed in their onset and desensitization rate. The most frequently encountered responses were insensitive to the action of strychnine and α‐BgT, and were reproduced by ACh and nicotine but not by cytisine. We conclude that SPNs of the lamina X express several classes of nAChRs and in particular α‐BgT‐sensitive nAChRs. This is the first demonstration in a mammalian spinal cord preparation of a fast cholinergic neurotransmission in which α‐BgT‐sensitive nicotinic receptors are involved.
Contributions of the non‐α subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor
Authors: Akk Gustav
Published: November 2002

The agonist binding site of the nicotinic acetylcholine receptor has a loop‐based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites δ57 and δ59, and the equivalent residues in the ε subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites δ57, δ59, ε55 and ε57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell‐attached single‐channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the δ57/ε55 and the δ59/ε57 site, a mutation in the ε subunit had a greater effect on channel gating than a mutation in the δ subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the δ57 and δ59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the ε subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.
Serotonin 5‐HT3 receptors in rat CA1 hippocampal interneurons: functional and molecular characterization
Authors: Sudweeks Sterling N. , van Hooft Johannes A. , Yakel Jerrel L.
Published: November 2002

The molecular makeup of the serotonin 5‐HT3 receptor (5‐HT3R) channel was investigated in rat hippocampal CA1 interneurons in slices using single‐cell RT‐PCR and patch‐clamp recording techniques. We tested for the expression of the 5‐HT3A (both short and long splice variants) and 5‐HT3B subunits, as well as the expression of the α4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co‐assemble with the 5‐HT3A subunit in heterologous expression systems. Both the 5‐HT3A‐short and α4‐nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5‐HT3B or the 5‐HT3A‐long subunits. Furthermore, there was a strong tendency for the 5‐HT3A‐short and α4‐nAChR subunits to be co‐expressed in individual interneurons. To assess whether there was any functional evidence for co‐assembly between the 5‐HT3A‐short and α4‐nAChR subunits, we used the sulphydryl agent 2‐aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5‐HT3Rs that contain the α4‐nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5‐HT3R‐mediated responses, which is consistent with the notion that the α4‐nAChR subunit co‐assembles with the 5‐HT3A subunit to form a native heteromeric 5‐HT3R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single‐channel properties of the 5‐HT3R were investigated in outside‐out patches. No resolvable single‐channel currents were observed. Using non‐stationary fluctuation analysis, we obtained an estimate of the single‐channel conductance of 4 pS, which is well below that expected for channels containing both the 5‐HT3A and 5‐HT3B subunits.
Regulation of the sodium bicarbonate cotransporter knbc1 function: role of Asp986, Asp988 and kNBC1‐carbonic anhydrase II binding
Authors: Gross Eitan , Pushkin Alexander , Abuladze Natalia , Fedotoff Olga , Kurtz Ira
Published: November 2002

The HCO3− : Na+ cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (Erev) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1‐Ser982 in the carboxy‐terminus of kNBC1 (kNBC1‐Ct), by cAMP‐protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1‐Ser982, or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate‐binding site. In the current study, we examined the role of the negative charge on Ser982‐phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)‐tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1‐Ser982. The data indicate that both kNBC1‐Asp986 and kNBC1‐Asp988, but not kNBC1‐Asp989, are required for the phosphorylation‐induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy‐terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1‐Ct with a KD of 160 ± 10 nm. Acetazolamide inhibited the short‐circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8‐Br‐cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1‐Ser982 is unphosphorylated.
Slow feedback inhibition in the Ca3 area of the rat hippocampus by synergistic synaptic activation of mGluR1 and mGluR5
Authors: Mori Masahiro , Gerber Urs
Published: November 2002

