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Biomedical subjects

H Zhang

Publications and source records attributed to H Zhang.

At least 181 records · Page 10Linked to original sources
PubMed2002

A vector with transcriptional terminators increases efficiency of cloning of an RNA virus by reverse transcription long polymerase chain reaction.

Full-length cDNA clones of RNA viruses are advantageous for maintaining the genomic sequence without the generation of diversity by accumulation of sequence mutations during productive virus replication. They permit in vitro manipulation of the genomic clone to test the effect of sequence changes on the phenotype of reactivated virus. Infectious cDNA clones have been produced by ligation of subgenomic clones but are sometimes difficult to generate in a single cloning operation. We used reverse-transcription to synthesize full-length cDNA from genomic RNA of Coxsackievirus B3 of the Picornavirus family and enzymatically amplified this by long PCR. Five different cloning vectors were used to clone the long PCR product, including the vector Lorist6 which contains transcriptional terminators on either side of the cloning site to prevent transcription of inserts in E. coli. No recombinant colonies were obtained from any of the vectors lacking transcriptional terminators but three full-length clones were obtained using Lorist6. The results suggest that transcriptional terminators increase the recovery of cDNA clones of the 7.4 kb Coxsackie virus genome in this cosmid vector, without resort to phage packaging, representing an advance over previous methods and advantages in the molecular manipulation of these viruses.

Animals↗
PubMed2001

(-)-Epicatechin inhibits nitration and dimerization of tyrosine in hydrophilic as well as hydrophobic environments.

The flavanol (-)-epicatechin is known to protect against peroxynitrite-induced nitration and oxidation reactions. This study investigated the protection afforded by (-)-epicatechin against both these reaction types on one target molecule, the aminoacid tyrosine, in a hydrophilic milieu as well as with a lipophilic tyrosine derivative, N-t-BOC l-tyrosine tert-butyl ester (BTBE), bound to liposomes. The flavanol efficiently attenuated both tyrosine nitration and tyrosine dimerization (which is based on an initial oxidation reaction) and was active in the hydrophilic and hydrophobic systems at similar IC(50) values, approximately 0.02-0.05 mol (-)-epicatechin/mol peroxynitrite. Related procyanidin oligomers of different chain-length (dimer to octamer) were also tested for their protective properties, and exhibited protection that, on a monomer basis, was in the same order of magnitude as those for (-)-epicatechin.

Antioxidants↗
PubMed2001

Characterization of human GRK7 as a potential cone opsin kinase.

PURPOSE: Homozygous inactivation of the mouse gene for GRK1 (G protein-coupled receptor kinase 1, or rhodopsin kinase) causes severe defects in the recovery of cone phototransduction. However, electroretinographic (ERG) analyses of human oguchi patients with defective GRK1 alleles showed normal or slightly abnormal photopic responses. It remains unclear why the loss of GRK1 yields such different phenotypes in the recovery of mouse and human cones. We examined the localization and enzyme activity of GRK7, the human ortholog of the seventh member of the GRK family, in an attempt to understand its potential role in photopic vision. METHODS: Bioinformatic approaches were used to identify the human GRK7 gene. Human and bovine GRK7 cDNAs were isolated by RT-PCR. Recombinant GRK7, expressed in insect cells, was used to phosphorylate activated rhodopsin. Antibodies raised against GRK7 peptides were used to examine the retina specific expression of GRK7 by immunoblotting and its subcellular localization by immunocytochemistry. RESULTS: The human GRK7 gene is located on chromosome 3q21, spans at least 10 Kb and consists of 4 exons. In human, GRK7 is expressed exclusively in the retina and is found in all retinal neurons, and specifically, in cone outer segments. Recombinant human GRK7 catalyzes rhodopsin phosphorylation in a light dependent manner. We provide evidence that GRK1 and GRK7 are co-expressed in human cones. In contrast, mouse GRK7 is expressed in many tissues including retina where photoreceptors apparently do not express GRK7. CONCLUSIONS: The presence of GRK7 in human, but not in mouse, cone outer segments suggests that GRK7 may function to provide the normal photopic vision reported by oguchi patients with a defective GRK1 gene. The absence of GRK7 expression in cone outer segments of mice is consistent with the notion that mouse cones rely solely on GRK1 to shutoff cone visual pigments.

