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H Huang

Publications and source records attributed to H Huang.

At least 199 records · Page 11Linked to original sources
PubMed2000

The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis.

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Amyotrophic Lateral Sclerosis↗
PubMed2000

APC mutations in sporadic medulloblastomas.

The cerebellar medulloblastoma (WHO Grade IV) is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Several molecular alterations appear to be involved, including isochromosome 17q and the p53, PTCH, and beta-catenin gene mutations. In this study, 46 sporadic medulloblastomas were screened for the presence of mutations in genes of the Wnt signaling pathway (APC and beta-catenin). Single-strand conformational polymorphism (SSCP) analysis followed by direct DNA sequencing revealed 3 miscoding APC mutations in 2 (4.3%) medulloblastomas. One case contained a GCA-->GTA mutation at codon 1296 (Ala-->Val), and another case had double point mutations at codons 1472 (GTA-->ATA, Val-->Ile) and 1495 (AGT-->GGT, Ser-->Gly). Miscoding beta-catenin mutations were detected in 4 tumors (8.7%). Three of these were located at codon 33 (TCT -->TTT, Ser-->Phe) and another at codon 37 (TCT-->GCT, Ser-->Ala). Adenomatous polyposis coli (APC) gene and beta-catenin mutations were mutually exclusive and occurred in a total of 6 of 46 cases (13%). Although germline APC mutations are a well established cause of familial colon and brain tumors (Turcot syndrome), this study provides the first evidence that APC mutations are also operative in a subset of sporadic medulloblastomas.

Adenomatous Polyposis Coli Protein↗
PubMed2000

Enhancement of gene expression in rat dental follicle cells by parathyroid hormone-related protein.

Recent studies indicate that parathyroid hormone-related protein (PTHrP) is required for tooth eruption in mice. Localized in the stellate reticulum, PTHrP might exert a paracrine effect on cells of the adjacent dental follicle to initiate eruption. The presence of a follicle is needed for eruption and, at the cellular level, there is an influx of mononuclear cells in the follicle early postnatally in the first mandibular molar of the rat. In turn, these mononuclear cells fuse to form osteoclasts, which erode the alveolar bone to form an eruption pathway. At the molecular level, the dental follicle cells of the rat molar maximally express the genes for monocyte chemotactic protein-1 (MCP-1) and colony-stimulating factor-1 (CSF-1) at day 3 postnatally. Because day 3 also is the time of maximal influx of the mononuclear cells into the follicle, MCP-1 and CSF-1 could be involved in the recruitment/maturation of these cells. To determine if PTHrP can modulate gene expression in the dental follicle, cultured follicle cells were immunostained to show the receptor for PTHrP. The gene expression of this receptor was enhanced by incubating the cells with interleukin-1alpha (IL-1alpha). Next, the ability of PTHrP itself to enhance gene expression of either MCP-1 or CSF-1 in the dental follicle cells was determined by incubating the cells with PTHrP in either a time- or concentration-course manner (1-15 h or 1-100 ng/ml). By reverse transcription-polymerase chain reaction, it was demonstrated that PTHrP enhanced MCP-1 expression in a concentration-dependent fashion, with 50 ng PTHrP/ml inducing maximal expression of either MCP-1 or CSF-1. In the time-dependent studies, PTHrP caused maximal expression within 30 min for either MCP-1 or CSF-1. Immunoblotting revealed that PTHrP also enhanced secretion of MCP-1 by the follicle cells. Thus, one of the actions of PTHrP in tooth eruption may be that it enhances MCP-1 and CSF-1 gene expression and secretion in the dental follicle. Moreover, IL-1alpha may accentuate its action by enhancing the expression for the PTHrP receptor in the follicle cells.

Animals↗
PubMed2000

Chemical kinetic modelling of PCDD formation from chlorophenol catalysed by incinerator fly ash.

