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Biomedical subjects

X Cheng

Publications and source records attributed to X Cheng.

At least 217 records · Page 12Linked to original sources
PubMed1996

Specific metal-oligonucleotide binding studied by high resolution tandem mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was used to study the binding of metal ions to two oligonucleotides, d(pGCTTGCATp) and d(TTGGCCCTCCTT). Collision-induced dissociation (CID) of the metal-oligonucleotide complex revealed that metal ions preferentially bound to the central thymine region of the sequence. The most probable binding sites were the phosphodiester backbone since the sum of the maximum number of charge addition from the metal ions and the charge state of the whole complex was found to be equal to the number of ionizable protons on the DNA backbone. Although site-specific and sequence-specific binding was observed for all three of the metal ions studied, the binding specificity of UO2(2+) ions was significantly greater than for Mg2+ and Na+. These experiments demonstrate that ESI-MS/MS can be applied to study the binding of metal ions and their complexes to oligonucleotides, providing not only information on the number of metal ions binding to the oligonucleotide, but also information related to the binding site(s) and binding specificity.

Cyclotrons↗
PubMed1996

Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts.

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or gamma-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21cipl/wafl/sdil remains inducible. The results demonstrate a growth-state and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells.

Aging↗
PubMed1996

Reversal by L- and D-enantiomers of NG-nitro-arginine of endotoxin-induced hypotension and vascular hyporesponsiveness.

We examined the effects of D-NNA (NG-nitro-D-arginine) and L-NNA (NG-nitro-L-arginine) on suppression of Escherichia coli lipopolysaccharide (LPS)-induced vascular hyporeactivity in pentobarbital-anesthetized rats. Mean arterial pressure (MAP) and pressor response to norepinephrine (NE) were reduced at 40 min (early phase) and 3.5-4 h (late phase) after i.v. injection of LPS (10 mg/kg). Pretreatment with either D-NNA (16 mg/kg) or L-NNA (8 mg/kg) abolished LPS-induced reduction in MAP and hyporesponsiveness to NE during the early phase but not the late phase of endotoxemia and increased mortality. In contrast, posttreatment with D-NNA and L-NNA at 3 h after the injection of LPS prevented further decreases of MAP and pressor response to NE during the late phase of endotoxemia. The restoration of vascular response by pretreatment with either D-NNA or L-NNA during the early phases or posttreatment with either of these two agents during the late phase of endotoxemia was abolished by i.v. infusion (10 mg/kg/min) of L-arginine (L-Arg), but not D-arginine (D-Arg), suggesting involvement of the L-Arg/ nitric oxide pathway.

Animals↗
PubMed1996

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Amino Acid Sequence↗
PubMed1996

Characterization of the murine antibody response to peptides representing the variable domains of the major outer membrane protein of Chlamydia pneumoniae.

In an attempt to gain more knowledge about the immunogenicity of the variable domains (VDs) of the major outer membrane protein (MOMP) of Chlamydia pneumoniae, peptides representing these areas were used to immunize BALB/c and C57BL/6 mice. Antisera to the peptides and to peptides conjugated to keyhole limpet hemocyanin (KLH) were characterized by their ability to recognize the immunizing peptide and elementary bodies (EBs) of C. pneumoniae by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot). In addition, antiserum was analyzed for its molecular specificity by a pepscan as well as its in vitro neutralizing ability. In general, results obtained with antisera to the peptides paralleled the results obtained with the antisera to the KLH-conjugated peptides except that the titers or strength of reaction in the assays was less. Antisera to the VDs in both strains of mice gave ELISA titers to the homologous VD peptide ranging from 1,000 to >64,000. The strength of reactivity with the reduced MOMP as judged by Western blot, in most cases, paralleled the ELISA titer to the peptide. However, only antisera raised in both strains of mice to the VD1 and VD4 peptides reacted strongly with the EBs, suggesting surface exposure of these VDs. In addition, antisera to VD3 from C57BL/6 mice gave strong reactivity to EBs. By pepscan analysis antisera from both strains of mice reacted with several VD1 and VD3 octameric peptides, with weaker reactivity being seen with the octameric peptides in the other two VDs. This was in contrast to antisera raised to EBs of C. pneumoniae TW-183, which identified two immunogenic regions, one in VD1 and the other mapped to VD4. While antisera raised to EBs strongly neutralized the infectivity of C. pneumoniae, none of the peptide antisera was able to neutralize. In addition, peptides to the VDs were not able to block the neutralizing ability of the antisera to EBs of C. pneumoniae. Therefore, these results suggest that the VDs of the MOMP of C. pneumoniae are surface exposed but do not elicit neutralizing antibodies when linear peptides representing them are used as the immunogen.

Animals↗
PubMed1996

[Simultaneous rhinoplasty and blepharoplasty].

