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Biomedical subjects

S Huang

Publications and source records attributed to S Huang.

At least 271 records · Page 15Linked to original sources
PubMed1998

The perinucleolar compartment and transcription.

The perinucleolar compartment (PNC) is a unique nuclear structure localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III and two hnRNP proteins have been localized in the PNC (Ghetti, A., S. Piñol-Roma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181- 1193; Timchenko, L.T., J.W. Miller, N.A. Timchenko, D.R. DeVore, K.V. Datar, L. Lin, R. Roberts, C.T. Caskey, and M.S. Swanson. 1996. Nucleic Acids Res. 24: 4407-4414; Huang, S., T. Deerinck, M.H. Ellisman, and D.L. Spector. 1997. J. Cell Biol. 137:965-974). In this report, we show that the PNC incorporates Br-UTP and FITC-conjugated CTP within 5 min of pulse labeling. Selective inhibition of RNA polymerase I does not appreciably affect the nucleotide incorporation in the PNC. Inhibition of all RNA polymerases by actinomycin D blocks the incorporation completely, suggesting that Br-UTP incorporation in the PNC is due to transcription by RNA polymerases II and/or III. Treatment of cells with an RNA polymerase II and III inhibitor induces a significant reorganization of the PNC. In addition, double labeling experiments showed that poly(A) RNA and some of the factors required for pre-mRNA processing were localized in the PNC in addition to being distributed in their previously characterized nucleoplasmic domains. Fluorescence recovery after photobleaching (FRAP) analysis revealed a rapid turnover of polypyrimidine tract binding protein within the PNC, demonstrating the dynamic nature of the structure. Together, these findings suggest that the PNC is a functional compartment involved in RNA metabolism in the cell nucleus.

Cell Nucleolus↗
PubMed1998

RIZ1, but not the alternative RIZ2 product of the same gene, is underexpressed in breast cancer, and forced RIZ1 expression causes G2-M cell cycle arrest and/or apoptosis.

The retinoblastoma protein-interacting zinc finger gene RIZ maps to the distal short arm of human chromosome 1 (1p36), a region thought to harbor tumor suppressor genes for a variety of human cancers including breast cancer. The RIZ gene normally produces two protein products of different length, RIZ1 and RIZ2. RIZ2 is generated by an internal promoter and lacks an NH2-terminal motif of RIZ1, the PR domain conserved in a subfamily of zinc finger genes that function as negative regulators of tumorigenesis. We have here studied whether the RIZ gene may play a role in human neoplasia. We found that expression of RIZ1 is commonly decreased or at undetectable levels in breast cancer tissues and cell lines. Decreased RIZ1 expression was also found in other tumor types including neuroblastoma and lung cancer. Remarkably, RIZ2 is normally expressed in all cases examined, suggesting that the abnormality observed for RIZ1 is specific. Forced RIZ1 expression in breast cancer cells caused cell cycle arrest in G2-M and/or programmed cell death. These observations suggest an exclusive negative selection for RIZ1 but not RIZ2 in breast cancer and a role for RIZ1 in tumor suppression.

Alternative Splicing↗
PubMed1998

Predictors of fluoroscopy time and estimated radiation exposure during radiofrequency catheter ablation procedures.

The objective of this study was to identify factors that predict fluoroscopy duration and radiation exposure during catheter ablation procedures. The patient population included 859 patients who participated in the Atakr Ablation System clinical trial at 1 of 9 centers (398 male and 461 female patients, aged 36 +/- 21 years). Each patient underwent catheter ablation of an accessory pathway, the atrioventricular junction, or atrioventricular nodal reentrant tachycardia using standard techniques. The duration of fluoroscopy was 53 +/- 50 minutes. Factors identified as independent predictors of fluoroscopy duration included patient age and sex, the success or failure of the ablation procedure, and the institution at which the ablation was performed. Catheter ablation in adults required longer fluoroscopy exposure than it did in children. Men required longer durations of fluoroscopy exposure than did women. The mean estimated "entrance" radiation dose was 1.3 +/- 1.3 Sv. The dose needed to cause radiation skin injury was exceeded during 22% of procedures. The overall mean effective absorbed dose from catheter ablation procedures was 0.025 Sv for female patients and 0.017 Sv for male patients. This degree of radiation exposure would result in an estimated 1,400 excess fatal malignancies in female patients and 2,600 excess fatal malignancies in male patients per 1 million patients.

