Biomedical subjects

B Ye

Publications and source records attributed to B Ye.

At least 19 recordsLinked to original sources

PubMed2006

Gamma-aminobutyric acid up- and downregulates insulin secretion from beta cells in concert with changes in glucose concentration.

AIMS/HYPOTHESIS: The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA(A)Rs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA(A)Rs in the pancreatic islets. However, the reported effects of GABA(A)R activation on insulin secretion from islet beta cells have been controversial. METHODS: This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. RESULTS: Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 micromol/l, induced a transmembrane current (I(GABA)) which was sensitive to the GABA(A)R antagonist bicuculline. The current-voltage relationship revealed that I(GABA) reversed at -42+/-2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V (M)), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V (M) of INS-1 cells was below the I(GABA) reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V (M) of INS-1 cells was above the I(GABA) reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). CONCLUSIONS/INTERPRETATION: These findings indicate that activation of GABA(A)Rs in beta cells regulates insulin secretion in concert with changes in glucose levels.

Animals↗

PubMed2006

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it.

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.

Animals↗

PubMed2002

Physiological and pathological caspase cleavage of the neuronal RasGEF GRASP-1 as detected using a cleavage site-specific antibody.

Caspases are proteases involved in various physiological and pathological processes in the nervous system, including development and pathogenesis. GRASP-1 is a recently identified neuronal substrate of caspase-3-subfamily caspases. It is a Ras-guanine nucleotide exchange factor (RasGEF) that interacts with the glutamate receptor interacting protein (GRIP). This alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor/GRIP protein complex has been proposed to be involved in AMPA receptor synaptic targeting. The caspase-3 cleavage of GRASP-1 separates the N-terminal RasGEF catalytic domain from the C-terminal GRIP-interacting region, potentially disrupting regulation of the RasGEF activity by GRIP. To examine the regulation and regional distribution of the caspase-3 cleavage of GRASP-1 in vivo, we generated a cleavage site-specific antibody, termed CGP, against the cleaved N-terminal fragment of GRASP-1. Using this antibody, we have examined the caspase cleavage of GRASP-1 during postnatal development and following ischemia in mice. We found that caspase cleavage of GRASP-1 occurs in specific brain regions in a time-dependent manner during development and ischemia. This data provides an important account of the brain areas that might require caspase-3 activity in postnatal development and ischemic damage, which has not been documented. It also demonstrates that the CGP antibody is a powerful tool for studying neuronal activity of the caspase-3-subfamily caspases in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2001

[In vitro culture of modified neonatal pig islets].

A cell-isolation-and-cell-culture-in-vitro method which can produce a mass of new-born pig islet-like clusters was introduced. The new-born pig pancreas were incubated in V-type collagenase (0.5-1.0 mg.ml-1) for 8-12 minutes, and then cultured in vitro for 7 days before large amount of highly purified porcine islets were produced. The neonatal pig islets had good insulin secretion and normal sub-cell structure. The results suggest that the neonatal pig islets are probably a fine donor source for clinical xenotransplantation of islets.

Animals↗

PubMed2001

Phospho-Azatyrosine, a less effective protein-tyrosine phosphatase substrate than phosphotyrosine.

Azatyrosine (AzaTyr, 4) is a natural product isolated from Streptomyces chibanesis, whose structure is characterized by a nitrogen atom in the aryl ring of a tyrosyl residue. This seemingly minor modification to the tyrosyl residue results in profound physiological effects, as AzaTyr has been shown to promote permanent reversion of ras-dependent transformed cells to the normal phenotype in culture and to inhibit chemical induction of carcinogenesis in transgenic mice bearing oncogenic human ras. The mechanisms underlying these effects are not known, however ras-pathways involve an intricate balance between both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). The present study was undertaken to examine the general utility of AzaTyr as a structural motif for PTP inhibitor design by examining the phospho-azatyrosine (pAzaTyr)-containing peptide Ac-Asp-Ala-Asp-Glu-pAzaTyr-Leu-amide (8) in a PTP1 enzyme system. Kinetic analysis indicated that 8 binds with a Km value of 210 microM and a catalytic turnover rate, kcat of 52 s(-1). This represents a greater than 50-fold reduction in binding affinity relative to the parent phosphotyrosine-containing peptide, indicating that the aryl nitrogen adversely affects binding affinity. The much lower PTP affinity of the pAzaTyr-containing peptide reduces the potential utility of the AzaTyr pharmacophore for PTP inhibitor design. These results are discussed from the point of view that incorporation of AzaTyr residues into proteins could result in perturbation of protein-tyrosine phosphorylation,dephosphorylation cascades that control signal transduction processes, including ras-dependent pathways.

