Search PubMed⌕ Search

Biomedical subjects

P Huang

Publications and source records attributed to P Huang.

At least 73 records · Page 4Linked to original sources
PubMed2000

The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors.

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu >> delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.

Amino Acid Sequence↗
PubMed2000

Nitric oxide mediates 17beta-estradiol-stimulated human and rodent osteoblast proliferation and differentiation.

Oestradiol can stimulate osteoblast activity. Osteoblast function is thought to be regulated by nitric oxide (NO). We hypothesised that the effect of 17beta-oestradiol (17beta-E(2)) on osteoblast activity is mediated by NO. This hypothesis was tested using osteoblasts isolated from human trabecular bone, calvariae of rats, endothelial NO synthase (eNOS) gene-deficient mice, and their wild-type counterparts. Our results show that 17beta-E(2) dose-dependently stimulated proliferation and differentiation of primary human, rat and wild-typeosteoblasts. The presence of N(G)-monomethyl-l-arginine (10(-3) M), an inhibitor of NOS activity, blocked the 17beta-E(2)-(10(-7) M)-induced increases in thymidine incorporation (P < 0.01), alkaline phosphatase activity (P < 0.01) and bone nodule formation (P < 0.01) of wild-type, human and rat osteoblasts, respectively. Moreover, 17beta-E(2) did not induce a response in eNOS gene-deficient osteoblasts. 17beta-E(2) also increased total eNOS enzyme expression in rat osteoblasts. These findings indicate 17beta-E(2) modulates osteoblast function by NO-dependent mechanisms mediated via the eNOS isoform.

Animals↗
PubMed2000

Superoxide dismutase as a target for the selective killing of cancer cells.

Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.

2-Methoxyestradiol↗
PubMed2000

Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus.

Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis.

Animals↗
PubMed2000

Acute and late outcome after use of 2.5-mm intracoronary stents in small (< 2.5 mm) coronary arteries.

We describe the high-pressure deployment of 2.5-mm stents in small (< 2.5 mm) coronary vessels. Forty-three lesions in 40 patients were treated. The mean reference vessel diameter was 2.3 +/- 0.2 mm. The mean % luminal stenosis was 90 +/- 9. The mean lesion length was 11. 7 +/- 9.1 mm. Sixteen lesions were pretreated with rotational atherectomy, and the remainder with PTCA. The rate of successful stent deployment was 41/43 (95%). The mean postintervention % stenosis was -1 +/- 10. There were no in-hospital deaths or procedure-related Q-wave MI. The patients were followed for a mean of 18 months. Eight patients (or 21%) developed recurrent chest pain and/or angiographically proven restenosis. One patient (3%) developed intermediate restenosis. Twenty-nine patients (or 76%) either remain symptom-free or have patent target sites on repeat angiography. It appears that reasonable acute and long-term results can be achieved with 2.5-mm stents in small coronary arteries using high-pressure deployment techniques. Cathet. Cardiovasc. Intervent. 49:121-126, 2000.

Adult↗
PubMed2000

Increased susceptibility to development of triggered activity in myocytes from mice with targeted disruption of endothelial nitric oxide synthase.

Nitric oxide generated by cardiac myocytes or delivered by drugs has been shown to regulate cardiac contractile function and has been implicated in suppressing some cardiac arrhythmias, although this remains controversial. We examined the ability of the soluble cardiac glycoside, ouabain, to trigger arrhythmic contractions in ventricular myocytes isolated from mice lacking a functional endothelial nitric oxide synthase gene (eNOS(null)). Arrhythmic activity, defined as aftercontractions, was induced with ouabain (50 micromol/L) and recorded using a video-motion detector in isolated, electrically driven single ventricular myocytes from adult eNOS(null)or from their wild-type (WT) littermates. The rate of ouabain-induced arrhythmic contractions was significantly higher in eNOS(null)myocytes than in WT myocytes. Application of the NO donor S-nitroso-acetylcysteine (SNAC) significantly diminished the frequency of arrhythmic contractions in eNOS(null)myocytes. The antiarrhythmic effect of NO, whether generated by eNOS in WT cells or by SNAC, could be partially reversed by 1H-[1,2,4]oxadiazolo-[4, 3-a]- quinoxalin-1-one (ODQ), a specific soluble guanylyl cyclase inhibitor. Ouabain significantly increased intracellular cGMP in WT but not eNOS(null)hearts, and this cGMP response was blocked by ODQ. Since cardiac glycoside- induced aftercontractions are activated by the transient inward current (I(ti)), the role of NO in ouabain (100 micromol/L)- induced I(ti)was examined using the nystatin-perforated patch-clamp technique. The frequency of ouabain-induced I(ti)was significantly higher in eNOS(null)myocytes than in WT myocytes, and this could be suppressed by SNAC. These data demonstrate that NO derived from myocyte eNOS activation suppresses ouabain-induced arrhythmic contractions by a mechanism that might involve activation of guanylyl cyclase and elevation of cGMP.