Amino Acid Sequence↗
PubMed2001

Identification, mapping, and genomic structural analysis of an immunoreceptor tyrosine-based inhibition motif-bearing C-type lectin from homozygous clones of rainbow trout (Oncorhynchus mykiss).

Utilizing a spleen-derived cDNA library and rapid amplification of cDNA 5' ends, we cloned a novel type II C-type lectin from two homozygous clones of rainbow trout. The cDNA is 2535 bp in length, and contains a 1017-bp open reading frame. From this sequence, a protein containing 339 amino acids (aa) was deduced. Using PSI-BLAST to search the GenBank database, the deduced protein is a C-type lectin, belonging to the type II membrane receptors. The protein contains four domains: an 87-aa N-terminal cytoplasmic domain, a 21-aa transmembrane domain, an 82-aa neck domain, and a 149-aa C-terminal C-type lectin domain. Two immunoreceptor tyrosine-based inhibition motifs (ITIMs) were located in the N-terminal cytoplasmic domain. RT-PCR results indicated that this gene is transcribed mainly in peripheral blood lymphocytes, spleen, kidney, and gill, and its expression in liver and intestine is weak. Monoclonal antibody 1.14 was used to isolate B cells from peripheral blood lymphocytes. Analysis revealed that this gene is highly expressed in B cells. Genomic DNA was amplified with long-template PCR and sequenced. The gDNA is 12.0 kb in length and contains nine exons and eight introns. The first intron of the genes from the OSU and AR clones differed in length. Based on this difference, the genotype of 69 doubled-haploid offspring of OSU and AR were screened. Subsequently, this gene was mapped on the rainbow trout linkage map to group XXI. Results of a Southern blot indicated that the gene ( TCL-2) exists as a single copy in the rainbow trout genome. The genomic structure, the deduced protein structure, the tissue expression pattern, as well as the phylogenetic analysis of the carbohydrate recognition domain based on the deduced amino acid sequence indicate that TCL-2 resembles CD72; however, the carbohydrate recognition domain sequences of TCL-2 and CD72 are highly diverged.

Amino Acid Motifs↗
PubMed2001

[Classification of intracranial dural arteriovenous fistula and its clinical signification].

OBJECTIVE: To develop a practical imaging calssification of intracranial dural arteriovenous fistula (DAVF) based on imaging and its clinical significance. METHOD: The lesion positions and drainage patterns of vein in 110 patients with 121 dural arteriovenous fistulas hospitalized in Xuanwu Hospital were analyzed and a new type of classification of DAVF was developed. RESULTS: The dural arteriovenous fistulas in 110 patients were classified accrding to lesion position into DAVF of dural sinus, cavernous sinus, tentorial incisure, venous plexus at skull base, and cerebral falx; or classified according to the pattern of venous drainiage into type I (draining directly into dural vein or dural sinus), type II (directly draining into dural sinus with retrograde venous drainage into cortical vein or spinal vcein), and type III (directly draining into cortical vein or spinal vein). Then, based on a combination of these two criteria, DAVF was claassified into cavernius sinus area type I (38 cases, 69.1%) and type II (17 cases, 30.9%); dural sinus area type I (11 cases, 33.3%), type II (16 cases, 56.4%), and type III (2 cases, 10.3%); and tentorial area (20 cases), skull base vein plexus area (6 cases), and cerebral falx area (1 case), all in type III. CONCLUSION: The newly developed classification system of intracranial dural arteriovenous fistula helps analyze clinical risk and determine therapeutic procedures.

Adolescent↗
PubMed2001

Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain.

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.

Amino Acid Sequence↗
PubMed2001

Phylogenetic comparison of classical swine fever virus in China.

