Biomedical subjects

D Wu

Publications and source records attributed to D Wu.

At least 469 records · Page 26Linked to original sources

PubMed1994

Localization of neuronal and glial glutamate transporters.

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.

Amino Acid Sequence↗

PubMed1994

Choline acetyltransferase: celebrating its fiftieth year.

It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein-protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic-specific elements and the characterization of the trans-acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three-dimensional structure of the enzyme.

Alzheimer Disease↗

PubMed1994

In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects.

The effect of in vitro glutathione (GSH) supplementation on mitogenic response, interleukin-1, interleukin-2 and prostaglandin E2 production, and cellular GSH level in peripheral blood mononuclear cells (PBMC) from healthy young and old human subjects was studied. In vitro addition of GSH increased cellular GSH level (P < 0.001). Glutathione supplementation at concentrations between 2 to 10 mmol/L enhanced lymphocyte proliferation but at low concentrations (0.5 and 1 mmol/L) decreased mitogenic response. Glutathione-induced enhancement of lymphocyte proliferation due to phytohemagglutinin or concanavalin A was greater in the PBMC from old subjects than in those from young subjects. At optimal concentration (5 mmol), GSH increased interleukin-2 production (P < 0.05) and decreased prostaglandin E2 and leukotriene B4 production (P < 0.01) in both age groups. Furthermore, decreased PBMC mitogenic response by in vitro addition of prostaglandin E2 was reversed by GSH supplementation. Glutathione did not have an effect on interleukin-1 production by PBMC from young subjects; however, GSH supplementation tended (P = 0.08) to increase interleukin-1 production by PBMC from old subjects. We conclude that GSH supplementation enhances T cell-mediated mitogenic response in young and old subjects. This effect is due at least in part to decreased eicosanoid production.

Adult↗

PubMed1994

Effects in vitro of new growth hormone releasing peptide (GHRP-1) on growth hormone secretion from ovine pituitary cells in primary culture.

Continuous perifusion of pituitary cells was used to study the effects of a newly synthesized GHRP (GHRP-1 or KP 101) on growth hormone (GH) secretion from ovine pituitary cells and these have been compared to effects of growth hormone-releasing factor (GRF) and the original growth hormone-releasing peptide (GHRP-6). GH was continuously released at a constant rate during perifusion and secretion was increased by KP 101, GHRP-6 and GRF in a dose-dependent manner. The half-maximal effective dose of KP 101 and GHRP-6 was 10(-7) M, an order of magnitude higher than that for GRF. The maximal effects of KP 101 and GHRP-6 were similar but significantly less than the maximal effect of GRF. Blockade of calcium channels with Cd2+ (2 mM) totally and reversibly abolished the releasing effects of all three peptides. Like GHRP-6, the GH release induced by KP 101 was not affected by a GRF antagonist ([Ac-Tyr1, D-Arg2]-GRF 1-29, 1 microM) which significantly reduced the effect of GRF on GH release. For each peptide, the response to a second application (1 h after the first application) was lower than the first response. When GRF (or KP 101, GHRP-6) was applied first and then KP 101 or GHRP-6 (or GRF) given 1 h later, the second response was not attenuated. Only a small additive effect on the release of GH by GRF was obtained by the co-administration of either KP 101 or GHRP-6. This result was achieved with maximal doses of the peptides, but not with half-maximal doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis.

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1. In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni is due to synergism among three or four of their major proteins.

Aedes↗

PubMed1994

Voltage-dependent potassium currents in ovine somatotrophs and their function in growth hormone secretion.

Sheep somatotroph-enriched cultures were obtained by means of enzyme dissociation and Percoll gradient separation. Nystatin-perforated-whole-cell recordings were performed on post-recording-identified somatotrophs after 4-14 days in vitro. Using Ca(2+)-free, tetrodotoxin-containing (1 microM) bath solution and K+ electrode solution, three types of voltage-dependent K+ currents were recorded as inward rectifying, outward transient and outward delayed rectifying K+ currents. The inward rectifying K+ current was very small at physiological extracellular K+ concentrations (5 mM) and enhanced by increasing the K+ concentration in the bath to 55 mM; it was blocked by tetraethylammonium (2 mM) but not by 4-aminopyridine (5 mM). A transient outward K+ current appeared at -50 mV and was selectively diminished by 4-aminopyridine (2 or 4 mM). A delayed rectifying outward K+ current was observed when the membrane potential was depolarized to -20 mV and was blocked by tetraethylammonium (2 mM) but not 4-aminopyridine (4 mM). Application of 4-aminopyridine but not tetraethylammonium (up to 5 mM) depolarized the cell membrane potential recorded under current clamp conditions and triggered action potentials when the bath solution contained Ca2+ (2 mM) but not tetrodotoxin. The intracellular Ca2+ concentration was increased by 4-aminopyridine as was growth hormone release. Therefore, the 4-aminopyridine-sensitive transient outward K+ current appears to be important in the determining the resting potential of ovine somatotrophs and plays a major role in regulating basal intracellular Ca2+ concentration and growth hormone secretion.