Interneurons are critical in regulating the excitability of principal cells in neuronal circuits, thereby modulating the output of neuronal networks. We investigated synaptically evoked inhibitory responses in CA3 pyramidal cells mediated by metabotropic glutamate receptors (mGluRs) expressed somatodendritically by interneurons. Although pharmacological activation of mGluRs in interneurons has been shown to enhance their excitability, the inability to record mGluR‐mediated synaptic responses has precluded detailed characterization of mGluR function in hippocampal interneurons. We found that a single extracellular pulse in CA3 stratum pyramidale was sufficient to induce disynaptic inhibitory responses mediated by postsynaptic mGluRs of the interneurons in CA3 pyramidal cells of hippocampal slice cultures. The disynaptic inhibitory response followed a short‐latency monosynaptic inhibitory response, and was observed at stimulus intensities evoking half‐maximal monosynaptic IPSCs. Synergistic activation of mGluR1 and mGluR5 was required to induce the full inhibitory response. When recordings were obtained from interneurons in CA3 stratum radiatum or stratum oriens, a single extracellular stimulus induced a slow inward cationic current with a time course corresponding to the slow inhibitory response measured in pyramidal cells. DCG IV, a group II mGluR agonist, which specifically blocks synaptic transmission through mossy fibres, had no effect on mGluR‐mediated synaptic responses in interneurons, suggesting that feed‐forward inhibition via mossy fibres is not involved. Thus, postsynaptic mGluR1 and mGluR5 in hippocampal interneurons cooperatively mediate slow feedback inhibition of CA3 pyramidal cells. This mechanism may allow interneurons to monitor activity levels from populations of neighbouring principal cells to adapt inhibitory tone to the state of the network.
The effect of tezosentan, a non‐selective endothelin receptor antagonist, on shear stress‐induced changes in arterial diameter of the anaesthetized dog
Authors: Markos F. , Hennessy B. A. , Fitzpatrick M. , O'Sullivan J. , Snow H. M.
Published: November 2002

The effects of changes in the mean (Sm) and pulsatile (Sp) components of arterial wall shear stress on arterial dilatation of the iliac artery of the anaesthetized dog were examined in the absence and presence of the endothelin receptor antagonist tezosentan (10 mg kg−1 I.V.; Ro 61‐0612; [5‐isopropyl‐pyridine‐2‐sulphonic acid 6‐(2‐hydroxy‐ethoxy)‐5‐(2‐methoxy‐phenoxy)‐2‐(2‐1H‐tetrazol‐5‐yl‐pyridin‐4‐yl)‐pyrimidin‐4‐ylamide]). Changes in shear stress were brought about by varying local peripheral resistance and stroke volume using a distal infusion of acetylcholine and stimulation of the left ansa subclavia. An increase in Sm from 1.81 ± 0.3 to 7.29 ± 0.7 N m−2 (means ± S.E.M.) before tezosentan caused an endothelium‐dependent arterial dilatation which was unaffected by administration of tezosentan for a similar increase in Sm from 1.34 ± 0.6 to 5.76 ± 1.4 N m−2 (means ± S.E.M.). In contrast, increasing the Sp from 7.1 ± 0.8 to a maximum of 11.5 ± 1.1 N m−2 (means ± S.E.M.) before tezosentan reduced arterial diameter significantly. Importantly, after administration of tezosentan subsequent increases in Sp caused arterial dilatation for the same increase in Sp achieved prior to tezosentan, increasing from a baseline of 4.23 ± 0.4 to a maximum of 9.03 ± 0.9 N m−2 (means ± S.E.M.; P < 0.001). In conclusion, the results of this study provide the first in vivo evidence that pulsatile shear stress is a stimulus for the release of endothelin from the vascular endothelium.
Identification of a site involved in the block by extracellular Mg2+ and Ba2+ as well as permeation of K+ in the Kir2.1 K+ channel
Authors: Murata Yoshimichi , Fujiwara Yuichiro , Kubo Yoshihiro
Published: November 2002

