Backbone resonance assignments of human UBC9.
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Amino Acid Sequence↗
Biomedical subjects
B Shen
Publications and source records attributed to B Shen.
Growth hormone as a cytokine.
1. The growth hormone (GH) receptor was the first of the class 1 cytokine receptors to be cloned. It shares a number of structural characteristics with other family members and common signalling mechanisms based on common usage of the Janus kinase 2 (JAK2). 2. Growth hormone receptor activation is initiated by GH-induced homodimerization of receptor molecules. This has enabled the creation of specific hormone antagonists that block receptor dimerization. 3. The details of the transcription factors used by the activated receptor are being revealed as a result of promoter analyses and electrophoretic mobility gelshift analysis. 4. Growth hormone receptors are widespread and their discovery in certain tissues has led to the assignment of new physiological roles for GH. Some of these involve local or paracrine roles for GH, as befits its cytokine status. 5. Four examples of such novel roles are discussed. These are: (i) the brain GH axis; (ii) GH and the vitamin B12 axis; (iii) GH in early pre-implantation development; and (iv) GH in development of the tooth. 6. We propose that the view that GH acts through the intermediacy of insulin-like growth factor-1 is simplistic; rather, GH acts to induce an array of growth factors and their receptors and the composition of this array varies with tissue type and, probably, stage of development.
Animals↗
Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals.
Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.
3T3 Cells↗
Roles of sugar alcohols in osmotic stress adaptation. Replacement of glycerol by mannitol and sorbitol in yeast.
For many organisms there is a correlation between increases of metabolites and osmotic stress tolerance, but the mechanisms that cause this protection are not clear. To understand the role of polyols, genes for bacterial mannitol-1-P dehydrogenase and apple sorbitol-6-P dehydrogenase were introduced into a Saccharomyces cerevisiae mutant deficient in glycerol synthesis. Sorbitol and mannitol provided some protection, but less than that generated by a similar concentration of glycerol generated by glycerol-3-P dehydrogenase (GPD1). Reduced protection by polyols suggested that glycerol had specific functions for which mannitol and sorbitol could not substitute, and that the absolute amount of the accumulating osmoticum might not be crucial. The retention of glycerol and mannitol/sorbitol, respectively, was a major difference. During salt stress, cells retained more of the six-carbon polyols than glycerol. We suggest that the loss of >98% of the glycerol synthesized could provide a safety valve that dissipates reducing power, while a similar high intracellular concentration of retained polyols would be less protective. To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient strain. One mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient line, probably due to a doubling of [K(+)] accumulating during stress. We compare these results to the "osmotic adjustment" concept typically applied to accumulating metabolites in plants. The accumulation of polyols may have dual functions: facilitating osmotic adjustment and supporting redox control.
Cations↗
Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons.
The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.
Chromosomes, Bacterial↗
Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease.
Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.
Base Sequence↗
[Distribution of six STR loci in Bai ethnic group in Yunnan province of China].
OBJECTIVE: This study was conducted to make known the distribution of six STR loci in Bai ethnic group in Yunnan province. METHODS: DAN extraction from blood samples (126 in number ) and multiplex amplification of CSF1PO,TPOX, TH01,F13A01, FESFPS and vWA were carried out. Using denaturing polyacrylamide gel electrophoresis and silver stain, the authors investigated the distribution of allele frequencies of CSF1PO, TPOX,TH01,F13A01,FESFPS and vWA loci in Bai ethnic group living in Yunnan province. RESULTS: 7 alleles and 19 genotypes of CSF1PO locus, 5 alleles and 11 genotypes of TPOX locus, 6 alleles and 16 genotypes of TH01 locus 6 alleles and 16 genotypes of F13A01 locus, 7 alleles and 15 genotypes of FESFPS locus,and 7 alleles and 21 genotypes of vWA locus were observed. CONCLUSION: The allele distribution of the loci was in good agreement with the Hardy-Weinberg equilibrium.
China↗
STR HUMARA locus gene and genotype frequencies in Han and Bei populations in China.