4-Aminopyridine↗

PubMed1994

Radiofrequency ablation therapy in idiopathic left ventricular tachycardia with no obvious structural heart disease.

BACKGROUND: The feasibility and efficacy of radiofrequency ablation therapy in idiopathic left ventricular tachycardia has not been assessed in a large group of patients. METHODS AND RESULTS: Twenty consecutive patients with idiopathic left ventricular tachycardia and without structural heart disease underwent electrophysiological study, pharmacological interventions with administration of verapamil and adenosine, and radiofrequency ablation therapy. There were 17 men and 3 women with a mean age of 28 +/- 8 years. The QRS configuration during tachycardia was of right bundle branch block and superior axis in 13 patients, indeterminate axis in 6 patients, and right axis in 1 patient. The tachycardia was electrically inducible and responsive to verapamil but not to adenosine. Thirteen patients demonstrated entrainment. Activation and pace-mapping studies disclosed that the tachycardia originated from the inferior apical septum in 15 patients, the midseptum in 4 patients, and the anterior lateral wall of the left ventricle in 1 patient. Radiofrequency ablation was successful in 17 of the 20 patients (85%). The successful ablation sites were characterized by an endocardial activation time 30 milliseconds earlier than the onset of QRS during tachycardia and by a pace-mapping QRS similar to or closely resembling the tachycardia. All patients displayed sharp spikes preceding the local ventricular electrogram at the ablation site. However, these sharp spikes also were noted in 15 control patients and were not specific for this tachycardia; they persisted after ablation. There were no complications. A follow-up of 7 +/- 8 months in the 17 successfully ablated patients showed no symptomatic tachyarrhythmias without medications. Six patients underwent repeat electrophysiological study, and no induction of tachycardia was revealed. CONCLUSIONS: Radiofrequency ablation therapy is effective and safe in patients with idiopathic left ventricular tachycardia. It should be considered as the primary therapeutic modality in these patients.

Adult↗

PubMed1994

Concertina-like phenomenon in ventricular preexcitation due to spontaneous atrioventricular nodal Wenckebach periodicity.

Electrophysiologic study and radiofrequency ablation therapy were performed in a 28-year-old male patient with the Wolff-Parkinson-White syndrome and electrocardiographic manifestation of concertina phenomenon. His bundle recordings showed a sinus cycle length of 720 = ms with a cyclic variation of QRS morphologies of every 3 beats and an antidromic atrial echo following the last fully preexcited QRS complex. After successful radiofrequency ablation of a left posterior accessory pathway, spontaneous 3:2 atrioventricular nodal Wenckebach periodicity was noted and the mechanism of the concertina phenomenon was unraveled.

Adult↗

PubMed1994

Worsening of vasovagal syncope after beta-blocker therapy.

Head-up tilt test was done in a 27-year-old man with recurrent syncope of unexplained cause. Severe sinus bradycardia and hypotension accompanied by light-headedness, cold sweating, and nausea occurred at 80 degrees head-up position during 4 micrograms/min isoproterenol infusion. Oral propranolol, 160 mg/d, in four divided doses, effectively prevented the above-mentioned abnormal vasovagal reflexes; diltiazem was only partially effective while disopyramide, aminophylline, or atropine was ineffective in preventing the abnormal vasovagal reflexes induced by head-up tilt with isoproterenol infusion. However, the patient experienced ten episodes of syncope in 2 weeks after he was discharged from the hospital on a regimen of atenolol, 50 mg/d. His symptoms ameliorated immediately after discontinuation of atenolol therapy and he became free of severe symptoms while receiving fludrocortisone. Thus, we have documented a patient with worsening of vasovagal syncope after beta-blocker therapy.

Adrenergic beta-Antagonists↗

PubMed1994

The effect of GH-releasing peptide-2 (GHRP-2 or KP 102) on GH secretion from primary cultured ovine pituitary cells can be abolished by a specific GH-releasing factor (GRF) receptor antagonist.