Alanine↗

PubMed2001

Zinc metallothionein imported into liver mitochondria modulates respiration.

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal beta-domain of MT; in this system, the isolated C-terminal alpha-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated beta-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

Animals↗

PubMed2001

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

Acetylene↗

PubMed2001

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes.

OBJECTIVE: To detect malaria DNA in mosquitoes. METHODS: A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used. RESULTS: In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121 bp band was seen with DNA templates from other malaria parasites or negative mosquitoes. CONCLUSION: Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

Animals↗

PubMed2001

[On cleanliness of hands in diminution of Ascaris lumbricoides infection in children].

OBJECTIVE: To study the relationship between cleanliness of children's hands and diminution of Ascaris lumbricoides infection. METHODS: Before the study all persons positive for ascaris eggs in the preliminary survey were treated with albendazole. Hand-washing habit before meal and after defecation was kept in children of experimental group, but not in the control group. Kato thick smear stool examination was done once every two months for one year to compare the new infection rates in children without ascaris infection in the two groups, and the reinfection rates in the cured negative cases were also compared between them in half a month after chemotherapy. RESULTS: All the new infection rates as well as reinfection rates of each reexamination in the experimental group were significantly lower than that of the control group (P < 0.001). Reexamination one year later showed that the ascaris infection rate of the experimental group was 35.2%, reducing by 48.5% as compared with 68.3% before the operation of the project; while ascaris infection rate of the control group was 73.7%, increasing by 78.0% as compared with 41.4% before the operation of the project. CONCLUSION: Washing hands with toilet soap to keep hands clean can significantly reduce ascaris infection rate.

Ascariasis↗

PubMed2000

[Chemically induced (streptozotocin-alloxan) diabetes mellitus in dogs].

To evaluate the efficiency and toxic and side effect of the combined chemical inducer for Type I diabetes mellitus, Type I diabetes in 9 adult dogs were induced with alloxan(50 mg.kg-1) and streptozotocin (30 mg.kg-1) with vein injection. After 7-14 days, the blood glucose of 5 male dogs was kept on high levels [mean value was (22.7 +/- 3.2) mmol.L-1], exogenous insulin was (21.4 +/- 2.4) U.day-1. Three female dogs died in 7 days after they had been injected with the combined inducers. Alloxan-streptozotocin combination administration reduced the dosage of each drug, and decreased the toxic and side effect of each drug. The results suggest that this method has a high rate of success to induce Type I diabetes male dogs.

Alloxan↗

PubMed2000

Transient, oxidant-induced antioxidant transcript and enzyme levels correlate with greater oxidant-resistance in paraquat-resistant Conyza bonariensis.

The elucidation of mechanisms plants use to overcome oxidative stress is facilitated where there is intra-specific genetic variability. The differential induction of higher levels of mRNAs, cytosol and chloroplast antioxidant enzyme activities, and proteins occurred after sub-lethal paraquat treatment of the oxidant-resistant biotype of Conyza bonariensis (L.) Cronq. By 6 h after sub-lethal paraquat treatment the activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), dehydroascorbate reductase (EC 1.8.5), monodehydroascorbate reductase (EC 1.6.5.4), and glutathione peroxidase (EC 1.11.19) had increased, peaking at 24 h and then slowly reverting back to the basal level. Similarly, the levels of mRNAs encoding these enzymes were enhanced by 12 h and peaked at 18-24 h after sub-lethal paraquat treatment. The time courses of the transient elevation of both transcript and antioxidant enzyme levels correlated with a further transient 2.5- to 3.0-fold increase of paraquat resistance, which occurred only in the constitutively resistant biotype. The individual enzymes seem to be part of a coordinately controlled oxidant tolerance in the resistant biotype, utilizing oxidant-induced, increasingly abundant transcript levels, upon which more antioxidant enzymes were synthesized.

Ascorbate Peroxidases↗

PubMed2000

GRASP-1: a neuronal RasGEF associated with the AMPA receptor/GRIP complex.

The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, we have identified several GRIP-associated proteins (GRASPs) that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduced the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 was rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity.

Amino Acid Sequence↗

PubMed1999

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ¿(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.

Benzylidene Compounds↗

PubMed1999

[Partial splenic embolotherapy in hypersplenism with portal hypertension].