For the purpose of the population genetics study of the HUMARA locus, the allele, and genotype frequencies were determined in two Chinese population samples (Han-101, Bei-113) using PCR, PAGE, and silver staining. Fourteen alleles were found. The size of amplified fragments were 258 bp-315 bp. The observed heterozygosities were 0.83 in the Han population and 0.73 in the Bei population respectively. The expected heterozygosities were 0.91 in the Han population and 0.97 in the Bei population respectively. Both populations meet Hardy-Weinberg expectation, Han population x2 = 17.7206, df = 11, p > 0.05; Bei population x2 = 7.4268, df = 10, p > 0.05. The discrimination power were 0.95 in females and 0.89 in males in the Han population, 0.94 in females and 0.88 in males in the Bei population. Thus, the allelic frequency data can be used in the personal identification and parentage testing in the forensic science practice. The PCR test established in this study is robust and reproducible.
Alleles↗
Relationship between pathogenesis of Guillain-Barre syndrome and Penner's serotypes of Campylobacter jejuni.
OBJECTIVE: To investigate the relationship between the pathogenesis of Guillain-Barre syndrome (GBS) and Campylobacter jejuni (C. jejuni) in China. METHODS: C. jejuni strains were isolated from fresh stools of 47 GBS patients in Beijing area from 1995 to 1997 by modified Skirrow's method. Serotyping of C. jejuni was performed with Penner's method in 47 GBS patients and 171 patients with C. jejuni enteritis in our hospital during the same period. The stools from which no C. jejuni strains could be isolated were typed by PCR-RFLP (restriction fragment length polymorphism) method. RESULTS: Six C. jejuni strains were isolated by Skirrow's method, including 3 strains of Penner serotype 2, 1 Penner serotype 4, 1 Penner serotype 19 and 1 Penner serotype 26. Three strains of DNA-I, which was equal to the same type of Penner serotype 19, were found by PCR-RFLP method. CONCLUSION: C. jejuni isolated from Beijing area are similar to those reported in literature, which once more confirms the molecular mimicry pathogenetic theory of GBS caused by C. jejuni infection in China. The similarities of serotypes between C. jejuni strains from GBS patients and those from C. jejuni enteritis patients may explain the high incidence of GBS following C. jejuni infection in China.
Campylobacter Infections↗
[The relationship between Guillain-Barre syndrome and serotypes of Campylobacter jejuni isolated from the patients].
OBJECTIVE: To investigate the relationship between Campylobacter jejuni (CJ) serotype and the high incidence of Guillain-Barre syndrome (GBS) following CJ enteritis in China. METHODS: CJ was isolated from fresh stool of 47 GBS patients in Beijing area from 1995-1997 with a combination of Skirrow method and micro-filtration method. Serotyping of the CJ strains was performed with passive hemagglutination method with standard serum from CDC. 171 CJ strains from CJ enteritis in our hospital in the same period were serotyped with the same method. The stool from which no CJ could be isolated was typed with PCR-RFLP method. RESULTS: 3 Penner 2, 1 Penner 4, 1 Penner 19 and 1 Penner 26 strains were isolated and serotyped. Three strains which could not be isolated were found by PCR-RFLP method to be DNA-I, which is equivalent to the type of Penner 19. There were some CJ strains which could be typed with serum from CDC. CONCLUSION: CJ from GBS patients in Beijing area are similar to those reported in the world; it is suggested that CJ may cause GBS by molecular mimicry mechanism in China. The most common serotype of CJ from GBS was reported to be Penner 19 in Japan, while it was rare in CJ enteritis. However, the similarity of the serotypes between CJ from GBS patients and CJ from enteritis patients may explain the high incidence of GBS following CJ infection in China. Penner 26 is reported to be isolated from GBS patients for the first time.
Campylobacter jejuni↗
[Clinical observation of phenytoin in promoting fracture healing].