Adolescent↗
PubMed1998

Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis.

Expression of the tyrosine kinase receptor, c-KIT, progressively decreases during local tumor growth and invasion of human melanomas. We have previously shown that enforced c-KIT expression in highly metastatic cells inhibited tumor growth and metastasis in nude mice. Furthermore, the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. Here we show that loss of c-KIT expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The c-KIT promoter contains three binding sites for AP-2 and EMSA gels demonstrated that AP-2 protein binds directly to the c-KIT promoter. Transfection of wild-type AP-2 into c-KIT-negative A375SM melanoma cells activated a c-KIT promoter-driven luciferase reporter gene, while expression of a dominant-negative AP-2B in c-KIT-positive Mel-501 cells inhibited its activation. Endogenous c-KIT mRNA and expression of proteins were upregulated in AP-2-transfected cells, but not in control cells. In addition, re-expression of AP-2 in A375SM cells suppressed their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of c-KIT is highly regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly through c-KIT transactivation and SCF-induced apoptosis. Therefore, loss of AP-2 expression might be a crucial event in the development of malignant melanoma.

Animals↗
PubMed1998

Procathepsin-L, a proteinase that cleaves human C3 (the third component of complement), confers high tumorigenic and metastatic properties to human melanoma cells.

We previously demonstrated that highly metastatic human melanoma cells secrete a 41 kDa proteinase that cleaves C3, the third component of complement, and shares antigenic determinants with procathepsin-L. Thus, we herein transfected the nonmetastatic DX-3 melanoma cells with the procathepsin-L cDNA. Three clones expressing and secreting high levels of procathepsin-L were selected. Conditioned medium and whole cell extracts from these clones, but not from control cells, carried a high C3-cleaving activity. The transfected clones displayed up to 60% resistance to complement-mediated lysis. Overexpression of procathepsin-L in melanoma cells increased their tumorigenicity and switched their phenotype from nonmetastatic to highly metastatic cells. This is the first report that demonstrates that enforced expression of procathepsin-L by human melanoma cells arms them with the ability to inactivate complement-mediated lysis and contributes to tumor growth and metastasis.

Animals↗
PubMed1998

The PR domain of the Rb-binding zinc finger protein RIZ1 is a protein binding interface and is related to the SET domain functioning in chromatin-mediated gene expression.

The PR domain, first noted as the PRDI-BF1-RIZ1 homologous region, defines a sub-class of zinc finger genes that appear to function as negative regulators of tumorigenesis. This family includes the MDS1-EVI1 gene inactivated in myeloid leukemia, the PRDI-BF1/BLIMP1 transcription repressor of c-myc involved in driving B-cell differentiation, and the RIZ gene, which encodes proteins capable of binding to the retinoblastoma tumor suppressor protein (Rb). The PR domain of MDS1-EVI1 is disrupted by translocations linked to myeloid leukemia, resulting in the activation of the PR-minus oncogenic product EVI1. Remarkably similar to MDS1-EVI1, RIZ gene also normally produces two protein products of different length, and the smaller protein RIZ2 lacks the PR domain of RIZ1 but is otherwise identical to RIZ1. These observations raise considerable interest to determine the function of PR. We show here that RIZ1 PR domain mediates protein-protein interaction. Recombinant fusion proteins of PR can bind to in vitro translated RIZ1 and RIZ2 proteins. The binding can be disrupted by amino acid substitutions at conserved residues of PR, suggesting that binding is specific. Of the three conserved exons of PR, the first two appear dispensable for binding, whereas the third exon is required. A region in the carboxyl terminus of RIZ proteins was mapped to be necessary and sufficient for PR binding. We also found that the PR domain shares significant sequence identity to the SET domain present in chromosomal proteins that function in modulating gene expression from yeast to mammals. Our data suggest that the PR domain is a derivative of SET domain and may function as protein binding interface in the regulation of chromatin-mediated gene expression.