Acetylcysteine↗
PubMed2000

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification.

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.

Amino Acid Sequence↗
PubMed2000

Protection against TNF-induced lethal shock by soluble guanylate cyclase inhibition requires functional inducible nitric oxide synthase.

Hypotension and shock observed in sepsis, SIRS, and tumor necrosis factor (TNF) or cytokine-based cancer treatment are the consequence of excessive nitric oxide (NO) production and subsequent soluble guanylate cyclase (sGC)-mediated vascular smooth muscle relaxation. We demonstrate here that, while NO synthase (NOS) inhibitors exacerbated toxicity, inhibitors of sGC activation protected against TNF-induced lethality, bradycardia, and hypotension. Importantly, sGC inhibition did not interfere with the antitumor activity of TNF. Using NOS inhibitors or iNOS-deficient animals, we furthermore observed that no protection against TNF toxicity could be obtained in the absence of NO. These data imply that iNOS- (and not eNOS-) derived NO is an endogenous protective molecule indispensable to survive a TNF challenge and exerting this beneficial effect via sGC-independent mechanisms.

Animals↗
PubMed2000

Inhibition of RNA transcription: a biochemical mechanism of action against chronic lymphocytic leukemia cells by fludarabine.

Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leukemia (CLL) and other indolent lymphocytic malignancies. Although the incorporation of fludarabine into DNA is a key event in causing cytotoxicity in proliferating leukemia cells, the precise mechanisms by which fludarabine kills CLL cells remain unclear because of the quiescent nature of this malignancy. The present study demonstrated that inhibition of RNA transcription correlated significantly with the cytotoxic action of fludarabine in CLL cells. In contrast, suppression of the low level of DNA synthesis did not affect the survival of the leukemia cells. In addition, inhibition of fludarabine incorporation into cellular DNA through repair synthesis in CLL cells did not alter the cytotoxicity of this drug. Rather, inhibition of RNA synthesis by fludarabine led to a specific diminishment of certain cellular proteins from CLL cells. The combination of fludarabine with another RNA synthesis inhibitor, actinomycin D, or with the protein synthesis inhibitor, puromycin, substantially enhanced the cytotoxic activity against CLL cells. These results suggest that termination of mRNA transcription and the consequent depletion of proteins required for cell survival may be a novel biochemical mechanism of action of fludarabine in CLL cells. Thus, inhibition of RNA/protein synthesis may provide a new therapeutic strategy for the treatment of CLL patients.

Antineoplastic Agents↗
PubMed2000

Decreased mRNA expression of TNF-alpha and IL-10 in non-stimulated peripheral blood mononuclear cells in myasthenia gravis.

Myasthenia gravis (MG) is characterized by T cell-dependent autoantibodies to the acetylcholine receptor on the post-synaptic membrane of the neuromuscular junction. The production of autoantibodies is regulated by T cells via cytokines. To investigate the involvement of interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4) and interleukin-10 (IL-10) in MG, the mRNA levels of these cytokines in peripheral blood mononuclear cells without stimulation in vitro were quantified by competitive reverse transcription polymerase chain reaction technique. The mRNA level of TNF-alpha was significantly lower (P = 0.0004) in the overall group of MG patients compared with controls. IL-10 was also lower in MG patients (P = 0.008), most markedly in non-thymectomized patients (P = 0.016). There were no significant differences in IFN-gamma and IL-4 between patients and healthy controls, but the mRNA levels of IL-4 in non-thymectomized patients was significantly lower than in controls (P = 0.02) and in thymectomized patients (P = 0.03). These results, reflecting the in vivo expression pattern of these cytokines in the periphery, suggest an altered cytokine expression at the systemic level in MG.

Adult↗
PubMed2000

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein.

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.

Alzheimer Disease↗
PubMed2000

PKA holoenzyme is functionally coupled to CFTR by AKAPs.

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.

Adenosine Triphosphate↗
PubMed2000

Impermeability of the GIRK2 weaver channel to divalent cations.

A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K(+) channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2wv channels contributes to a rise in internal calcium in the GIRK2wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2wv conductance to Ca(2+). Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2wv is expressed in mammalian cells it is impermeable to Ca(2+).

Animals↗
PubMed2000

Different vasculoprotective roles of NO synthase isoforms in vascular lesion formation in mice.