The inward rectifier potassium channel Kir2.1 is more sensitive to the weakly voltage‐dependent block by extracellular Mg2+ (Mg2+0) than Kir2.2 and Kir2.3. We identified Glu125 in an extracellular loop before the pore region of Kir2.1 as a site responsible for this sensitivity to M2+0 block, based on the observations that the Glu125Gln (E125Q) mutation strongly decreased the sensitivity, while a mutation to Glu at the corresponding sites of Kir2.2 and 2.3 led to an increase. The negative charge proved to be crucial since the Glu125Asp (E125D) mutant showed similar properties to the wild type (WT). A similar weakly voltage‐dependent block was also caused by extracellular Ca2+ and La3+ in Kir2.1 WT but not in the E125Q mutant. The sensitivity to block by extracellular Ba2+ (Ba2+0) was also decreased in the E125Q mutant, although the voltage dependency of half‐inhibition concentration was not changed, as reported previously. We additionally observed that the speed of Ba2+0 block and recovery was decelerated by the presence of Mg2+0 in WT, but not in the E125Q mutant. The sensitivity to the block by Mg2+0 was increased by lowering extracellular K+ (K+0), suggesting a competitive interaction of Mg2+0 and K+0. The single‐channel conductance of the WT in 140 mm K+ was 39.6 pS (0 mm Mg2+0) and 11.5 pS (10 mm), while that of the E125Q mutant was 26.0 pS (0 mm) and 19.6 pS (10 mm). These results demonstrate that Mg2+ competes with K+ permeation in the WT and that E125 is required for efficient K+ permeation in the absence of Mg2+0. We conclude that E125 in an extracellular loop of Kir2.1 is a site which facilitates K+ permeation and entry of Ba2+ toward a deeper plugging site, and that Mg2+0 competes with K+0 and Ba2+0 at this site.
Large Deflection of Thin Plates in Pressure Sensor Applications
Authors: Tong P. , Huang W.
Published: November 2002

The present paper examines the large deflections of a clamped circulate thin plate for pressure sensor applications and establishes a simple solution using the singular perturbation technique. The perturbation solution for the slope of the lateral deflection is in a closed form in terms of the load-induced radial stress resultant. The nonlinearity has a strong stiffened effect. The nondimensional load-induced stress resultants are functions of the nondimensional initial stress resultant, lateral load, and Poisson’s ratio.
Wet chemical etching studies of Zr and Hf-silicate gate dielectrics
Authors: Quevedo-Lopez M. A. , El-Bouanani M. , Wallace R. M. , Gnade B. E.
Published: November 2002

The etching properties of alternate gate dielectric candidates Hf and Zr silicate in hydrofluoric acid (HF) solutions are presented. As-deposited Hf silicate films were found to be more difficult to etch when compared with as-deposited Zr silicate films. After annealing, both Hf and Zr silicate are harder to etch than as-deposited films. Annealed Zr silicate films were the most difficult to remove in either concentrated, or diluted HF solutions. Film densification, along with crystallization of the silicate films near the Si interface are thought to be responsible for the etch rate change in these silicate systems. Alternate processes to remove remnant metal from the silicon surface after gate dielectric removal are also discussed. After annealing and dielectric film removal, remnant Zr and Hf concentrations near the Si surface of ∼1019/cm3 and ∼1016/cm3, respectively, were observed.
Structural, electronic and magnetic properties of chalcopyrite magnetic semiconductors: A first-principles study
Authors: Picozzi Silvia , Continenza Alessandra , Zhao Yu-Jun , Geng Wen-Tong , Freeman Arthur J.
Published: November 2002

Stimulated by recent experimental observations of room-temperature ferromagnetism of MnxCd1−xGeP2 and MnxZn1−xGeP2, we investigate the structural, electronic, and magnetic properties of this class of systems (II–Ge–V2, II=Zn, Cd, and V=As, P) as a function of Mn concentration and chemical constituents by means of first-principles density-functional- theory-based codes. Our calculations indicate that, for Mn substituting the II element, the antiferromagnetic alignment is the most stable ordering for all the systems studied. For Zn- and Cd-rich systems, the total magnetic moments per Mn atom of the ferromagnetic phase is very close to the ideal value of 5 μB, since the Mn 3d states in the minority spin channel are nearly empty; on the other hand, for Mn rich compounds, the stronger p–d hybridization lowers the total magnetic moment to about 4.4 μB.
Conductance calculation of a long tube with equilateral triangle cross section
Authors: Fan Ping , Chu Ji-Guo , Shao Jian-Da
Published: November 2002

