Biomedical subjects
Publications and source records attributed to Y Lu.
A rapid auxanographic method for biotin determination was established by means of a biotin auxotrophic yeast strain, and a computer program was designed to calculate the data obtained. Its determination concentration ranges from 0.3 to 5.0 micrograms/ml. The time-tested routine work in our laboratory proved that it is rapid, precis and reliable.
A new 3,7-dioxabicyclo[3, 3, O] octan-6-one named zhepeiresinol was isolated from Fritillaria thumbergii Miq. The spectroscopic and X-ray diffraction analysis established the structure as 2-(3',5'-dimethoxy-4'-hjydroxyphenyl)-3,7-dioxabicyclo[3, 3, O]octan-6-one.
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The promoter of the human gene encoding the p65 subunit of the transcription factor NF-kappa B was cloned and the nucleotide sequence determined. The p65 promoter lacks both TATA and CCAAT consensus sequences. The p65 promoter contains three consensus binding sites of the transcription factor SP1. In contrast to the promoter of the p50 subunit of NF-kappa B, no sequences predicted to bind NF-kappa B are present in the p65 promoter. Phorbol ester (PMA) and phytohemagglutinin (PHA) treatment of Jurkat cells did not activate the p65 promoter in transient transfection experiments. Using different deletion mutants of the p65 promoter, essential promoter elements were mapped.
We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.
AraC protein regulates the transcription of arabinose transport and catabolic operons in Escherichia coli through interaction with specific DNA sequences in the promoter regions of the operons. The interaction of AraC protein with two binding sites in the araFGH promoter was determined and compared to previously studied AraC binding sites in the araBAD promoter. Methylation and ethylation interference assays show that AraC protein binds along one side of the DNA to four adjacent major groove regions at each of the araFG1 and araFG2 sites. Mutations within any of the four regions of araFG1 greatly reduce protein binding in vitro. The promoter function in vivo is also greatly reduced, indicating that all four regions of the binding site are required. The chemical interference and genetic data, combined with the consensus sequence for AraC protein binding to ara promoters, support a binding motif in which two directly repeated units each span two adjacent turns of the DNA helix. The function of the two AraC binding sites was also examined. The proximal araFG1 site is required for promoter activation, whereas the distal araFG2 site has only a slight effect on the promoter activity.
The differences in the molecular properties of the nuclear aryl hydrocarbon (Ah) receptor from human Hep G2 and mouse Hepa 1c1c7 cells were investigated by time-dependent partial proteolysis with chymotrypsin or trypsin followed by column chromatographic and velocity sedimentation analysis. The sedimentation coefficients, Stokes radii and apparent molecular weights of the untreated human and mouse Ah receptor complexes were similar. Treatment of the nuclear Ah receptor complexes from both cell lines with chymotrypsin for 10 or 60 min gave lower molecular weight proteolytic products which also exhibited comparable molecular properties and salt gradient elution profiles from Sepharose columns linked to DNA. Treatment of the human and mouse nuclear Ah receptor complexes with trypsin (5 micrograms/mg protein) for 10 or 60 min gave a minor low molecular weight (29.7- or 25.7-kDa) proteolysis product which was detected only with the mouse Hepa 1c1c7 Ah receptor complex. The time- and concentration-dependent proteolytic digest maps of the human and mouse Ah receptor were determined using receptor preparations which were photoaffinity labeled with [125I]7-iodo-2, 3-dibromodibenzo-p-dioxin. The human Ah receptor was significantly more resistant to proteolysis by trypsin or chymotrypsin than the mouse Ah receptor. At a low concentration of chymotrypsin (1 microgram/mg protein) the Hepa 1c1c7 receptor was degraded to two lower molecular weight fragments with apparent M(r) values at 71- and 48-kDa whereas the Hep G2 Ah receptor was relatively stable under these conditions. Although the human Ah receptor was more slowly hydrolyzed than the mouse receptor by trypsin, the major photolabeled breakdown products for the Ah receptor from both cell lines were observed at M(r) 48- and 45-kDa. The results of this study demonstrate that there were subtle but significant differences in the human and mouse Ah receptor complex; however, the proteolysis studies suggest that there are common structural features in their ligand binding sites.
A high-performance liquid chromatographic method to measure the concentration of 3-methylhistidine in human urine with o-phthaldialdehyde precolumn derivatization is described. A clear separation of the 3-methylhistidine derivative from other derivatives in urine was achieved within 25 min. The linearity, recovery and coefficients of variation were determined. The method was successfully applied to clinical assays and used for diagnostic purposes.
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Physiological measurements suggest that the inhibition of primate spinothalamic tract cells by serotonin is mediated by myelinated axons. Previous morphologic studies emphasize that most serotonin-containing axons in the spinal cord are unmyelinated. Accordingly, the possibility that some serotonin-containing axons in the primate dorsolateral funiculus of the spinal cord are myelinated was investigated. Macaque monkeys were given L-tryptophan and the monoamine oxidase inhibitor, nialamide, intraperitoneally 1 h prior to sacrifice to increase axonal stores of serotonin. The animals were perfused (0.05 or 0.5% glutaraldehyde, 4% paraformaldehyde), and transverse sections of the thoracic cord were reacted with antibody against serotonin and then prepared for electron microscopy. Many of the immunostained axons in the dorsolateral funiculus included fine, myelinated fibers with diameters of 0.7-2.2 microns. Unmyelinated serotonin-containing axons were also observed. The observation of myelinated serotonin-containing axons in the white matter of the monkey dorsolateral funiculus contradicts the view that the descending serotoninergic projection consists entirely of unmyelinated fibers, particularly since the conduction velocity of the fine fibers would be too slow to account for the earliest latency of descending inhibition following stimulation in the brainstem. The presence of myelinated serotoninergic axons presumably accounts for the latencies reported for the inhibition of primate spinothalamic cells following stimulation of the periaqueductal gray, an inhibition that can be blocked with serotonin antagonists and that is associated with the release of serotonin in the dorsal horn.