A kinetic model is developed for PCDD formation from chlorophenol catalysed by incinerator fly ash. The key step in the model is a Langmuir-Hinshelwood type elementary step for the coupling of two adsorbed chlorophenol species to PCDD. Kinetic expression is derived which can relate PCDD formation rates with process variables including temperature, precursor concentration, fly ash loading and number of active sites in fly ash. Calculated PCDD formation rates based on this kinetic model are in good agreement with laboratory measurements reported in the literature. When the model is applied to industrial incinerator conditions, at maximum a PCDD yield of 10(-3) microg/N m3 is calculated.

Air Pollution↗
PubMed2000

Influence of cetane improvers on the air quality in an urban street canyon.

The concentration distributions of NOx, PM, HC and CO in an urban street canyon have been estimated using a two-dimensional air quality numerical model based on the k-epsilon turbulent model and the atmospheric convection diffusion equation when various cetane improvers were used in diesel fuels. A wind vortex can be found within the street canyon, and the pollutants emitted from the bottom of the street canyon tend to follow the course of the wind field, moving circularly. The addition of cetane improvers can improve the air quality in a street canyon, all of the pollutants were found to decrease with increasing centane number.

Air↗
PubMed2000

Circulating adhesion molecules and neutral endopeptidase enzymuria in patients with urolithiasis and hydronephrosis.

OBJECTIVES: To measure the concentrations of adhesion molecules (intercellular adhesion molecule-1 ¿ICAM-1, E-selectin, and L-selectin) in the serum of patients with renal stones and patients with hydronephrosis caused by obstructive ureteral stones. Renal tubular enzymes were examined from their urine samples to evaluate whether neutral endopeptidase (NEP) behaved as traditional tubular enzyme markers (N-acetyl-beta-glucosaminidase ¿NAG and beta-galactosidase ¿beta-GAL) in their disease state. METHODS: Three groups were studied. Group 1 included 15 normal volunteers, group 2 included 12 patients with ureteral stones and ipsilateral hydronephrosis, and group 3 included 17 patients with renal stones in one kidney without hydronephrosis or hydrocalycosis. A single, overnight fasting blood and urine sample was collected from each subject. Serum levels of ICAM-1, E-selectin, and L-selectin were measured by enzyme-linked immunosorbent assay. NEP, NAG, and beta-GAL were measured from urine samples, and the enzyme activities were expressed per gram of creatinine. RESULTS: Serum levels of ICAM-1 were higher in groups 2 and 3 (522 +/- 95 and 329 +/- 42 ng/mL, respectively), but the differences were only significant between group 2 and group 1 (263 +/- 32 ng/mL) and group 2 and group 3 (P <0.05). Serum levels of L-selectin were lower in group 3 and were significantly different when compared with groups 1 and 2 (P <0. 05). The serum levels of E-selectin were not significantly different among these three groups. Urinary levels of NEP were lower in group 2, although the levels of NAG and beta-GAL were more elevated than in group 1. CONCLUSIONS: Serum levels of ICAM-1 were elevated in patients with unilateral hydronephrosis caused by a ureteral stone, and E-selectin and L-selectin levels did not change significantly. These findings suggest that ICAM-1 may play a role in renal immune injury to ureteral obstruction. The urinary NEP values were lower in patients with hydronephrosis caused by ureteral stones, and the traditional lysosomal enzymes were increased. The lower urinary NEP values might suggest impairment of ipsilateral renal function. Renal stones per se resulted in no significant changes in serum adhesion molecule levels, although the levels of L-selectin were significantly decreased.

Cell Adhesion Molecules↗
PubMed2000

A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo.

A highly specific monoclonal antibody, which recognises an epitope between amino acids (aa) 408 and 446 of EBNA-1, was found to immunoprecipitate this protein with great efficiency. By adding a 39-aa tag, the BGLF4 protein product of EBV was shown to express in the cytoplasm of transfected cells. This EBNA-1 tag could be used for the detection of specific protein expression by immunoblotting, immunofluorescence and, especially, immunoprecipitation assays. A plasmid vector encoding this EBNA-1 tag sequence was therefore designed for efficient expression both in vitro and in vivo. The efficient translational start signal of black beetle virus (BBV) was placed under the control of the SV40 or T7 promoters, and the beta-globin splicing signal used to enhance the transportation of mRNA from the nucleus to the cytoplasm.