Using the stratified gas flow model for calculating the conductance of long tubes with constant cross section, an analytical expression for calculating the conductance of a long tube with equilateral triangle cross section has been derived. The formula given is applicable to the full pressure range. A minimum in the conductance in the intermediate flow state is shown.
Dramatic change of electrical properties in La–Ba–Mn–O thin films prepared using bias sputtering
Authors: Lee Jong Cheol , Ie Sang Yub , Park Seung Iel , Yi Yeonjin , Jang Gyu In , Song Ho Shik , You Dong Gyun , Jeong Kwangho
Published: November 2002

Polycrystalline La–Ba–Mn–O thin films have been grown using rf magnetron sputtering on Si(100) substrates from a stoichiometric La0.7Ba0.3MnO3 target. Four films were grown with substrate biased such as −5, −10, −15, and −25 V and compared with the film grown with grounded substrate. Bias effects on the stoichiometry and physical properties of the films are studied. The dc resistivity was measured in the temperature range from 85 to 300 K for the films with substrate bias of 0, −5, −10, −15, and −25 V. The magnetoresistance was measured in the magnetic field of 0–10 000 Oe and at 77 K. The film with grounded substrate showed semiconductor behavior (dρ/dT<0) over the whole temperature range. The films with biased substrate, however, showed the maximum resistance at each metal–insulator transition temperature TMI. In order to find out the origin of the extreme change of electrical resistivity, x-ray photoemission spectroscopy (XPS) and x-ray diffraction (XRD) measurements were carried out. From the analysis of XPS and XRD data, bias sputtering increased La and Ba concentration in the La–Ba–Mn–O thin film and led to an increase of Mn–O–Mn bond angle. It changed films from semiconductor behavior to metal–insulator transition behavior.
Immune Responses to Bile-Tolerant
Helicobacter
Species in Patients with Chronic Liver Diseases, a Randomized Population Group, and Healthy Blood Donors
Authors: Ananieva Olga , Nilsson Ingrid , Vorobjova Tamara , Uibo Raivo , Wadström Torkel
Published: November 2002

Bile-tolerant
Helicobacter
species such as
Helicobacter pullorum
,
Helicobacter bilis
, and
Helicobacter hepaticus
are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD) in humans. Antibody responses to cell surface proteins of
H. pullorum, H. bilis
, and
H. hepaticus
in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to
Helicobacter pylori
. For analysis of a possible cross-reactivity between bile-tolerant
Helicobacter
species and
H. pylori
, sera from a subpopulation of each group were absorbed with a whole-cell extract of
H. pylori
and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for
H. pullorum
was significantly higher in patients with CLD than in healthy blood donors (
P
= 0.01). Antibody reactivity to cell surface protein of
H. hepaticus
was also significantly higher in the CLD patients than in the healthy blood donors and the population group (
P
= 0.005 and
P
= 0.002, respectively). Following the absorption, antibody responses to
H. pullorum
decreased significantly in all three groups (
P
= 0.0001 for CLD patients,
P
= 0.0005 for the population group, and
P
< 0.0001 for the blood donors), indicating that cross-reactivity between
H. pylori
and other
Helicobacter
spp. occurs. The antibody responses to
H. hepaticus
and
H. bilis
in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance of this finding requires further investigations.
Phagocytosis and Killing of Bacteria by Professional Phagocytes and Dendritic Cells
Authors: Nagl Markus , Kacani Laco , Müllauer Brigitte , Lemberger Eva-Maria , Stoiber Heribert , Sprinzl Georg M. , Schennach Harald , Dierich Manfred P.
Published: November 2002

Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroyStaphylococcus aureusandEscherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.
Change of Mouse CD5+B1 Cells to a Macrophage-Like Morphology Induced by Gamma Interferon and Inhibited by Interleukin-4
Authors: Koide Naoki , Sugiyama Tsuyoshi , Mori Isamu , Mu Mya Mya , Hamano Teruaki , Yoshida Tomoaki , Yokochi Takashi
Published: November 2002

The in vitro effects of gamma interferon (IFN-γ) on the mouse CD5+B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-γ, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-γ. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-γ-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-γ and IL-4 exhibited contradictory actions on morphological change of CD5+B1 cells into macrophage-like cells. Differential regulation of CD5+B1 cells by IFN-γ, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.
Lipoarabinomannan-Induced Cell Signaling Involves Ceramide and Mitogen-Activated Protein Kinase
Authors: Sirkar Madhumita , Majumdar Subrata
Published: November 2002