An N-terminal fragment of the E2 gene of classical swine fever (CSF) virus encoding major immunogenic sites was amplified by RT-PCR directly from 110 clinical specimens representing 109 epizootic sites during the last decade in China. Phylogenetic relationships between these viruses as well as 20 reference strains were determined by comparison of their nucleotide sequences. A phylogenetic tree showed that 103 of the 110 field viruses (93.6%) were clustered within group 2 and subdivided into three subgroups, while the remaining seven viruses (6.4%), along with two Chinese reference strains, Shimen and HCLV (attenuated vaccine strain), were clustered into subgroup 1.1 within group 1. However, none of the Chinese CSF viruses were members of subgroup 1.2 (represented by reference strain Brescia). This is the first report on the distribution of CSF virus genotypes in China. Results indicated that CSF viruses predominating in recent epizootics within China are genetically divergent from the reference strain Shimen and the vaccine strain HCLV.

Animals↗
PubMed2001

Complement 1 inhibitor is a regulator of the alternative complement pathway.

We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B-depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti-C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.

Absorption↗
PubMed2001

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.

Animals↗
PubMed2001

Crystal structure of YecO from Haemophilus influenzae (HI0319) reveals a methyltransferase fold and a bound S-adenosylhomocysteine.

The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2-A resolution. The structure reveals a fold typical of S-adenosyl-L-methionine-dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S-adenosyl-L-homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent-excluded substrate binding cavity of 162 A(3) in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho-substituted benzoic acids fit best in the active site.

Binding Sites↗
PubMed2001

Response to hypoxia involves transforming growth factor-beta2 and Smad proteins in human endothelial cells.

Oxygen deprivation (hypoxia) is a consistent component of ischemia that induces an inflammatory and prothrombotic response in the endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O(2)) increases messenger RNA and protein levels of transforming growth factor-beta2 (TGF-beta2), a cytokine with potent regulatory effects on vascular inflammatory responses. Messenger RNA levels of the TGF-beta2 type II membrane receptor, which is a serine threonine kinase, also increased. The stimulatory effect of hypoxia was found to occur at the level of transcription of the TGF-beta2 gene and involves Smad proteins, a class of intracellular signaling proteins that mediates the downstream effects of TGF-beta receptors. Transient transfection studies showed that the region spanning -77 and -40 base pairs within the TGF-beta2 promoter (harboring a Smad-binding "CAGA box") is activated in hypoxic cells compared with nonhypoxic controls (P <.01). Hypoxia also stimulated transcription from another promoter, 3TP-Lux, a reporter construct responsive to Smads and TGF-beta. In addition, specific binding to a Smad-binding oligonucleotide was observed with nuclear extracts from hypoxic endothelial cells but not from nonhypoxic cells. It is concluded that Smad proteins, which can regulate endothelial responses to mechanical and inflammatory stress, also may play an important role in vascular responses to hypoxia and ischemia.

Binding Sites↗
PubMed2001

Synthesis of isoquinolines and pyridines by the palladium-catalyzed iminoannulation of internal alkynes.

A wide variety of substituted isoquinoline, tetrahydroisoquinoline, 5,6-dihydrobenz[f]isoquinoline, pyrindine, and pyridine heterocycles have been prepared in good to excellent yields via annulation of internal acetylenes with the tert-butylimines of o-iodobenzaldehydes and 3-halo-2-alkenals in the presence of a palladium catalyst. The best results are obtained by employing 5 mol % of Pd(OAc)(2), an excess of the alkyne, 1 equiv of Na(2)CO(3) as a base, and 10 mol % of PPh(3) in DMF as the solvent. This annulation methodology is particularly effective for aryl- or alkenyl-substituted alkynes. When electron-rich imines are employed, this chemistry can be extended to alkyl-substituted alkynes. Trimethylsilyl-substituted alkynes also undergo this annulation process to afford monosubstituted heterocyclic products absent the silyl group.

Alkaloids↗
PubMed2001

Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines.