Simultaneous rhinoplasty and blepharoplasty are the commonest facial cosmetic operation. The paper presents 73 cases who underwent this procedure with satisfactory results from Jan. 1989 to Nov. 1993. The authors described operation steps, e.g., making lacuna in the dorsum, lengthening nasal columella, nasal tip plasty. It was emphasized that the pretarsal flap should contain complete network of the superficial fascia, and the soft tissue underneath be trimmed. All the dissection should be kept in the same cleavage, which would reduce intraoperative bleeding and postoperative edema.

Adolescent↗
PubMed1996

[An experimental study of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in aqueous humor after intraocular lens implantation].

OBJECTIVE: The tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels in aqueous humor after intraocular lens (IOL) implantation were studied in rabbit eyes and their effects on post-operative anterior ocular inflammation were discussed. METHODS: 27 adult pigmented rabbits were divided into three groups: (1) Lens extraction and intraocular lens (IOL) implantation in the capsular bag. (2) Simple extracapsular lens extraction, and (3) normal control group without surgical intervention. Aqueous humor samples were aspirated on days 1, 3, 7 and 14 postoperatively. A modified double-antibody indirect sandwich ELISA was used to detect for the presence of TNF. MTT colorimetry was used to detect for the presence of IL-1. The data were analysed statistically. RESULTS: TNF and IL-1 levels in the aqueous humor were significantly higher (P < 0.05) at 1, 3, 7, and 14 days postoperatively in IOL implanted group than that in the control group, and TNF levels in aqueous humor at 3, 7 and 14 days postoperatively and IL-1 levels at 1 and 3 days postoperatively were significantly higher (P < 0.05) in IOL implanted group than that in simple extracapsular lens extraction group (P < 0.05). In the postoperative 7-14 days, the TNF and in the 3 to 14 days the IL-1 in the aqueous humor reached their maximum levels in IOL implantation group. CONCLUSIONS: It is reasonable to consider that the increases of TNF and IL-1 levels may play critical roles in the anterior ocular inflammation response after IOL implantation.

Animals↗
PubMed1995

Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes.

Previous X-ray crystallographic studies have revealed that the catalytic domain of a DNA methyltransferase (Mtase) generating C5-methylcytosine bears a striking structural similarity to that of a Mtase generating N6-methyladenine. Guided by this common structure, we performed a multiple sequence alignment of 42 amino-Mtases (N6-adenine and N4-cytosine). This comparison revealed nine conserved motifs, corresponding to the motifs I to VIII and X previously defined in C5-cytosine Mtases. The amino and C5-cytosine Mtases thus appear to be more closely related than has been appreciated. The amino Mtases could be divided into three groups, based on the sequential order of motifs, and this variation in order may explain why only two motifs were previously recognized in the amino Mtases. The Mtases grouped in this way show several other group-specific properties, including differences in amino acid sequence, molecular mass and DNA sequence specificity. Surprisingly, the N4-cytosine and N6-adenine Mtases do not form separate groups. These results have implications for the catalytic mechanisms, evolution and diversification of this family of enzymes. Furthermore, a comparative analysis of the S-adenosyl-L-methionine and adenine/cytosine binding pockets suggests that, structurally and functionally, they are remarkably similar to one another.

Amino Acid Sequence↗
PubMed1995

Probing the mechanism of CRP activation by site-directed mutagenesis: the role of serine 128 in the allosteric pathway of cAMP receptor protein activation.

Upon activation by cAMP, Escherichia coli cAMP receptor protein (CRP) controls the expression of a network of catabolite sensitive genes. The activation of CRP by cAMP involves conformational changes such as realignments between subunits and domains within the protein. To understand the molecular events that lead to the activation of CRP, point mutations at position 128 were introduced via site-directed mutagenesis in an attempt to specifically affect the subunit interfacial interactions, as well as the ligand-binding reaction. The biochemical and biophysical properties of these mutants were rigorously tested with the goal of identifying the partial reactions in the activation pathway that are perturbed by this specific mutation. Results from this study suggest that mutation of Ser 128 to Ala or Pro does not significantly disturb the overall secondary structure as monitored by circular dichroism. The energetics of subunit-subunit interaction and protein stability were monitored by sedimentation and spectroscopic techniques. Although these mutants were designed to interrupt intersubunit interactions, the energetics of subunit association and protein stability remain quantitatively the same as those of the wild-type CRP. Nevertheless, the ability of the subunit to be realigned to the DNA-binding form is significantly affected as reflected by the pronounced decrease in the susceptibility of mutant CRP to proteolytic digestion in the presence of cAMP. In addition, the binding affinity of cAMP to the first ligand site in mutants S128A and S128P is the same as that of the wild type, but the affinity to the second ligand site is reduced. This observation indicates that mutation at position 128 affects ligand binding by amplifying the magnitude of negative cooperativity. Mutation at residue 128 does not impair the ability of interdomain interactions as indicated by the quantitative response of a spectroscopic probe in the DNA-binding domain to the binding of cAMP to the ligand-binding domain. The S128A mutant binds to a specific DNA sequence about 50-fold weaker than the wild-type CRP, while the mutant S128P has no measurable DNA affinity under the same conditions. This observation is consistent with the in vivo result that both mutants display an inactive CRP phenotype (CRP-). In summary, these results suggest that communication between domains induced by cAMP binding can be dissociated from the proper subunit realignment of the CRP dimer that is crucial for the activation of CRP.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1995

Characterization of cytochrome c variants with high-resolution FTICR mass spectrometry: correlation of fragmentation and structure.

