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Biomedical subjects

B Shen

Publications and source records attributed to B Shen.

At least 127 records · Page 7Linked to original sources
PubMed1998

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.

Amino Acid Sequence↗
PubMed1998

[Distribution of three STR loci in Jingpo ethnic group in Yunnan province].

OBJECTIVE: This study was aimed at the use of old blood stains for investigating the distribution of three STR loci in Jingpo ethnic group. METHODS: DNA extraction from old blood stains (106 in number) and multiplex amplification of CSF1PO, TPOX and TH 01 were carried out. Using denaturing polyacrylamide gel electrophoresis and silver stain, the authors investigated the distribution of allele frequencies of CSF1PO, TPOX and TH01 loci in a Jingpo ethnic group in the southwestern part of Yunnan province. RESULTS: 7 alleles and 26 genotypes of CSF1PO locus,7 alleles and 19 genotypes of TPOX locus, and 6 alleles and 18 genotypes of TH01 locus were observed. CONCLUSION: The satisfactory results demonstrate that multiplex amplification of CSF1PO, TPOX and TH01 is sensitive and the old stain of a drop of blood is sufficient for such amplification.

Asian People↗
PubMed1998

Phosphorylation modulates direct interactions between the Toll receptor, Pelle kinase and Tube.

Determination of dorsal/ventral polarity in Drosophila requires 12 genetically defined, maternally encoded proteins. These include Toll, a transmembrane receptor, Pelle, a ser/thr protein kinase and Tube, all of which function intracytoplasmically to initiate the cascade that ultimately activates Dorsal, an NF-kappaB family transcription factor. Here we describe biochemical interactions between recombinant Toll, Pelle and Tube that provide insights into early events in activation of the signaling cascade. We first show that Pelle binds directly to a region within the Toll intracytoplasmic domain, providing the first evidence that these two evolutionarily conserved molecules physically interact. We then demonstrate that Pelle can be autophosphorylated, and that this prevents binding to Toll as well as Tube. Autophosphorylation occurs in the N-terminal, death-domain-containing region of Pelle, which is dispensable for binding to Toll but required for enzymatic activity. We also show that Pelle phosphorylates Toll, within the region required for Pelle interaction, but this phosphorylation can be blocked by a previously characterized inhibitory domain at the Toll C terminus. These and other results allow us to propose a model by which multiple phosphorylation-regulated interactions between these three proteins lead to activation of the Dorsal signaling pathway.

Animals↗
PubMed1998

[Cloning and expression of leptospiral protective antigen gene OmpL1 in BCG].

This study was intended to produce a new living vaccine against leptospirosis using BCG as vector. Leptospiral outer envelop antigen gene OmpL1 was amplified from the genome of pathogenic leptopira serova Lai 017 by PCR, and cloned in E. coli-BCG shuttle plasmid pY6002. Recombinant plasmids were isolated by dot blotting with Digoxigeninlabeled OmpL1 gene. After transforming the recombinant plasmids in BCG (Shanghai strain) by electroporation, the genomic DNA of all 21 transformants were prepared and hybridized with OmpL1. It showed that 6 of the 21 transformants were recombinants in which the OmpL1 gene had been integrated into the genome of BCG. By immunoblotting with OmpL1 infected rabbit antiserum, which was preabsorbed to remove antibody against E. coli and SPA-HRP, three recombinants, pLI1, pLI2 and pLI3, were detected to express OmpL1 protein. The ability of expression is in the order of pLI2 > pLI1 >> plI3. These studies provide the possibility of further research on the development of highly efficient recombinant vaccines against leptospirosis.

Cloning, Molecular↗
PubMed1998

[Analysis of MDMA and its metabolites in urine by GC and GC/MS].

