Biomedical subjects

X Cheng

Publications and source records attributed to X Cheng.

At least 55 records · Page 3Linked to original sources

PubMed2000

Serologic response to Blastocystis hominis infection in asymptomatic individuals.

The pathogenic potential of Blastocystis hominis in the human intestine is subject to controversy because the organism has been found in both symptomatic and asymptomatic individuals. To help clarify this issue, we monitored the serologic response to the organism in B. hominis-infected individuals free of gastroenterologic disorders. 1) Serum antibodies to B. hominis were detected in 70% of infected asymptomatic individuals by an indirect immunofluorescence (IFA) test. 2) IFA and immunoelectron microscopy revealed that the antibody response was directed against a surface antigen(s) of the organism. 3) Analysis by immunoblotting implicated a 12 kDa protein of B. hominis. 4) The strongest positive reaction was obtained in an individual chronically infected for more than 2 years. It may be that long exposure to the parasite is necessary for a serologic response.

Animals↗

PubMed1999

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom.

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.

Agkistrodon↗

PubMed1999

Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch.

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (R(I) and R(II)) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the R(I) and R(II) subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-myristylation and R(II) subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R(II) subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the R(I) and R(II) subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the R(II) subunit, but not the R(I) subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R(II) subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. R(II) subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.

Cyclic AMP-Dependent Protein Kinases↗

PubMed1999

Isolation and identification of fxsA, an Escherichia coli gene that can suppress F exclusion of bacteriophage T7.

A selection for mutants of Escherichia coli that survive coexpression of bacteriophage T7 gene 10 and plasmid F pifA has allowed the identification of a newly defined genetic locus, fxsA. fxsA is located at 94.1 min on the E. coli chromosome; the gene is monocistronic and non-essential for growth. Overexpression of fxsA is necessary for resistance to the toxicity of T7 gene 10 in the presence of pifA; the original mutant strain contains a promoter-up mutation, changing a G residue to the "invariant" T in the -10 hexamer of a sigma(70)promoter. This chromosomal mutation causes a 25-fold increase in the level of fxsA mRNA. The initiation codon of fxsA is shown to be UUG, and the FxsA protein is then deduced to consist of 158 amino acid residues. A similar mutant selection that demanded cell survival to a challenge of T7 gene 1.2 and pifA also resulted in the isolation of the identical promoter-up mutation that affects expression of fxsA. The increased levels of FxsA resulting from the promoter-up mutation allow phage T7 to avoid exclusion by the F plasmid, presumably by protecting the cell from premature death due to gene 10 or to gene 1.2 expression in the presence of the PifA protein.

Amino Acid Sequence↗

PubMed1999

Crystal structure of the conserved core of the herpes simplex virus transcriptional regulatory protein VP16.

On infection, the herpes simplex virus (HSV) virion protein VP16 (Vmw65; alphaTIF) forms a transcriptional regulatory complex-the VP16-induced complex-with two cellular proteins, HCF and Oct-1, on VP16-responsive cis-regulatory elements in HSV immediate-early promoters called TAATGARAT. Comparison of different HSV VP16 sequences reveals a conserved core region that is sufficient for VP16-induced complex formation. The crystal structure of the VP16 core has been determined at 2.1 A resolution. The results reveal a novel, seat-like protein structure. Together with the activity of mutant VP16 proteins, the structure of free VP16 suggests that it contains (1) a disordered carboxy-terminal region that associates with HCF, Oct-1, and DNA in the VP16-induced complex, and (2) a structured region involved in virion assembly and possessing a novel DNA-binding surface that differentiates among TAATGARAT VP16-response elements.

Amino Acid Sequence↗

PubMed1999

Structure of a binary complex of HhaI methyltransferase with S-adenosyl-L-methionine formed in the presence of a short non-specific DNA oligonucleotide.

We have determined a structure for a complex formed between HhaI methyltransferase (M.HhaI) and S-adenosyl-L-methionine (AdoMet) in the presence of a non-specific short oligonucleotide. M.HhaI binds to the non-specific short oligonucleotides in solution. Although no DNA is incorporated in the crystal, AdoMet binds in a primed orientation, identical with that observed in the ternary complex of the enzyme, cognate DNA, and AdoMet or S-adenosyl-L-homocysteine (AdoHcy). This orientation differs from the previously observed unprimed orientation in the M.HhaI-AdoMet binary complex, where the S+-CH3 unit of AdoMet is protected by a favorable cation-pi interaction with Trp41. The structure suggests that the presence of DNA can guide AdoMet into the primed orientation. These results shed new light on the proposed ordered mechanism of binding and explains the stable association between AdoMet and M.HhaI.

Crystallization↗

PubMed1999

Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin.

