PubMed Health⌕ Search

Biomedical subjects

K C Cheng

Publications and source records attributed to K C Cheng.

At least 73 records · Page 4Linked to original sources
PubMed1988

N-terminal amino acid sequences of the hemopexins from chicken, rat and rabbit.

The N-terminal amino acid sequences of the hemopexins purified from the plasma of rat, rabbit and chicken were compared with each other and with that of human hemopexin. Although the N-terminal sequences differ among these species, residues 2, 3 and 14 are identical in all four hemopexins. Ten of the first 28 residues are identical in all but the chicken protein. When introducing gaps into the sequence, a much greater homology is observed between the human and rat or rabbit hemopexins (60%) than when the sequences were compared directly (40%).

Amino Acid Sequence↗
PubMed1988

Peptide-specific antibodies employed in determining the interspecies immunological cross-reactivity of haemopexin.

1. Antibodies were raised in rabbits against nine peptides analogous to sequences of the human serum beta-glycoprotein haemopexin, and seven peptides were very antigenic. 2. One of these affinity-purified peptide-specific antibodies interacted with a highly conserved sequence of the haemopexin of five of the seven species tested. 3. Another antibody bound pig haemopexin even better than human haemopexin. 4. The overall, arbitrarily assessed, immunological cross-reactivity between the haemopexin of human and other species follows the order: rabbit greater than mouse greater than chicken greater than pig greater than rat greater than cow.

Animals↗
PubMed1987

Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli.

Chi, 5' G-C-T-G-G-T-G-G 3', stimulates coliphage lambda recombination mediated by the major recombination pathway of Escherichia coli, the RecBC pathway. Purified RecBCD enzyme makes a single strand endonuclease cleavage four to six nucleotides to the 3' side of the chi sequence. Three sequences similar to chi, 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', have partial recombinational hotspot activity in genetic crosses. We report here that purified RecBCD enzyme preferentially cuts four nucleotides to the right of the two most active chi-like octamers. The degree of cutting correlated with the genetic activities of these sequences; this result indicates that these cleavages are essential to the genetic activity of chi and chi-like sequences.

Bacteriophage lambda↗
PubMed1987

Selective synthesis and secretion of particles composed of the hepatitis B virus middle surface protein directed by a recombinant vaccinia virus: induction of antibodies to pre-S and S epitopes.

Selective synthesis in mammalian cells of the hepatitis B virus middle surface (MS) protein, which is 55 amino acids longer than the major surface (S) protein, was achieved by using a recombinant vaccinia virus. The 33-kilodalton MS polypeptide was glycosylated and secreted as particles that resembled human hepatitis B surface antigen as well as particles composed solely of S protein with regard to antigenicity, buoyant density, size, and electron micrographic appearance. The MS particles differed from S particles, however, by binding to polymerized human albumin and inducing antibodies that reacted with a pre-S peptide and inhibited the binding of human plasma-derived hepatitis B surface antigen to polymerized human albumin.

Animals↗
PubMed1986

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.

Amino Acid Sequence↗
PubMed1986

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450.

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.

Amino Acid Sequence↗
PubMed1986

Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine.

Animals↗
PubMed1984

Recombinational hotspot activity of Chi-like sequences.

Chi sites, consisting of the nucleotide octamer 5' G-C-T-G-G-T-G-G 3', stimulate coliphage lambda recombination mediated by the Escherichia coli RecBC recombination pathway. In a sensitive genetic assay using phage lambda crosses, three of four Chi-like sequences tested, namely 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', had about 6%, 11% and 38% of full Chi activity, respectively. We conclude that certain Chi-like sequences manifest a spectrum of recombinational hotspot activities and may account for RecBC-mediated generalized recombination of lambda lacking Chi sites.

Alleles↗
PubMed1984

Detection and purification of cytochromes P-450 in animal tissues with monoclonal antibodies.

Monoclonal antibody (MAb)-based radioimmunoassay (RIA) and immunopurification procedures were used to probe the immunochemical relatedness of cytochromes P-450 in tissues from different species. RIAs based on MAb 1-7-1 and MAb 1-31-2, both to the major 3-methylcholanthrene (MC)-induced rat liver microsomal cytochrome P-450, detected antigenically related forms of cytochrome P-450 in liver microsomes from MC-induced rats, C57BL/6 and DBA/2 mice, hamsters, and guinea pigs, and in lung and kidney microsomes of MC-induced rats. Individual cytochromes P-450 were also isolated from these microsomes by MAb-directed immunopurification. When bound to Sepharose, MAb 1-7-1 adsorbed two species of Mr 56,000 and 57,000 from liver microsomes from rats and C57BL/6 mice, while MAb 1-31-2 adsorbed only the Mr 57,000 polypeptide. These results reveal that livers from both rats and C57BL/6 mice contain a cytochrome P-450 (Mr 56,000) with the epitope for 1-7-1 and a cytochrome P-450 (Mr 57,000) with epitopes for both 1-31-2 and 1-7-1. Additional immunochemical relatedness between the cytochromes P-450 in different tissues was demonstrated by MAb-directed immunopurification of cytochromes P-450 from DBA/2 mouse liver (Mr 56,000), guinea pig liver (Mr 53,000), hamster liver (Mr 57,000), and rat lung (Mr 57,000). These results demonstrate the efficacy of MAb-based RIA and immunopurification procedures for simple and rapid detection and purification of cytochromes P-450 from a variety of tissues.

Animals↗
PubMed1984

Amino-terminal sequence analysis of six cytochrome P-450 isozymes purified by monoclonal antibody directed immunopurification.