A newly synthesised GH-releasing peptide, KP 102 (also named GHRP-2), was studied in an in vitro perifusion system of primary cultured ovine anterior pituitary cells. Application of KP 102 to the perifusion medium caused a dose-dependent increase in GH secretion. Dose-response relationships indicated that KP 102 had similar potency to GRF and was 10-fold more potent than earlier generations of GH-releasing peptide (GHRP-6 and GHRP-1) tested in same system. The response to a second application of KP 102 given within 1 h of initial application was significantly lower than the response to the first application. When KP 102 (or GRF) was applied first and then GRF (or KP 102) given 1 h later, the second response was not attenuated. When GRF and KP 102 were coadministered, an additive effect on release of GH was obtained. The effect of maximal dose of KP 102 (100 nM) on GH release was totally abolished by [Ac-Tyr1,D-Arg2] GRF 1-29 (1 microM) which is believed to be a specific antagonist for the GRF receptor. Blockade of Ca2+ channels by Cd2+ (2 mM) diminished the basal GH secretion and abolished the increase in GH release in response to KP 102 (100 nM). These data suggest that the action of KP 102 is blocked by a GRF receptor antagonist and therefore acts through a different receptor to that employed by earlier generations of GH-releasing peptides. GH release in response to KP 102 involves an increase in Ca2+ influx and there is no cross-desensitization between KP 102 and GRF responses.

Animals↗

PubMed1994

[Effect of methylmercury chloride on brain muscarinic receptor in rat].

In this study, we found that methylmercury chloride inhibited the binding of 3H-QNB to muscarinic receptor of rat brain tissue in vitro with IC50 values of 0.0137 +/- 0.0037mol/L. It also decreased the densities of muscarinic receptor and affinity to 3H-QNB. In vivo test system, the rats were exposed to methylmercury chloride (ip) 0, 1.5, 2.5, 3.5 mg/kg.d on the 7-12th day of gestation. On the 7, 14 and 21st day after birth, the offsprings were killed, and the cerebrum and cerebellum were immediately dissected on ice and homogenizated. The ligand assays were carried out. The result showed the effect of inhibition on the binding to cerebrum and cerebellum with significant correlation.

Animals↗

PubMed1994

Characterization of pyrazole and 4-methylpyrazole induction of cytochrome P4502E1 in rat kidney.

Pyrazole and 4-methylpyrazole induce cytochrome P4502E1 (P4502E1) in the liver. It is not known whether induction occurs in nonhepatic tissue such as kidney and lung. Rats were treated with saline, pyrazole or 4-methylpyrazole and assayed for the activity and content of P4502E1 and mRNA in liver, lung and kidney. Treatment with these agents resulted in increases in P4502E1 content as detected by immunoblots in liver and kidney, but not lung, microsomes. Oxidation of relatively specific substrates for P4502E1 was also significantly increased with liver and kidney microsomes after pyrazole or 4-methylpyrazole treatment. P4502E1 mRNA levels in liver, kidney and lung were not increased by treatment with pyrazole or 4-methylpyrazole. Associated with the induction of P4502E1 was an elevated production of reactive oxygen intermediates such as superoxide radical and H2O2 by kidney and liver, but not lung. microsomes. Lipid peroxidation induced by CCI4 was also increased with kidney microsomes after treatment with pyrazole or 4-methylpyrazole. Anti-P4502E1 IgG inhibited the increased oxidation of substrates and the increased production of H2O2 by the kidney microsomes found after treatment with pyrazole and 4-methylpyrazole. These results show that pyrazole and 4-methylpyrazole, which induce P4502E1 in liver, are also effective in inducing this enzyme in the kidney, whereas the lung is not sensitive to induction by these agents. The mechanism of induction of kidney P4502E1, similarly to that of liver, appears to reflect a post-transcriptional effect-probably stabilization of the protein against degradation.

Animals↗

PubMed1993

Localized invasive pulmonary aspergillosis in patients with neutropenia. Effectiveness of surgical resection.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in patients with neutropenia. Two severe complications with poor outcome can be observed after apparently successful IPA medical treatment: severe hemoptysis and IPA relapse during subsequent cytotoxic treatments. Early surgical therapy has not been considered routinely in the management of localized IPA. METHODS: Six consecutive patients (four women, two men; median age, 52 years) with localized cavitating IPA diagnosed during chemotherapy-induced aplasia were treated with early surgical resection after hematologic recovery. RESULTS: All patients received a lobectomy. Surgery was uneventful. This procedures allows patients to proceed with further intensive chemotherapy and/or bone marrow transplantation without IPA reactivation. CONCLUSIONS: For selected patients, surgical resection of localized IPA with unique cavitating lesion, which prevents hemoptysis and IPA recurrence and allows for subsequent cytotoxic treatment, may be recommended.