Vasoactive GTP-binding protein-coupled receptor agonists such as angiotensin II (AII), endothelin-1 (ET-1), and alpha-thrombin (alpha-Thr) have been reported to indirectly stimulate vascular smooth muscle cell (VSMC) proliferation by regulating the expression of one or more autocrine growth factors. Using ion-exchange, gel-filtration, and reverse-phase chromatographic purification methods, we isolated a major mitogenic protein present in AII-stimulated rat aortic smooth muscle (RASM) cell conditioned medium. Twenty N-terminal amino acids of the purified peptide were identified, and they had 75% amino acid sequence identity with mouse epiregulin, an epidermal growth factor (EGF)-related growth factor. We cloned the cDNA for rat epiregulin to determine its pattern of expression in G-protein-coupled receptor agonist-stimulated cells and confirm its activity as a mitogen. After treatment of RASM cells with AII, ET-1, or alpha-Thr for 1 h, induction of two epiregulin transcripts was observed, including a 4.8-kb transcript and a novel transcript of approximately 1.2 kb. Recombinant rat epiregulin was strongly mitogenic for RASM cells, stimulating DNA synthesis to levels similar to those induced by serum or platelet-derived growth factor and approximately 3-fold above that observed with saturating concentrations of EGF. In addition, epiregulin caused rapid EGF receptor activation in RASM cells. However, relative levels of EGF receptor tyrosine phosphorylation stimulated by epiregulin were less than those induced by EGF or betacellulin. Taken together, these results indicate that epiregulin is a potent VSMC-secreted mitogen, induced in common by AII, ET-1, and alpha-Thr, that may contribute to VSMC proliferation and vascular remodeling stimulated by vasoactive agonists.

3T3 Cells↗

PubMed1999

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation.

Animals↗

PubMed1999

Bilateral kidney ligation abolishes pressor response to N(G)-nitro-D-arginine.

We have shown that N(G)-nitro-D-arginine (D-NNA) is 50% as potent as N(G)-nitro-L-arginine (L-NNA) in causing pressor response and 2-3% as potent as L-NNA in inhibiting endothelium-dependent relaxation in vitro. These results suggest in vivo activation of D-NNA. Furthermore, the potency of D-NNA was markedly increased after it had been incubated with homogenate of the kidney, but not plasma or homogenate of the aorta, lungs or liver. This study examined if bilateral ligation of the kidneys attenuated the biological action of D-NNA. I.v. bolus of D-NNA (16 mg/kg), L-NNA (3 mg/kg) and norepinephrine (0.25-16 microg/kg) increased arterial pressure in sham-operated rats. Bilateral ligation of the kidneys abolished pressor response to D-NNA, but not L-NNA and norepinephrine. I.v. bolus D-NNA in sham-operated rats, but not kidney-ligated rats, inhibited relaxation response to acetylcholine in pre-constricted aortic rings ex vivo. These results indicate that the kidney is the primary organ which activates D-NNA.

Acetylcholine↗

PubMed1999

Effects of several drugs on the liver injury induced by delayed-type hypersensitivity to picryl chloride by regulating suppressor or helper T cells.

The effects of histamine, cimetidine, and diphenhydramine on picryl chloride (PCl)-induced ear contact sensitivity, as well as liver injury, were examined in mice. Histamine was found to produce less response in mice to PCl. In contrast, cimetidine, a selective antagonist of histamine type 2 receptor, significantly enhanced the response, while diphenhydramine, a selective antagonist of histamine type 1 receptor showed no effect. The pre-treatment of 2,4, 6-trinitrobenzene sulphonic acid (TNBS) significantly caused a tolerance to the formation of the liver injury induced by delayed-type hypersensitivity (DTH) to PCl. Against the tolerance, the single intravenous administration of 150 mg kg-1 cyclophosphamide (Cy) at 3 days before the TNBS-treatment recovered the response and induced a remarkable elevation of serum transaminases. On the other hand, cyclosporin A protected the liver injury. These observations revealed that the development of acute PCl-DTH liver injury was regulated by the functional state of suppressor and helper T cells.

Animals↗

PubMed1999

Coiling phagocytosis is the predominant mechanism for uptake of the colonic spirochetosis bacterium Serpulina pilosicoli by human monocytes.

Serpulina pilosicoli is a newly identified pathogenic spirochete that establishes persistent colonic infections in human beings and animals. Macrophages are one of the key defenses against invasion of mucosal surfaces by bacterial pathogens. Macrophages engulf many bacteria by conventional phagocytosis; however recent studies indicate coiling phagocytosis as a new and important mechanism for internalization of Legionella pneumophila and spirochetes of the genus Borrelia, Leptospira, and Treponema. In this study, THP-1 human monocytic cells were incubated with the human S. pilosicoli strain SP16 and the contribution of coiling and conventional phagocytosis to the total number of phagocytic events were determined by sequential ultrastructural examination between 5 and 45 minutes. The frequency of phagocytosis increased over time from 5.1% after 5 minutes up to 21.9% after 45 minutes with greater than 70% of the events involving coiling phagocytosis. The data indicate that coiling phagocytosis may be a universal mechanism for uptake of pathogenic spirochetes.

Brachyspira↗

PubMed1999

Molecular cloning and sequence analysis of cDNA encoding haemorrhagic toxin acutolysin A from Agkistrodon acutus.