Antibodies, Monoclonal↗
PubMed2000

3-Aryl-2-carbomethoxybicyclo[3.2.1]oct-2-enes inhibit WIN 35,428 binding potently and selectively at the dopamine transporter.

The search for medications for cocaine abuse has focused upon the design of potential cocaine antagonists or cocaine substitutes which interact at the dopamine transporter of mammalian systems. This manuscript describes the synthesis and biological evaluation of 8-substituted 2-carbomethoxy-3-arylbicyclo[3.2.1]oct-2-enes. These compounds prove potent and selective inhibitors of the dopamine transporter. Their selectivity results primarily from a reduced inhibitory potency toward the serotonin transporter. This work supports the notion that the orientation of the 3-aryl ring in the bicyclo[3.2.1]octane system affects the interaction of these molecules with the serotonin transporter far more markedly than it affects the interaction with the dopamine transporter.

Animals↗
PubMed2000

Trapping of a catalytic HIV reverse transcriptase*template:primer complex through a disulfide bond.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for the treatment of acquired immunodeficiency syndrome (AIDS). Resistance mutations in RT compromise treatment, however. Efforts to understand the enzymatic mechanism of RT and the basis for mutational resistance to anti-RT drugs have been hampered by the failure to crystallize a catalytically informative RT-substrate complex. RESULTS: We present here experiments that allow us to understand the reason for the failure to crystallize such a complex. Based on this understanding, we have devised a new approach for using a combinatorial disulfide cross-linking strategy to trap a catalytic RT*template:primer*dNTP ternary complex, thereby enabling the growth of co-crystals suitable for high-resolution structural analysis. The crystals contain a fully assembled active site poised for catalysis. The cross-link itself appears to be conformationally mobile, and the surrounding region is undistorted, suggesting that the cross-link is a structurally passive device that merely acts to prevent dissociation of the catalytic complex. CONCLUSIONS: The new strategy discussed here has resulted in the crystallization and structure determination of a catalytically relevant RT*template:primer*dNTP complex. The structure has allowed us to analyze possible causes of drug resistance at the molecular level. This information will assist efforts to develop new classes of nucleoside analog inhibitors, which might help circumvent current resistance profiles. The covalent trapping strategy described here may be useful with other protein-DNA complexes that have been refractory to structural analysis.

Catalysis↗
PubMed2000

Delay of tooth eruption in null mice devoid of the type I IL-1R gene.

Interleukin-one alpha (IL-1alpha) is located in the stellate reticulum and its receptor, type I IL-1R (IL-1R), is present in the adjacent dental follicle. IL-1alpha may play a role in initiating tooth eruption because of its ability to enhance the gene expression in the dental follicle of putative tooth eruption molecules colony-stimulating factor-one (CSF-1). monocyte chemotactic protein-1 (MCP-1), and nuclear factor kappa B (NFkappa-B). To directly examine the effect of IL-1alpha and IL-1R on tooth eruption, we observed the times of tooth eruption in null mice devoid of the type I IL-1R gene. The results showed that the time of eruption in the null mice was delayed by 2 d for the first mandibular molar and 1 d for incisors as compared to wild type controls. Reverse transcription-polymerase chain reaction (RT-PCR) techniques confirmed the absence of the IL-1R gene in the null mice, but the genes for CSF-1 and NFkappaB were still expressed. Thus, a molecule(s) other than IL-1alpha also may enhance the expression of CSF- and NFkappaB in the dental follicle. Because there is a slight delay of tooth eruption in IL-1R null mice, IL-1alpha may normally play a role in eruption. However, eruption eventually can occur without the signaling from IL-1alpha.

Animals↗
PubMed2000

Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement.

Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.

Amino Acid Sequence↗
PubMed2000

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis.