Six hepatic microsomal cytochromes P-450 were isolated from 3-methylcholanthrene induced animals by immunopurification using two monoclonal antibodies. Two forms of cytochromes P-450 (MW 56K and 57K) were from Sprague-Dawley rats, two from C57BL/6 mice (56K and 57K), one form from DBA/2 mice (56K) and one form from guinea pigs (53K). NH2-terminal sequences of the first ten amino acids of these cytochromes P-450 were determined by automated Edman degradation. The 56K polypeptides from rats, C57BL/6 mice, and DBA/2 mice were shown to have identical NH2-terminal sequences. The 57K polypeptides from rats and C57BL/6 mice are homologous to each other but exhibit no homology to 56K polypeptides. The 53K polypeptide from guinea pigs has a unique NH2-terminal sequence with no apparent homology to the other five cytochromes P-450.

Amino Acid Sequence↗
PubMed1983

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification.

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.

Animals↗
PubMed1982

Purification and characterization of two constitutive forms of rat liver microsomal cytochrome P-450.

Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum molecular weights of 50,000 and 51,000, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute oxidized spectra indicate that RLM3 is essentially all in the low spin state, whereas RLM5 contains some high spin component. The CO-reduced difference spectral maximum of RLM3 is at 449 nm and RLM5 is at 451 nm. RLM3 preferentially hydroxylates testosterone at 6 beta and 7 alpha positions and forms an even higher amount of an as yet unidentified polar metabolite, whereas RLM5 hydroxylates testosterone preferentially at 2 beta and 16 alpha positions, and exhibits 6 beta-hydroxylase activity similar to RLM3. RLM5 metabolizes aminopyrine, ethylmorphine, and benzphetamine all faster than does RLM3. When RLM3 and RLM5 were digested with proteolytic enzymes, peptide maps showed that these two cytochrome P-450 isozymes have different primary structures. Cyanogen bromide digestion breaks both isozymes into fragments too small to compare on polyacrylamide gels. Amino acid analyses indicate that RLM3 and RLM5 have very similar but not identical compositions. NH2-terminal residues were determined by Edman degradation and high pressure liquid chromatography separation of phenylthiohydantoin-derivatives. The first four residues of both enzymes are identical, NH2-Met-Asp-Pro-Val, but the fifth residue of RLM5 is -Leu-while that of RLM3 is -Val-. These sequences differ dramatically from all other P-450 isozymes reported to date.

Amino Acids↗
PubMed1981

Postsynaptic inhibition of neuromuscular transmission by trifluoperazine.

The effect of trifluoperazine (TFP) on neuromuscular transmission was investigated on chick biventer cervicis and frog cutaneous pectoris and sartorius nerve-muscles. In the chick, TFP inhibited indirectly elicited twitches in a frequency-dependent manner. Inhibition was much more rapid at higher frequencies of stimulation. Directly elicited twitches, KCl contracture and action potentials of desheathed frog sciatic nerve and sartorius muscles were unaffected by TFP, suggesting an action of TFP on neuromuscular transmission. TFP depressed end plate potential amplitude and miniature end plate potential (MEPP) amplitude without affecting MEPP frequency. When MEPP frequency was increased by high Na+ Ringer, depression of MEPP amplitude was much more rapid. Similarly, at high frequencies of stimulation (100 Hz), TFP rapidly depressed end plate currents. TFP inhibited contractures induced by bath-applied acetylcholine (ACh); depressed ACh potentials produced by iontophoretically applied ACh; decreased ionic current and time constant of decay of end-plate currents of transected muscle; and inhibited [alpha-125I]bungarotoxin binding to ACh receptor. These data suggest that TFP acts postsynaptically in a frequency-dependent manner to inhibit neuromuscular transmission. Based on recent evidence that TFP is a potent antagonist of calmodulin and that calmodulin is localized mainly to postsynaptic regions, we postulate that the postsynaptic inhibitory actions of TFP may be mediated through antagonism of calmodulin, which in turn may regulate ACh receptor function.

Acetylcholine↗
PubMed1981

Dietary fat and 3-MC induction of hepatic nuclear and microsomal cytochrome P-450.

Hepatic microsomes from male Holtzman albino rats fed a synthetic fat-free diet for 21 days had significantly less cytochrome P-450 and exhibited less binding capacity (delta Amax/mg protein) for aniline and octylamine than microsomes from similar rats fed a diet containing 10% corn oil. Treatment with 3-methylcholanthrene (3-MC) increased the concentrations of cytochrome P-450 (as measured by CO binding spectra) to nearly equal levels in both dietary groups, but the binding of aniline and octylamine to microsomes of rats fed the fat diet exceeded the increase in cytochrome P-450 concentration. Nuclear envelope concentrations of cytochrome P-450 were unaffected by diet. The administration of 3-MC to rats fed a fat-free diet failed to induce nuclear envelope P-450; however, in rats fed the corn oil diet, 3-MC increased this CO binding pigment over twofold. The affinity of nuclear envelope P-450 towards type II substrates was at least equal to that of microsomes, except in control rats fed the fat-free diet. In general, 3-MC pretreatment increased the binding affinity of nuclear envelop and microsomes toward aniline, while increasing affinity for SKF 525-A binding only to nuclear envelope. Molecular weight species in the region known to contain the cytochrome P-450 were quantified by fluorescence gel electrophoresis. Molecular weight species of 48,000 and 53,000 in the nuclear envelope had their counterparts in the microsomal preparation, but a 50,000 dalton component of nuclear envelope was not detected in microsomes. 3-Methylcholanthrene increased only a species with molecular weight 45,500 in the microsomal and nuclear envelope preparations. Rats fed the diet containing corn oil had microsomes with increased capacity for binding CO, but this was not accompanied by increased cytochrome P-450 protein concentration, as measured by quantitative fluorescence gel electrophoresis.

Animals↗