Twenty-one cases of hypersplenism with portal hypertension were treated with partial splenic embolization. The results were that spleens were shrink and the condition of white blood cells, platelets, and prothrombin time of the patients were obviously improved. We conclude that the appropriate antibiotics, superselective catheterization, and the management of postoperation are necessary; the volume of embolism is limited from 50% to 75% of the spleen. This technique has the features of steady therapeutic effect, safety, and microtrauma.

Embolization, Therapeutic↗

PubMed1998

The enzymatic domain of Clostridium difficile toxin A is located within its N-terminal region.

Clostridium difficile, an anaerobic pathogen encountered in human enteric disease, produces two major virulence factors, toxins A and B, which are members of a clostridial family of large cytotoxins. These are glucosyltransferases, which use a UDP-sugar as co-substrate to glucosylate and inactivate small GTPases of the Rho or Ras families, culminating in cytotoxicity. Clinically, toxin A is perhaps the most important family member, because it causes major tissue damage in the course of disease, leading to a potentially lethal, pseudomembranous colitis. The location of the enzymatic domain of toxin A and mechanistic details of its action are not yet known, so we wished to localize this domain using gene deletion constructions from the full-length gene and by monitoring glucosylation activity of encoded protein products. Toxin A deletions were obtained by successively truncating the C-terminal coding region. These were transformed into E. coli, cell lysates were prepared and they were assayed for their ability to glucosylate Rho A protein, using an in vitro enzymatic assay. We report that the UDP-glucose binding site, the catalytic site for glucose transfer and the Rho A interaction site occur within the first 659 N-terminal amino acids of toxin A, i.e., less than 25% of the length of holotoxin A. Localization of the enzymatic domain of toxin A to these 659 N-terminal amino acids should greatly simplify studies on mechanistic details of this clinically important toxin.

Bacterial Toxins↗

PubMed1998

Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.

Action Potentials↗

PubMed1998

Structure-based design and synthesis of small molecule protein-tyrosine phosphatase 1B inhibitors.

Protein-tyrosine phosphatase (PTP) inhibitors are attractive as potential signal transduction-directed therapeutics which may be useful in the treatment of a variety of diseases. We have previously reported the X-ray structure of 1,1-difluoro-1-(2-naphthalenyl)methyl] phosphonic acid (4) complexed with the human the protein-tyrosine phosphatase 1B (PTP1B) and its use in the design of an analogue which binds with higher affinity within the catalytic site (Burke, T. R., Jr. et al. Biochemistry 1996, 35, 15989). In the current study, new naphthyldifluoromethyl phosphonic acids were designed bearing acidic functionality intended to interact with the PTP1B Arg47, which is situated just outside the catalytic pocket. This residue has been shown previously to provide key interactions with acidic residues of phosphotyrosyl-containing peptide substrates. Consistent with trends predicted by molecular dynamics calculations, the new analogues bound with 7- to 14-fold higher affinity than the parent 4, in principal validating the design rationale.

Drug Design↗

PubMed1998

Temperature dependence of early and late currents in human cardiac wild-type and long Q-T DeltaKPQ Na+ channels.

Na+ current (INa) through wild-type human heart Na+ channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. INa kinetics depend strongly on temperature, but INa for hH1 has been studied previously only at room temperature. We characterized early INa (the peak and initial decay) and late INa of the wild-type hH1 channel and a mutant channel (DeltaKPQ) associated with congenital long Q-T syndrome. Channels were stably transfected in HEK-293 cells and studied at 23 and 33 degreesC using whole cell patch clamp. Activation and inactivation kinetics for early INa were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for DeltaKPQ. For early INa (<24 ms), DeltaKPQ decayed faster than the wild type for voltages negative to -20 mV but slower for more positive voltages, suggesting a reduced voltage dependence of fast inactivation. Late INa at 240 ms was significantly greater for DeltaKPQ than for the wild type at both temperatures. The majority of late INa for DeltaKPQ was not persistent; rather, it decayed slowly, and this late component exhibited slower recovery from inactivation compared with peak INa. Additional kinetic changes for early and peak INa for DeltaKPQ compared with the wild type at both temperatures were 1) reduced voltage dependence of steady-state inactivation with no difference in midpoint, 2) positive shift for activation kinetics, and 3) more rapid recovery from inactivation. This study represents the first description of human Na+ channel kinetics near physiological temperature and also demonstrates complex gating changes in the DeltaKPQ that are present at 33 degreesC and that may underlie the electrophysiological and clinical phenotype of congenital long Q-T Na+ channel syndromes.

Cell Line↗