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana. However, the leaf development that is specific to plants has been little studied. With the aim of contributing to the genetic dissection of leaf development, we have performed a large-scare screening for mutants with abnormal leaves. Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethyl-methae sulfonate (EMS) mutagenesis, four independent mutant lines have been identified and studied genetically. Phenotypes of these mutant lines represent the defects of four novel nuclear genes designated LL1 (LOTUS LEAF 1), LL2 (LOTUS LEAF 2), URO (UPRIGHT ROSETTE). and EIL (ENVIRONMENT CONDITION INDUCED LESION). The phenotypic analysis indicates that these genes play important roles during leaf development. For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2, we have mapped these genes to chromosome regions with an efficient and rapid mapping method.

Arabidopsis↗
PubMed2000

PIR: a new resource for bioinformatics.

UNLABELLED: The Protein Information Resource (PIR) has greatly expanded its Web site and developed a set of interactive search and analysis tools to facilitate the analysis, annotation, and functional identification of proteins. New search engines have been implemented to combine sequence similarity search results with database annotation information. The new PIR search systems have proved very useful in providing enriched functional annotation of protein sequences, determining protein superfamily-domain relationships, and detecting annotation errors in genomic database archives. AVAILABILITY: http://pir.georgetown.edu/. CONTACT: mcgarvey@nbrf.georgetown.edu

Animals↗
PubMed2000

Trypanosoma cruzi infection (Chagas' disease) of mice causes activation of the mitogen-activated protein kinase cascade and expression of endothelin-1 in the myocardium.

Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore. in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mitogen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice. These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection.

Animals↗
PubMed2000

A comparative map of the zebrafish genome.

Zebrafish mutations define the functions of hundreds of essential genes in the vertebrate genome. To accelerate the molecular analysis of zebrafish mutations and to facilitate comparisons among the genomes of zebrafish and other vertebrates, we used a homozygous diploid meiotic mapping panel to localize polymorphisms in 691 previously unmapped genes and expressed sequence tags (ESTs). Together with earlier efforts, this work raises the total number of markers scored in the mapping panel to 2119, including 1503 genes and ESTs and 616 previously characterized simple-sequence length polymorphisms. Sequence analysis of zebrafish genes mapped in this study and in prior work identified putative human orthologs for 804 zebrafish genes and ESTs. Map comparisons revealed 139 new conserved syntenies, in which two or more genes are on the same chromosome in zebrafish and human. Although some conserved syntenies are quite large, there were changes in gene order within conserved groups, apparently reflecting the relatively frequent occurrence of inversions and other intrachromosomal rearrangements since the divergence of teleost and tetrapod ancestors. Comparative mapping also shows that there is not a one-to-one correspondence between zebrafish and human chromosomes. Mapping of duplicate gene pairs identified segments of 20 linkage groups that may have arisen during a genome duplication that occurred early in the evolution of teleosts after the divergence of teleost and mammalian ancestors. This comparative map will accelerate the molecular analysis of zebrafish mutations and enhance the understanding of the evolution of the vertebrate genome.

Animals↗
PubMed2000

Genetic linkage mapping of zebrafish genes and ESTs.

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This "doubled haploid" strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes.

Animals↗
PubMed2000

Zebrafish comparative genomics and the origins of vertebrate chromosomes.

To help understand mechanisms of vertebrate genome evolution, we have compared zebrafish and tetrapod gene maps. It has been suggested that translocations are fixed more frequently than inversions in mammals. Gene maps showed that blocks of conserved syntenies between zebrafish and humans were large, but gene orders were frequently inverted and transposed. This shows that intrachromosomal rearrangements have been fixed more frequently than translocations. Duplicated chromosome segments suggest that a genome duplication occurred in ray-fin phylogeny, and comparative studies suggest that this event happened deep in the ancestry of teleost fish. Consideration of duplicate chromosome segments shows that at least 20% of duplicated gene pairs may be retained from this event. Despite genome duplication, zebrafish and humans have about the same number of chromosomes, and zebrafish chromosomes are mosaically orthologous to several human chromosomes. Is this because of an excess of chromosome fissions in the human lineage or an excess of chromosome fusions in the zebrafish lineage? Comparative analysis suggests that an excess of chromosome fissions in the tetrapod lineage may account for chromosome numbers and provides histories for several human chromosomes.

Animals↗