The dissociation of cytochrome c ions (15+ charge state) generated by electrospray ionization has been studied by Fourier transform ion cyclotron resonance mass spectrometry (FTICR) using a sustained off-resonance irradiation/collision-induced dissociation (SORI-CID) technique. Over 95% of the fragment ions can be accurately assigned (to better than 10 ppm), yielding information on the primary sequences of the various proteins. Up to four stages of mass spectrometry (MS4) have been achieved without the need for quadrupole excitation/collisional cooling of the product ions. The subtle structural differences among the cytochrome c variants (from bovine, tuna, rabbit, and horse) are clearly reflected in their fragmentation patterns: replacing 3 out of 104 residues of the cytochrome c is shown to dramatically change the dissociation pattern. Of particular importance are a variety of results indicating that the dissociation of the cytochrome c's is influenced by higher-order structure and charge location, in addition to the primary structure (i.e., sequence). No fragmentation is observed in the region between residues 10-20 and little dissociation between residues 70-90. This is most likely due to the interactions of the heme group with the polypeptide chain, and such a heme "footprinting" pattern is analogous to the protein conformation in solution. These studies demonstrate that electrospray ionization-FTICR using SORI-CID can be a useful tool to probe not only the small differences in the primary sequences of proteins but also suggest the potential for probing their higher-order structures and yielding information not readily available from H/D exchange or circular dichoism studies.

Amino Acid Sequence↗
PubMed1995

Metabolism of 1,3-butadiene to butadiene monoxide in mouse and human bone marrow cells.

1,3-Butadiene (BD), a gas used in the production of rubber and plastics, induces a high incidence of leukemias and lymphomas in B6C3F1 mice. Because of the potential involvement of the hematopoietic system in response to BD, we have examined metabolism of BD by B6C3F1 mouse and human bone marrow and by purified human myeloperoxidase (MPO), an enzyme rich in bone marrow. BD was metabolized to butadiene monoxide (BMO) by MPO and by mouse and human bone marrow cells. In all of these systems metabolism was stimulated by hydrogen peroxide suggesting a peroxidase-mediated process. In B6C3F1 mouse bone marrow cell lysates, hydrogen peroxide but not NADPH stimulated metabolism suggesting that cytochrome P450 was not involved in BMO formation. Metabolism of BD to BMO in hydrogen peroxide-fortified mouse bone marrow cell lysates was more than two orders of magnitude lower than in either NADPH-fortified rat or mouse hepatic microsomes. Experiments using both mouse and human bone marrow cells showed that cells from both sources could generate BMO from BD. These data show that BD can be converted to BMO in a target organ of BD carcinogenicity.

Animals↗
PubMed1995

Identification and characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC and presence in related mycoplasmas.

IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp. mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP). IS1296 is 1485-bp long and has 30-bp inverted repeats. It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli. IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2. It seems to transpose at low frequency in Mmm SC. IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp. 'bovine group 7' reference strain PG50. It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp. of ruminants.

Amino Acid Sequence↗
PubMed1995

Structure-based sequence alignment of three AdoMet-dependent DNA methyltransferases.

M.HhaI, M.TaqI and COMT are DNA methyltransferases (MTases) which catalyze the transfer of a methyl group from the cofactor AdoMet to C5 of cytosine, to N6 of adenine and to a hydroxyl group of catechol, respectively. The larger catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have an alpha/beta structure containing a mixed central beta-sheet. These domains have very similar folding. By allowing appropriate 'insertions' or 'deletions' in the backbones of the three structures, it was possible to find more conserved motifs in M.TaqI and COMT. The similarity in protein folding and the equivalence of amino-acid sequences revealed by the structural alignment indicate that many AdoMet-dependent MTases may share a common catalytic domain structure.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the PvuII restriction endonuclease.

Crystal structures have now been determined for the R.PvuII restriction endonuclease as a protein-DNA complex [Cheng et al., EMBO J. 13 (1994) 3927-3935; this report] and in apo-form [Athanasiadis et al., Nature Struc. Biol. 1 (1994) 469-475; our unpublished result]. The structures indicate how the interaction with DNA might proceed [Riddihough, Nature 370 (1994) 78].

Binding Sites↗