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression, referred to as RNA interference (RNAi). In invertebrates, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-nt-long short interfering (si) duplex RNAs, acting as effectors of RNAi. siRNAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are impeded by its nonspecific effects, mediated by dsRNA-dependent protein kinase PKR and RNase L. Here, we report that the RNAi response can be induced effectively by long dsRNA in nondifferentiated mouse cells grown in culture. Transfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results in a sequence-specific decrease in the level of proteins expressed from either exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediated by siRNAs, which are generated by cleavage of dsRNA by the RNaseIII-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into approximately 23-nt fragments and that Dicer localizes to the cytoplasm of EC and HeLa cells.

Animals↗
PubMed2001

Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.

Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.

Amino Acid Substitution↗
PubMed2001

Assessment of membrane potentials of mitochondrial populations in living cells.

Mitochondrial membrane potentials (MMP) reflect the functional status of mitochondria within cells. Fluorescent probes to estimate these potentials within cells have been available for some time, but measurements of populations of mitochondria are not possible by existing methods. Therefore, comparisons between different cell types (e.g., fibroblasts and neuroblastoma), fibroblast cell lines from different patients, or even the same cell following various experimental paradigms are not feasible. The current approach estimates populations of MMP within living cells at 37 degrees C using the combination of conventional fluorescence microscopy and three-dimensional deconvolution by exhaustive photon reassignment. With this method, raw images are acquired rapidly with low-intensity (nonlaser) light with minimal concentrations of fluorescent dye. The method uses the fluorescent dye tetramethylrhodamine methyl ester, which equilibrates in cells according to the Nernst equation and provides a numerical, replicable estimate of MMP for populations of cellular mitochondria. This method can detect either increases or decreases in MMP as small as 5%. Furthermore, MMP in different cell types appear distinct. Values in fibroblasts (-105 +/- 0.9 mV) and N2a cells (-81 +/- 0.7 mV) were very different by this method. This approach bridges investigations of individual mitochondria to those that assess MMP by examining global fluorescence from cells.

Cell Line↗
PubMed2001

Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.

14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with diverse ligands and are involved in the regulation of mitogenesis, cell cycle progression, and apoptosis. However, whether different 14-3-3 isoforms are responsible for distinct functions remains elusive. Here we report that multiple isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad, Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3 isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in regulating one of its ligands, Bad, is conserved among mammalian isoforms. We addressed whether 14-3-3 isoforms are differentially expressed in tissues, which may in part determine isoform-specific interactions. In situ hybridization revealed that 14-3-3zeta was present in most tissues tested, but sigma was preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can interact and control the function of selected protein ligands, and differential tissue distribution of 14-3-3 isoforms may contribute to their specific interactions and subsequent downstream signaling events.

14-3-3 Proteins↗
PubMed2001

DGT as an in situ tool for measuring radiocesium in natural waters.

The application of diffusive gradients in thin-films (DGT) samplers for the measurement of cesium radionuclides in solution, using an ammonium molybdophosphate (AMP) binding agent, was tested under both laboratory and field conditions. In the former they proved able to reproduce known 134Cs concentrations (60 Bq L(-1)) with a high degree of accuracy and precision over periods up to approximately 1 d, in freshwaters over a wide range of pH and temperature, and in saline water. In field trials in a freshwater lake receiving nuclear power station discharges, mean concentrations of 137CS (47-61 mBq L(-1)) were measured over periods from 5 d to 1 month. These agreed, within error, with mean concentrations determined from grab samples but rigorous field validation of long-term (month) deployments of DGT devices proved impossible using conventional sampling procedures, due to loss of 137Cs to container walls. Identified limitations of the DGT technique included probable AMP degradation over longer periods and calibration problems if large changes in temperature and concentration occurred together. Potential limitations due to biofilm growth were considered not to be significant. Despite the limitations, the technique appears to measure concentrations accurately for deployment times of 1 month or less. It has several advantages over traditional sampling methods for monitoring radionuclides in the solution/dissolved phase, including its simplicity, provision of time-averaged mean concentrations, and automatic in-situ concentration onto a medium with ideal counting geometry for gamma spectrometry.

Cesium Radioisotopes↗