Amino Acid Sequence↗
PubMed1998

Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

p38, a subfamily of the mitogen-activated protein kinase, regulates gene expression in response to various extracellular stimuli. The pyridinyl imidazoles like SB202190 are specific inhibitors of p38alpha and p38beta and have been widely used in investigation of the biological functions of p38. Here we show that SB202190 by itself was sufficient to induce cell death, with typical apoptotic features such as nucleus condensation and intranucleosomal DNA fragmentation. SB202190 stimulated the activity of CPP32-like caspases, and its apoptotic effect was completely blocked by the protease inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and expression of bcl-2. In addition, SB202190 was able to potentiate apoptosis induced by Fas(APO-1) ligation or UV irradiation. Expression of p38beta attenuated the apoptotic effect of SB202190 and the cell death induced by Fas ligation and UV irradiation. In contrast, expression of p38alpha induced cell death mildly. These results indicate that SB202190 induces apoptosis through activation of CPP32-like caspases and suggest that distinct members of the p38 subfamily of mitogen-activated protein kinase have different functions in apoptosis.

Apoptosis↗
PubMed1998

Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells.

MCAM/MUC18 is a cell-surface glycoprotein of 113 kDa, originally identified as a melanoma antigen, whose expression is associated with tumor progression and the development of metastatic potential. We have previously shown that enforced expression of MCAM/MUC18 in primary cutaneous melanoma led to increased tumor growth and metastatic potential in nude mice. The mechanism for up-regulation of MCAM/MUC18 during melanoma progression is unknown. Here we show that up-regulation of MCAM/MUC18 expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The MCAM/MUC18 promoter contains four binding sites for AP-2, and electrophoretic mobility shift assay gels demonstrated that the AP-2 protein bound directly to the MCAM/MUC18 promoter. Transfection of AP-2 into highly metastatic A375SM melanoma cells (AP-2-negative and MCAM/MUC18-positive) inhibited MCAM/MUC18 promoter-driven chloramphenicol acetyltransferase reporter gene in a dose-dependent manner. MCAM/MUC18 mRNA and protein expression were down-regulated in AP-2-transfected but not in control cells. In addition, re-expression of AP-2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of MCAM/MUC18 is regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly by down-regulating MCAM/MUC18 gene expression. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as c-KIT, E-cadherin, MMP-2, and p21(WAF-1), we propose that loss of AP-2 is a crucial event in the development of malignant melanoma.

Animals↗
PubMed1998

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Adenosine Triphosphate↗
PubMed1998

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

Anti-Bacterial Agents↗
PubMed1998

Methionine synthase exists in two distinct conformations that differ in reactivity toward methyltetrahydrofolate, adenosylmethionine, and flavodoxin.

Methionine synthase (MetH) from Escherichia coli catalyzes the synthesis of methionine from homocysteine and methyltetrahydrofolate via two methyl transfer reactions that are mediated by the endogenous cobalamin cofactor. After binding both substrates in a ternary complex, the enzyme transfers a methyl group from the methylcobalamin cofactor to homocysteine, generating cob(I)alamin enzyme and methionine. The enzyme then catalyzes methyl transfer from methyltetrahydrofolate to the cob(I)alamin cofactor, forming methylcobalamin cofactor and tetrahydrofolate prior to the release of both products. The cob(I)alamin form of the enzyme occasionally undergoes oxidation to an inactive cob(II)alamin species; the enzyme also catalyzes its own reactivation. Electron transfer from reduced flavodoxin to the cob(II)alamin cofactor is thought to generate cob(I)alamin enzyme, which is then trapped by methyl transfer from adenosylmethionine to the cobalt, restoring the enzyme to the active methylcobalamin form. Thus the enzyme is potentially able to catalyze two methyl transfers to the cob(I)alamin cofactor: methyl transfer from methyltetrahydrofolate during primary turnover and methyl transfer from adenosylmethionine during activation. It has recently been shown that methionine synthase is constructed from at least four separable regions that are responsible for binding each of the three substrates and the cobalamin cofactor, and it has been proposed that changes in positioning of the substrate binding regions vis-à-vis the cobalamin binding region could allow the enzyme to control which substrate has access to the cofactor. In this paper, we offer evidence that methionine synthase exists in two different conformations that interconvert in the cob(II)alamin oxidation state. In the primary turnover conformation, the enzyme reacts with homocysteine and methyltetrahydrofolate but is unreactive toward adenosylmethionine and flavodoxin. In the reactivation conformation, the enzyme is active toward adenosylmethionine and flavodoxin but unreactive toward methyltetrahydrofolate. The two conformations differ in the susceptibility of the substrate-binding regions to tryptic proteolysis. We propose a model in which conformational changes control access to the cobalamin cofactor and are the primary means of controlling cobalamin reactivity in methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1998

1-Methoxy-agroclavine from Penicillium sp. WC75209, a novel inhibitor of the Lck tyrosine kinase.