Lipoarabinomannan (LAM) is a major cell wall-associated lipoglycan, produced in large amounts (15 mg/g of bacteria) in different species of mycobacteria. Our laboratory has previously reported that LAM fromMycobacterium smegmatisexerts its cytotoxic activity via inhibition of protein kinase C, a key signaling molecule inside the mononuclear cells (S. Ghosh, S. Pal, S. Das, S. K. Dasgupta, and S. Majumdar, FEMS Immunol. Med. Microbiol. 21:181-188, 1998). In this study we report that LAM fromMycobacterium tuberculosisinduces a signal transduction pathway in favor of survivability of the host cells via the generation of ceramide, a novel second messenger. The endogenous ceramide level in mononuclear cells was found to be enhanced during LAM treatment. The effects of LAM on protein tyrosine phosphorylation in human peripheral blood mononuclear cells were examined. LAM enhanced the tyrosine phosphorylation of p42 mitogen-activated protein kinase and phosphoinositol 3-kinase (PI3 kinase) and dephosphorylation of stress-activated protein kinase. LAM-induced phosphorylation of p42 (extracellular signal-regulated kinase 2) was further enhanced by wortmannin, a PI3 kinase inhibitor. To examine whether these effects are due to elevation of endogenous ceramide, we exposed the cells to cell-permeative C2-ceramide exogenously and studied the activities of different protein kinases. Fluorescence-activated cell sorter analysis and morphological studies showed that LAM induces cell survival. Therefore, these results suggest the ability of LAM to induce ceramide in the altered signaling pathway and help in cell survival.
In Vitro and In Vivo Detection of Mx Gene Products in Bovine Cells following Stimulation with Alpha/Beta Interferon and Viruses
Authors: Müller-Doblies Doris , Ackermann Mathias , Metzler Alfred
Published: November 2002

This study focused on products of the bovine Mx1 gene as specific markers for acute viral infections. The rationale for this is the fact that viral infections are commonly paralleled by the synthesis, release, and remote action of alpha/beta interferons (IFN-α/β). Released IFN-α/β act through specific receptors present on nucleated cells to transduce signals for the transcription of numerous IFN-regulated genes, such as the ones for double-stranded-RNA-dependent protein kinase, 2′-5′-oligoadenylate synthetase, or the Mx proteins. In this study, cultured MDBK cells and bovine white blood cells (WBC) were treated with recombinant IFN-α or infected with either bovine herpesvirus 1 (BHV-1) or bovine rotavirus (BRV). Treatment of cultured cells with IFN-α was followed within 4 h by a time- and dose-dependent accumulation of intracytoplasmic Mx protein as revealed by immunostaining and Western blot immunoassay. This was preceded by a distinct rise of Mx mRNA in similarly treated cells, as revealed by a newly established quantitative TaqMan PCR technique. The two viruses displayed a cell-dependent in vitro ability to induce Mx proteins, which was limited to bovine WBC with BHV-1 and to MDBK cells with BRV. The established methods were successfully used to show that infection of calves with a noncytopathic strain of bovine viral diarrhea virus, a pestivirus, was followed within 2 days postinfection by strong expression of both Mx mRNA and Mx proteins in WBC.
Expression in Bacteria of the Gene Encoding the gp43 Antigen of
Paracoccidioides brasiliensis
: Immunological Reactivity of the Recombinant Fusion Proteins
Authors: Diniz Susana N. , Carvalho Kátia C. , Cisalpino Patrícia S. , Silveira José F. , Travassos Luiz R. , Puccia Rosana
Published: November 2002

gp43 is the major diagnostic antigen of
Paracoccidioides brasiliensis
, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (
PbGP43
) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione
S
-transferase (GST) gene, and expressed in
Escherichia coli
as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.
Nef Protein of Human Immunodeficiency Virus and Lipopolysaccharide Induce Expression of CD14 on Human Monocytes through Differential Utilization of Interleukin-10
Authors: Creery David , Angel Jonathan B. , Aucoin Susan , Weiss William , Cameron William D. , Diaz-Mitoma Francisco , Kumar Ashok
Published: November 2002

We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.