Biomedical subjects

X Cheng

Publications and source records attributed to X Cheng.

At least 19 recordsLinked to original sources

PubMed2000

Synthesis of the C-glycoside analogue of a novel sialyl Lewis X mimetic.

Sialyl Lewis X (sLex) mimetics that can function as selectin antagonists have received considerable attention in connection with the development of novel antiinflammatory therapies. An interesting structure that emerged from the studies of the Wong group is the 1,1-Gal-Man disaccharide 2, reported to bind E-selectin 5 times more strongly than sLex. The C-glycoside derivative 3 is of interest both as a conformational probe for selectin binding and as a hydrolytically stable analogue. Herein we illustrate a novel methodology for beta-C-galacto-disaccharides in the synthesis of 3. The protocol has as a key step a novel oxocarbenium ion-enol ether cyclization to give a C1-substituted galactal.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

Conformational differences between O- and C-glycosides: the alpha-O-man-(1-->1)-beta-Gal/alpha-C-Man-(1-->1)-beta-Gal case--a decisive demonstration of the importance of the exo-anomeric effect on the conformation of glycosides.

The conformational behavior of alpha-O-Man-(1-->1)-beta-Gal (1) and its C-analogue (2) has been studied using J/NOE NMR data, molecular mechanics, molecular dynamics, and ab initio calculations. The population distribution around the glycosidic linkages of 1 and 2 is rather different, especially for the alpha-Man linkage. A lower limit for the exo-anomeric effect in water has been experimentally determined.

Carbohydrate Conformation↗

PubMed2000

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes.

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.

Amino Acid Sequence↗

PubMed2000

High frequency of allelic loss in dysplastic lichenoid lesions.

Oral lichen planus (OLP) is a common mucosal condition that is considered premalignant by some, whereas others argue that only lichenoid lesions with epithelial dysplasia are at risk of progressing into oral carcinoma. A recent study from this laboratory used microsatellite analysis to evaluate OLP for loss of heterozygosity (LOH) at loci on three chromosomal arms (3p, 9p, and 17p) (Am J Path 1997;Vol151:Page323-Page327). Loss on these arms is a common event in oral epithelial dysplasia and has been associated with risk of progression of oral leukoplakia to cancer. The data showed that, although dysplastic epithelium demonstrated a high frequency of LOH (40% for mild dysplasia), a significantly lower frequency of LOH was noted in OLP (6%), which is even lower than that in hyperplasia (14%). Such results do not support OLP as a lesion at risk for malignant transformation. As a second step of the research, we determined LOH frequencies in 61 dysplastic lichenoid lesions (mild 35; moderate 19; severe 7) using the same microsatellite markers and compared these results with data obtained from the first study and from 13 normal mucosal specimens. Dysplastic lichenoid lesions showed a high frequency of loss (54% for lichenoid lesions with mild dysplasia), but values did not differ significantly from those observed in dysplasia of similar degree without lichenoid appearance. None of the normal mucosa demonstrated LOH. Epithelial dysplasia is a sign of malignant risk, independent of lichenoid changes. Such results suggest that pathologists should search for dysplasia carefully in lesions that otherwise qualify as OLP and that caution should be used when discounting dysplasia as being merely a reactive condition in lichenoid lesions.

Adult↗

PubMed2000

Affinity adsorbents for the vancomycin group of antibiotics.

The vancomycin group of antibiotics kill Gram-positive bacteria by binding to nascent bacterial cell-wall peptidoglycan bearing the C-terminal sequence-D-Ala-D-Ala. In this paper, affinity adsorbents for the vancomycin group of antibiotics were prepared by immobilizing the peptidoglycan analogues -D-Ala-D-Ala, -succinyl-D-Ala and -succinyl-Gly on to crosslinked poly(N, N-dimethylacrylamide). The adsorption capacities of the three adsorbents for demethylvancomycin were 0.59, 0.35 and 0.29 mmol/g, respectively. The adsorption capacity of the adsorbent with-D-Ala-D-Ala for vancomycin was 0.53 mmol/g. In contrast, the adsorbent bearing -succinyl-L-Ala hardly adsorbed demethylvancomycin. Aqueous sodium carbonate (0.4 M)/acetonitrile (7/3, v/v) completely desorbed demethylvancomycin adsorbed on the adsorbents.

Adsorption↗

PubMed2000

Respiratory syncytial virus that lacks open reading frame 2 of the M2 gene (M2-2) has altered growth characteristics and is attenuated in rodents.

The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2DeltaM2-2). Recombinant virus rA2DeltaM2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2DeltaM2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2DeltaM2-2-infected cells. By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2DeltaM2-2. rA2DeltaM2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2DeltaM2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV. rA2DeltaM2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRC5 cells but replicated efficiently in Vero and LLC-MK2 cells. Replication of rA2DeltaM2-2 in the upper and lower respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2DeltaM2-2 was able to provide protection against challenge with wild-type RSV A2. The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2DeltaM2-2 suggested that rA2DeltaM2-2 may be a potential candidate for use as a live attenuated vaccine.

Amino Acid Sequence↗

PubMed2000

Use of allelic loss to predict malignant risk for low-grade oral epithelial dysplasia.

One of the best approaches to identifying genetic changes critical to oral cancer progression is to compare progressing and nonprogressing oral premalignant lesions. However, such samples are rare, and they require long-term follow-up. The current study used the large archive network and clinical database in British Columbia to study loss of heterozygosity (LOH) in cases of early oral premalignancies, comparing those with a history of progression to carcinoma in situ or invasive cancer and those without a history of progression (referred to as nonprogressing cases). Each of 116 cases was analyzed for LOH at 19 microsatellite loci on seven chromosome arms (3p, 4q, 8p, 9p, 11q, 13q, and 17p). The progressing and nonprogressing cases showed dramatically different LOH patterns of multiple allelic losses. An essential step for progression seems to involve LOH at 3p and/or 9p because virtually all progressing cases showed such loss. However, LOH at 3p and/or 9p also occurred in nonprogressing cases. Individuals with LOH at 3p and/or 9p but at no other arms exhibit only a slight increase of 3.8-fold in relative risk for developing cancer. In contrast, individuals with additional losses (on 4q, 8p, 11q, or 17p), which appeared uncommon in nonprogressing cases, showed 33-fold increases in relative cancer risk. In conclusion, analysis of LOH at 3p and 9p could serve as an initial screening for cancer risk of early premalignancies. Follow-up investigation for additional losses would be essential for predicting cancer progression.

Chromosome Mapping↗

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↗