A high-throughput screen was developed and implemented to identify inhibitors of the Lck tyrosine kinase. This report describes the identification of a specific inhibitor of this enzyme from the solid fermentation culture of the Penicillium sp., WC75209. The active compound was isolated and structurally characterized as 1-methoxy-5R, 10S-agroclavine, a new member of the ergot alkaloid family.

Enzyme Inhibitors↗
PubMed1998

Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

Adenosine Diphosphate↗
PubMed1998

The effects of social isolation on the forced swim test in Fawn hooded and Wistar rats.

Although the forced swim test (FST) has long been used as a preclinical screen of antidepressant efficacy, locomotor stimulants are known to produce confounding effects using the traditional dependent measure in this test: immobility. It has recently been suggested that measurement of struggling behavior may be a better index of antidepressant activity. The present experiments examined behavior in the forced swim test in two potential animal models of depression: the Fawn hooded rat, and the isolation-reared rat. No evidence was found to support these assertions, indeed immobility was decreased in Fawn hooded compared to Wistar rats, however this appeared to be caused by increased struggling behavior in Fawn hooded socials and increased swimming in Fawn hooded isolates. Although these differential results are highly suggestive of different underlying causes of decreased immobility in Fawn hooded rats depending on rearing conditions, the data suggests that the underlying psychological functions assumed to be represented by behavior assessed in this paradigm may not be adequately discriminated.

Animals↗
PubMed1998

Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family.

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38 beta, and was suppressed by the p38 beta dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38 alpha, and was suppressed by the dominant negative p38 alpha mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.

Adenoviridae↗
PubMed1998

Nitric oxide-mediated cGMP synthesis in Helix neural ganglia.

The central nervous system of the mollusc Helix pomatia was stimulated with NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine, in the presence of a phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX). Radioimmunoassay revealed that all of the three NO donors significantly increased cGMP levels by 22-27-fold above basal levels. Compared with controls, strong cGMP immunoreactivity was observed in axons and cytoplasm of the stimulated neurons. About 80% of cGMP-immunoreactive neurons colocalized with NADPH-diaphorase activity. Some glial cells and giant neurons were not stained by NADPH-diaphorase histochemistry but were cGMP-immunoreactive. The results suggest the existence of a NO/cGMP pathway and indicate NO as an intra- and intercellular signaling molecule in the Helix central nervous system.

Animals↗
PubMed1998

Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN-gamma receptor gene.

To investigate the role of interferon-gamma (IFN-gamma) in experimental autoimmune thyroidits (EAT), H-2k mice with a disrupted IFN-gamma receptor (IFN-gamma R) gene were immunized with porcine thyroglobulin (pTg). We observed that EAT occurred on day 19 and remitted on day 35 in IFN-gamma R-deficient (IFN-gamma R(0/0)) mice, whereas in wild-type mice, EAT occurred on day 21 and remitted on day 42-49. Moreover, EAT in the mutant mice was attenuated and accompanied by diminished Tg-specific cytotoxic and proliferative responses and decreased titers of anti-Tg antibodies, notably of the IgG2a and IgG2b isotypes. In contrast, Tg-specific IgG1 was increased in the IFN-gamma R(0/0) mice. In supernatants from T cells further stimulated in vitro by Tg, IFN-gamma levels were higher in IFN-gamma R(0/0) than in wild-type mice throughout the course of the disease, whereas interleukin-10 was transiently increased prior to EAT onset in both groups of mice. Finally, using IFN-gamma R(0/0) mice, we demonstrate that induction of EAT does not require an intact IFN-gamma system, while progression to full-blown disease depends on the action of IFN-gamma.

Adjuvants, Immunologic↗