Adult↗

PubMed1993

The transforming activity of activated G alpha 12.

Heterotrimeric GTP-binding proteins transduce receptor-mediated extracellular signals to their effectors. Several activated mutations of Gs alpha and Gi alpha have been shown to be associated with endocrine tumors. In this report, we have evaluated the transforming activity of an putative activated form of G alpha 12 subunit. We found that transient expression in NIH3T3 cells of the G alpha 12 mutant with substitution of glutamine-229 for leucine could lead to focus formation and that stably transfected NIH3T3 cells could form colonies in soft agar and tumors in nude mice.

3T3 Cells↗

PubMed1993

Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes.

We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.

CD8 Antigens↗

PubMed1993

Native protein separations and enzyme microassays by capillary zone and gel electrophoresis.

Native protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for native proteins with molecular weights from 20,000 to 47,000. The separation of native proteins in gel-filled capillaries is based solely on the charge of the protein as in normal zonal electrophoresis. Retention of protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix. Alkaline phosphatase (ALP) and beta-galactosidase assays were conducted in both C18-PF108-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potential and zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection limit achieved was 5.2 x 10(-20) mol (7.6 x 10(-12) M sample injected) of ALP. The dual enzyme assay of ALP and beta-galactosidase was achieved in gel-filled capillaries simultaneously.

Electrophoresis↗

PubMed1993

G protein-coupled signal transduction pathways for interleukin-8.

Interleukin-8 (IL-8) is one of the major mediators of the inflammatory response. The pathways by which IL-8 activates inositide-specific phospholipase C (PLC) were investigated by co-expression of different components of the guanosine triphosphate binding protein (G protein) pathway in COS-7 cells. Two distinct IL-8 receptors reconstituted ligand-dependent activation of endogenous PLC when transfected together with the G protein alpha subunits G alpha 14, G alpha 15, or G alpha 16. However, reconstitution was not observed with cells that overexpressed G alpha q or G alpha 11. Furthermore, IL-8 receptors interacted with endogenous pertussis toxin-sensitive G proteins or with the recombinant G protein Gi to release free beta gamma subunits that could then specifically activate the beta 2 isoform of PLC. These findings suggest that IL-8 acts through signal-transducing pathways that are limited to specific heterotrimeric G proteins and effectors. These may provide suitable targets for the development of anti-inflammatory agents.

Animals↗

PubMed1993

Sphingosine metabolism induces Ca2+ oscillations in rat pancreatic acinar cells.

The possibility that sphingosine or a related metabolite could mediate inositol-1,4,5-triphosphate (1,4,5-IP3)-independent Ca2+ release in pancreatic acinar cells was investigated. In intact rat pancreatic acini superfused at 37 degrees C, sphingosine (10-100 microM) evoked, after a delay of several minutes, an increase in the cytosolic [Ca2+]i which was oscillatory below concentrations of 50 microM. At room temperature no increase in [Ca2+]i was observed. The increase in [Ca2+]i evoked by sphingosine was not inhibited by U73122, and thus was unlikely to be due to phosphatidylinositol (PI) hydrolysis. Furthermore, no PI hydrolysis or 1,4,5-IP3 production was detected on incubation with sphingosine. As metabolism of sphingosine could explain the temperature-dependent action, the effects of sphingosine and related sphingoid bases were tested in permeabilized cells. After a delay of 30 s at 37 degrees C, sphingosine evoked a slow increase in [Ca2+] into the medium. In contrast, addition of sphingosylphosphorylcholine (SPC) resulted in a rapid increase in [Ca2+]. This response was mimicked by 1,4,5-IP3 but not by addition of sphingosine phosphate (1-10 microM), ceramide (30 microM), or sphingomyelin (50 micrograms/ml). SPC appeared to induce release of Ca2+ from the same store as 1,4,5-IP3, as SPC failed to evoke any further increase in Ca2+ from cells previously depleted by addition of a maximal concentration of 2,4,5-IP3 or thapsigargin. 1,4,5-IP3 but not SPC-induced Ca2+ release was blocked by heparin, indicating that SPC-induced Ca2+ release was not mediated through the IP3 receptor-channel complex. This study demonstrates that SPC, a sphingosine metabolite, could be responsible for IP3-independent Ca2+ oscillations previously seen in pancreatic acinar cells.

Animals↗