NO is known to have several important vasculoprotective actions. Although NO is synthesized by 3 different NO synthase (NOS) isoforms, the vasculoprotective action of individual NOS isoforms remains to be clarified. Permanent ligation of the left common carotid artery was performed in control, endothelial NOS (eNOS) knockout (eNOS-KO), and inducible NOS (iNOS) knockout (iNOS-KO) mice. Four weeks after the procedure, neointimal formation and reduction of cross-sectional vascular area (constrictive remodeling) were noted in the left carotid artery. In the eNOS-KO mice, the extent of neointimal formation was significantly larger than in the control or iNOS-KO mice, whereas the extent of vascular remodeling was the highest in the iNOS-KO mice compared with other 2 strains. Antiplatelet therapy with aspirin or antihypertensive treatment with bunazosin failed to inhibit the accelerated neointimal formation in the eNOS-KO mice. These results indicate that eNOS and iNOS have different vasculoprotective actions against the vascular lesion formation caused by blood flow disruption in vivo: NO derived from eNOS inhibits neointimal formation, whereas NO derived from iNOS suppresses the development of constrictive remodeling.

Adrenergic alpha-Antagonists↗
PubMed2000

Atorvastatin upregulates type III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral ischemia in normocholesterolemic mice.

BACKGROUND AND PURPOSE: Thrombosis superimposed on atherosclerosis causes approximately two thirds of all brain infarctions. We previously demonstrated that statins protect from cerebral ischemia by upregulation of endothelial type III nitric oxide synthase (eNOS), but the downstream mechanisms have not been determined. Therefore, we investigated whether antithrombotic effects contribute to stroke protection by statins. METHODS: 129/SV wild-type and eNOS knockout mice were treated with atorvastatin for 14 days (0.5, 1, and 10 mg/kg). eNOS mRNA from aortas and platelets was measured by reverse-transcriptase polymerase chain reaction. Platelet factor 4 (PF 4) and beta-thromboglobulin (beta-TG) in the plasma were quantified by ELISA. Transient cerebral ischemia was induced by filamentous occlusion of the middle cerebral artery followed by reperfusion. RESULTS: Stroke volume after 1-hour middle cerebral artery occlusion/23-hour reperfusion was significantly reduced by 38% in atorvastatin-treated animals (10 mg/kg) compared with controls. Serum cholesterol levels were not affected by the treatment. eNOS mRNA was significantly upregulated in a dose-dependent manner in aortas and in thrombocytes of statin-treated mice compared with controls. Moreover, indices of platelet activation in vivo, ie, plasma levels of PF 4 and beta-TG, were dose-dependently downregulated in the treatment group. Surprisingly, atorvastatin-treatment did not influence PF 4 and beta-TG levels in eNOS knockout mice. CONCLUSIONS: The synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin upregulates eNOS in thrombocytes, decreases platelet activation in vivo, and protects from cerebral ischemia in normocholesterolemic mice. Antithrombotic and stroke-protective effects of statins are mediated in part by eNOS upregulation. Our results suggest that statins may provide a novel prophylactic treatment strategy independent of serum cholesterol levels.

Animals↗
PubMed2000

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis.

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT - PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P<0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder, an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.

Adult↗
PubMed2000

Role of p53 in cellular response to anticancer nucleoside analog-induced DNA damage.

Anticancer nucleoside analogs (e.g., ara-C, gemcitabine, fludarabine) induce apoptosis by incorporation into DNA. Removal of incorporated analogs from DNA by 3'-5' exonucleases is presumably a mechanism of drug resistance. Based on our previous observation that the 3'-5' exonuclease activity of wild-type (wt) p53 protein is able to preferentially remove mismatched nucleotides from DNA, in the present study we further investigated the ability of p53 to recognize and remove incorporated therapeutic analogs from DNA and its role in analog-induced apoptosis. We demonstrated that although the 3'-5' exonuclease of wt p53 protein was able to bind and excise the nucleoside analog residues from DNA in vitro, removal of the drug molecules from cellular DNA was slow in whole cells with wt p53 cells, and not detectable in mutant p53 cells. Furthermore, the wt p53 were more sensitive to the cytotoxic effect of the drugs compared to the p53-null or mutant cells. Incubation of ML-1 cells (wt p53) with gemcitabine caused an accumulation of p53 protein in their nuclei and preferentially induced apoptosis in the p53-positive cells, whereas the p53-negative cells remained intact. Transfection of p53-null cells with wt p53 expression vector enhanced the sensitivity of the cells to gemcitabine. Gel mobility shift assay using synthetic DNA containing gemcitabine as the probe suggests that p53 protein is likely to participate in the binding of the analog-containing DNA. Our study suggests that recognition of the incorporated nucleoside analogs in DNA by wt p53 did not confer resistance to the drugs, but it facilitated the apoptotic cell death process.

Antineoplastic Agents↗