The purpose of this study is to investigate the metabolism of MDMA in the person and establish the methods for the determination of MDMA and its metabolites in urine. MDMA and its metabolite were isolated from urine by liquid-liquid extraction after acidic or enzymatic hydrolysis and were determined by GC/MS(EI, PCI) and GC/FID. The results showed that MDMA was metabolized to MDA and HMMA in man. MDA in the urine after MDMA administration was approximately 0.10-0.14 that of the parent drug. The sensitivity limits of methods were 2-50 ng/ml, and the recoveries were greater than 85%(CV < 10%). It is concluded that the methods were simple, quick and accurate for determining urine concentration of MDMA addicts. MDA/MDMA Ratio can be used to indicate whether the parent drug is taken alone or in combination with its active metabolite.

3,4-Methylenedioxyamphetamine↗
PubMed1998

[Decomposition kinetics of methamidophos in preserved blood of rabbit].

Decomposition kinetics of methmidophos in preserved rabbit blood poisoned by methamidophos was studied. The blood was preserved in a room (temperature 12-15 degrees C) and in a refrigerator (temperature 2-5 degrees C). The concentration of methamidophos in preserved blood was determined by GC/FPD. The results showed that the decomposition of methamidophos in preserved blood of rabbits was fast for the first three days and got slow with the time prolonged. The decomposition speed of methamidophos in room was higher than in refrigerator.

Animals↗
PubMed1998

[Abuse of "pheticol" and its interference to the analysis of methamphetamine].

The study was to eliminate interference from ephedrine in the analysis of methamphetamine. The extraction procedure for methamphetamine was modified to include an oxidation step (2 ml urine specimen was treated with 0.5 ml of 1 mol/L phosphate buffer (pH 6.8) and 0.5 ml 0.3 mol/L sodium periodate). Results showed that ephedrine could be oxidized in the presence of periodate ions into smaller fragments while leaving methamphetamine intact. It is recommended that specimens be treated with sodium periodate prior to extraction in order to eliminate any interference caused by ephedrine.

Central Nervous System Stimulants↗
PubMed1998

[Study of the circular dichroism for tribasic complex of amine derivative of beta-cyclodextrindien-Cu2+ -benzoic acid].

The circular dichroism spectra of beta-cyclodextrindien, beta-cyclodextrindien-Cu2+ complex, beta-cyclodextrindien-benzoic acid complex, tribasic complex of beta-cyclodextrindien-Cu2+ -benzoic acid were determined by using JASCO J-20C automatic recording spectropolarimeter. The attribution of the bands obtained were made according to the order of orbit energy of Cu2+ complex and benzoic acid, and the coordination structure of tribasic complex of amine derivative of beta-cyclodextrindien-Cu2+ -benzoic acid were decided acording to the structure character of Cu2+ complex and KAJART sector rule of inclusion complex of beta-cyclodextrindien with aromatic compounds.

Amines↗
PubMed1998

[Distribution of CSF1PO, TPOX and TH01 loci in Han Chinese].

The application of short tandem repeat (STR) polymorphisms has stimulated progress in the elaboration of linkage maps, forensic identification and paternity determination, characterization of disease genes, and simplification and precision of DNA typing. In this report. We have carried out DNA extraction from blood multiplex amplification of CSF1PO, TPOX and TH01. By using denaturing polyacrylamide gel electrophoresis and silver stain, we investigated the allele frequencies of CSF1PO, TPOX and TH01 loci in Han group living in Yunnan provice of China. In this study, 9 alleles and 22 genotypes of CSF1PO locus were observed; In the locus TPOX, 6 alleles and 14 genotypes were observed. In the TH01 locus, 6 alleles and 19 genotypes were observed.

Alleles↗
PubMed1997

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.

Amino Acid Sequence↗
PubMed1997

Integration of local pattern elements into a global shape in human vision.