By means of reverse transcription polymerase chain reaction, a full-length cDNA of 1632 bp is amplified from the snake venom gland total RNA of Agkistrodon acutus. Analysis of the nucleotide sequence indicates that the amplified cDNA contains a complete open reading frame encoding 413 amino acid residues including signal peptide sequence, zymogen sequence and proteinase domain. The zymogen sequence contains PKMCGVT motif which is highly conserved in almost all venom metalloproteinases. The metalloproteinase domain contains the conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region. The predicted amino acid sequence of the metalloproteinase domain is identical to the crystallographic sequence of acutolysin A and also shares high homology with other class I snake venom haemorrhagic toxins.

Agkistrodon↗

PubMed1999

Antithrombotic effects of low-molecular-weight heparin calcium (LMWH-Ca) in experimental models.

The antithrombotic activity of low-molecular-weight heparin calcium (LMWH-Ca) was studied in venous and arterial thrombosis models, arterial thrombosis model in rats, arterio-venous shunt model and venous thrombosis model in rabbits. The data showed that LMWH-Ca reduced thrombus formation in a dose-dependent manner. It suggests that LMWH-Ca is a potent antithrombotic agent for venous thrombosis, and also may be a beneficial therapeutic agent in arterial thrombosis.

Animals↗

PubMed1999

Local tumor recurrence or emergence of a new primary lesion? A molecular analysis.

The distinction between a new primary oral tumor and recurrence may bear significant prognostic implications. Currently, this differentiation relies mainly on tumor location: when both lesions are at or near the same site, the new one is regarded as a recurrence; when the two are at different sites, the second lesion is regarded as a new primary. Recent investigations using molecular analysis have demonstrated that some oral squamous cell carcinomas (SCC) arising from different sites show the same clonogenical changes. In this case report, we studied the clonality of three SCC (one primary, two apparent recurrences) from the right lateral tongue of a young, non-smoking woman by using microsatellite analysis for loss of heterozygosity. The results showed that while the first two tumors were clonogenically similar, the third tumor was clonogenically different and was consistent with the development of a new primary. This result indicates that location of tumors alone is not always reliable in determining whether a new tumor is a recurrence or a new primary lesion.

Adult↗

PubMed1999

Construction of La-tPA vector and expression in the mammary gland of transgenic mice.

Expression vectors of long acting human tissue plasminogen activator (La-tPa) in the mammary gland was constructed using the promoters of sheep beta-lactoglobulin gene (BLG, 5 kb) containing the first and the second introns obtained by PCR. Transgenic mice were established by microinjection. 540 fertilized eggs were injected and 6 transgenic mice were screened out from the offspring by PCR and Southern Blot analysis. The foreign gene integrating rate was 32%. The expression of La-tPA in mammary glands of transgenic mice was confirmed by milk assay and Northern blot analysis. Expression level of La-tPA reached 6 ug/ml in milk of transgenic mice.

Animals↗

PubMed1999

[Allotransplantation of cryopreserved fetal cartilage in plastic surgery].

OBJECTIVE: To study the applicability of homologous fetal cartilage in plastic surgery. METHODS: Limb and costal cartilage were taken from the dead fetus. The fetal cartilage was then frozen at -80 degrees C and preserved in -196 degrees C liquid nitrogen with a cryoprotective agent. A total of 38 patients received fetal cartilage transplantation. Of them there were 24 saddle nose, 4 auricle defects, 6 secondary deformities of cheiloschisis, 4 mandible deformities. RESULTS: Through follow-up of three and a half years, it was found that none of the transplanted cartilage was absorbed, deformed or rejected due to immunological reaction. The contours were satisfactory except 2 cases with auricle reconstruction. CONCLUSION: The clinical application of homologous fetal cartilages is of value in plastic surgery.

Adolescent↗

PubMed1998

Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.

The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.

Aspartic Acid↗

PubMed1998

Recombinant human respiratory syncytial virus (RSV) from cDNA and construction of subgroup A and B chimeric RSV.

Infectious human respiratory syncytial virus (RSV) was produced from a cDNA clone that contains 15,222 nucleotides of RSV genome derived from the A2 strain of subgroup A. Recovery of infectious RSV from cDNA required cotransfection of only three expression plasmids encoding the nucleoprotein (N), the phosphoprotein (P), and the major polymerase protein (L). Inclusion of the M2-1 plasmid was not required in the transfection reaction and if included did not significantly increase the rescue efficiency. However, a single nucleotide substitution in the RSV leader region (C to G at position 4 in the antigenomic sense), greatly increased the amount of infectious virus recovered from cDNA. A recombinant RSVA2 virus that expresses an additional structural G protein derived from a subgroup B RSV was also obtained. Both A2 and B strain G glycoproteins were expressed in cells infected with the chimeric RSV. A chimeric RSV that expresses a heterologous subgroup antigen in a live attenuated vaccine candidate may be important for prevention of diseases associated with both RSV subgroup A and subgroup B infection.

Animals↗