CD28 is a 44-kDa homodimeric receptor expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with PMA causes the induction of lymphokine/cytokine production and proliferation in resting T cells via signal transduction pathways independent of the TCR. The precise nature of the biochemical events that occur after perturbation of the CD28 receptor remain unclear. We report evidence for the coupling of CD28 to a protein-tyrosine kinase pathway. Multivalent cross-linking of the CD28 receptor or stimulation by soluble CD28 mAb plus PMA, but not PMA or soluble CD28 mAb alone, reproducibly caused the rapid (within 2 min) tyrosine phosphorylation of a 100-kDa cellular substrate. In some experiments, additional cellular substrates of 110, 85, 74, 68, 56, 43, and 29 kDa were also observed. The tyrosine phosphorylation of these substrates was completely inhibited by 12 h pretreatment of T cells with herbimycin A, a selective inhibitor of src-family protein-tyrosine kinases. Pretreatment of T cells with herbimycin was without effect on CD28 surface expression but did inhibit CD28 mAb plus PMA-induced IL-2 mRNA levels, IL-2R(CD25) up-regulation, and cell proliferation. The inhibition of IL-2 mRNA levels was likely at the level of transcription, because herbimycin inhibited NF-AT, AP-1, and CD28RC but not NF-kappa B or OCT-1 binding activities to their respective IL-2 enhancer region sequences. Herbimycin did not inhibit PMA-dependent events including CD69 surface expression, NF-kappa B nuclear binding activity or the level of CD25 induced by PMA alone, supporting the notion that herbimycin is acting to inhibit a CD28 initiated or regulated protein-tyrosine kinase pathway(s).
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Mutations at the aecA locus of Bacillus subtilis lead to derepression of the lysC operon, which encodes aspartokinase II, and analysis of three independent aecA mutations has shown them to be nucleotide substitutions in the lysC leader region (Y. Lu, N.Y. Chen and H. Paulus (1991) J. Gen. Microbiol. 137, 1135-1141). DNA sequence analysis of the lysC control region of nine other mutants with derepressed levels of aspartokinase II revealed each of the mutations to be associated with changes in one or a few nucleotide residues. The nucleotide substitutions were clustered at two sites in the lysC leader: in a region of imperfect dyad symmetry about 40 base pairs from the transcription start site, and in the open reading frame for a putative leader peptide, which starts about 40 residues further downstream. The effect of nucleotide substitutions at the two sites differed in that those at the upstream site gave twice the degree of derepression. A mutant with a small deletion in the leader peptide coding region potentially affecting RNA secondary structure also had a higher level of lysC derepression. These results suggest that the lysC leader region contains at least two cis-acting control sites that play important and perhaps independent roles in the repression of the lysC operon by lysine.
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We report purification of an 18-kDa heparin-binding growth factor secreted from human cancer cells which is homologous to a developmentally regulated, neurotrophic factor, heparin-binding growth-associated molecule/pleiotrophin (HB-GAM/PTN; Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724; Li, Y. S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). We have purified the protein from tissue culture supernatants of human breast cancer cells (MDA-MB 231) and have used soft agar cloning of an epithelial cell line (SW-13) to detect its growth stimulating activity. A 32,000-fold purification was achieved by isoelectric focusing, heparin affinity chromatography, and reversed phase high pressure liquid chromatography. The molecular mass of the protein was confirmed by gel filtration chromatography in the presence of detergent and bioassay of the fractions. The N-terminal sequence was homologous to HB-GAM/PTN, and polymerase chain reaction amplification and DNA sequencing confirmed that the respective transcript was present in the cancer cells. We conclude that HB-GAM/PTN can function as a tumor growth factor in addition to its role during neuronal development.
Studies of cognitive function in subjects with human immunodeficiency virus type 1 (HIV-1) infection who remain relatively asymptomatic (ie, Centers for Disease Control stages II and III) have provided widely variable estimates of cognitive impairment. In view of the finding that approximately 25% of asymptomatic HIV-1-infected subjects demonstrate either marginal or overt vitamin B12 deficiency, we have investigated plasma vitamin B12 status as a potential cofactor in studies of HIV-1-related cognitive impairment. When cognition was assessed in asymptomatic (Centers for Disease Control stages II and III) HIV-1-infected participants taking into consideration vitamin B12 status, those subjects with low plasma vitamin B12 levels (less than 180 pmol/L) performed more poorly than did those with normal (greater than or equal to 180 pmol/L) vitamin B12 status on specific measures of information processing speed and visuospatial problem-solving skills. These findings suggest that concurrent vitamin B12 deficiency may be a cofactor in subtle cognitive changes observed in the asymptomatic stages of HIV-1 infection. These differences in prevalence of low plasma vitamin B12 levels may help to explain differences among studies in the proportion of HIV-1-infected subjects showing cognitive impairment.
Some of the relevant biological properties of a rhabdovirus isolated from penaeid shrimps (RPS) were examined. The virus replicated in an established fish cell line, epithelioma papulosum cyprini (EPC) which allowed for the development of a quantitative plaque assay protocol. Virus replication was not inhibited by the DNA antagonist, 5-bromo-2'-deoxyuridine (20 micrograms/ml). Virus infectivity was sensitive to 20% ethyl ether, low pH, and to 37 degrees C. The virus showed marked lability to repeated freezing and thawing and storage at -10 degrees C, but was stable at -70 degrees C for several weeks. The virus particle to infectious unit ratio in EPC was found to be 30.