The spatial extent of the cortical filters selective for different spatial frequencies and orientations is limited. We studied psychophysically how information from the local filters is integrated into global pattern shapes, i.e., whether performance in the identification of a global pattern consisting of small, locally oriented Gabor elements depends on the orientations of those elements. The observer was presented with an E-like stimulus pattern shape comprised of oriented Gabor patches on a blank background, and the performance measure was the threshold contrast for identifying the orientation of the E pattern (four possible rotated orientations). The results showed that contrast thresholds were significantly lower when the local elements all shared the same orientation (e.g., all horizontal) compared with the condition in which the elements had mixed orientations (both horizontal and vertical). The enhancement effect due to uniform local orientations can be explained by two factors: One is local facilitatory interactions between the orientation selective filters, and the other is second-order information integration across the filters.

Humans↗
PubMed1997

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Amino Acid Sequence↗
PubMed1997

A structural change occurs upon binding of syntaxin to SNAP-25.

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

Amino Acid Sequence↗
PubMed1997

Dietary manipulation of plasma carotenoid concentrations of squirrel monkeys (Saimiri sciureus).

Primate retinas accumulate the dihydroxy xanthophylls, lutein and zeaxanthin, from the diet via the plasma. Control of plasma concentrations of these carotenoids may be useful for prevention of retinal disease by manipulating carotenoid content of the retina. We have measured the plasma response of male squirrel monkeys to changes in the carotenoid content of a nonpurified diet. We have also supplemented the diet with zeaxanthin and beta-carotene. Plasma responses to dietary changes were rapid. Within one week, most of the change in plasma concentrations had already occurred. Within two weeks of increasing zeaxanthin intake, plasma zeaxanthin concentrations were at a new, relatively stable level. beta-carotene concentrations in the plasma were low while the monkeys were consuming a standard laboratory diet, and were only slightly increased by supplementation. Plasma lutein concentrations were unaffected by zeaxanthin supplementation. Our results suggest that it should be possible to manipulate plasma concentrations of each of the retinal carotenoids with little impact on the plasma concentrations of the other. This will facilitate exploration of the rates of accumulation of lutein and zeaxanthin in the retina, as well as exploration of the possibility of bioconversion from one xanthophyll to another.

Animals↗
PubMed1997

Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts.

To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide. Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 mumol/g fresh weight. Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.

Carbon Dioxide↗
PubMed1997

Protection by alpha G-rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate.

The protective effect of alpha G-Rutin against ferric nitrilotriacetate (Fe-NTA)-induced renal damage was studied in male ICR mice. Fe-NTA induces renal lipid peroxidation, leading to a high incidence of renal cell carcinoma in rodents. Administration of alpha G-Rutin (50 mumol as rutin/kg) by gastric intubation 30 min after i.p. injection of Fe-NTA (7 mg Fe/kg) most effectively suppressed renal lipid peroxidation. Repeated i.p. injection of Fe-NTA (2 mg Fe/kg/day for the first 3 days and 3 mg Fe /kg/day for 12 days, 5 days a week) causes subacute nephrotoxicity as revealed by induction of karyomegalic cells in renal proximal tubules. A protective effect was observed in mice given alpha G-Rutin 30 min after each Fe-NTA treatment. To elucidate the mechanism of protection by alpha G-Rutin, the pharmacokinetics and hydroxyl radical-scavenging effect of alpha G-Rutin were investigated by HPLC analysis and by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), respectively. When mice were given alpha G-Rutin (50 mumol as rutin/kg) by gastric intubation, rapid absorption into the circulation was observed. The plasma concentration of alpha G-Rutin reached the highest level 30 min after oral administration and then decreased to the control level within 60 min, alpha G-Rutin inhibited the formation of DMPO-OH in a concentration-dependent manner. Further, chelating activity of alpha G-Rutin to ferric ions was shown by spectrophotometric analysis. These results suggest that absorbed alpha G-Rutin works as an antioxidant in vivo either by scavenging reactive oxygen species or by chelating ferric ions and this serves to prevent oxidative renal damage in mice treated with Fe-NTA.

Animals↗