OBJECTIVE: To observe the promoting effect of phenytoin on fracture healing. METHODS: Fourty cases with close fractures of tibia and fibula were included and divided into two groups randomly: the experimental group was administrated with phenytoin and Chinese traditional drug-Jiegudan orally, while the control group was given Jiegudan only. Longitudinal percussion pain, clinical healing time of fracture, growth of calus in X-ray film were detected to evaluate the clinical result. RESULTS: All the fractures were healed in 3 months. But the experimental group was superior to the control group in all indexes. CONCLUSION: Administration of phenytoin orally can markedly promote fracture healing.
Adult↗
[Distribution of three STR loci in Dai ethnic group in China].
OBJECTIVE: This study was conducted to investigate the distribution of three STR loci in Dai ethnic group. METHODS: Multiplex amplifications of CSF1PO, TPOX and TH01 were carried out. Using denaturing polyacrylamide gel electrophoresis and silver staining the authors investigated the distribution of allele frequencies of CSF1PO, TPOX and TH01 loci in Dai ethnic group living in Yunnan province. RESULTS: 7 alleles and 15 genotype of CSF1PO locus, 7 alleles and 18 genotypes of TPOX locus, 6 alleles and 17 genotypes of TH01 locus were observed. CONCLUSION: The allele distribution of the loci is in good agreement with the Hardy-Weinberg equilibrium.
China↗
Partial functional deficiency of E160D flap endonuclease-1 mutant in vitro and in vivo is due to defective cleavage of DNA substrates.
To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.
Base Sequence↗
Intracellular association of FGF-2 with the ribosomal protein L6/TAXREB107.
By using the yeast two-hybrid system, we identified the ribosomal protein L6/TAXREB107 as an intracellular partner for FGF-2. L6/TAXREB107 also mediates the DNA binding of the HTLV-1 transactivator Tax. In vitro binding experiments indicated that both the high-molecular-weight forms (HMW) and the 18-kDa form of FGF-2 bind to L6/TAXREB107. Deletion analysis suggested that L6/TAXREB107 has two binding sites for HMW FGF-2 and one binding site for 18-kDa FGF-2, implying that the unique N-terminal extension of the HMW FGF-2 is one of the binding domains for L6/TAXREB107. Transfection assays showed that high expression of either HMW or 18 kDa FGF-2 stimulates Tax-mediated transactivation in NIH 3T3 cells. This result suggests a possible role of FGF-2 in Tax-mediated HTLV-1 transformation as well as FGF-2 binding to ribosomes and/or their precursors.
Animals↗
Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.
Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.
Acids↗
Molecular events after antisense inhibition of hMSH2 in a HeLa cell line.
To establish a cause-effect relationship between the human mismatch repair pathway deficiency and the observed phenotypes, a hMSH2 deficient HeLa cell line (HeLa-MSH2-) was established by transfecting the HeLa cells with an antisense RNA expression plasmid. The expression plasmid was constructed by inserting an 851 bp fragment of hMSH2 cDNA into the polyclonal site of the vector pREP9 in a reversed orientation. The production of the mismatch binding protein, hMSH2, was inhibited in HeLa-MSH2- cells, as demonstrated by Western blotting and band shift assay of its whole cell extract. The growth rate of this cell line was not different from the parental HeLa cells soon after transfection. However, the rate was faster after 10 subcultures. The spontaneous mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus increased markedly, but no N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) tolerance appeared in this cell line. Our results clearly demonstrated several molecular events happened after the inhibition of a major mismatch recognition protein, hMSH2, in the mismatch repair pathway, mimicking carcinogenesis processes.
Base Pair Mismatch↗
Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.
Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.
Amino Acid Sequence↗
Delta T 14/Delta D 15 Azotobacter vinelandii ferredoxin I: creation of a new CysXXCysXXCys motif that ligates a [4Fe-4S] cluster.
In clostridial-type ferredoxins, each of the two [4Fe-4S]2+/+ clusters receives three of its four ligands from a CysXXCysXXCys motif. Azotobacter vinelandii ferredoxin I (AvFdI) is a seven-iron ferredoxin that contains one [4Fe-4S]2+/+ cluster and one [3Fe-4S]+/0 cluster. During the evolution of the 7Fe azotobacter-type ferredoxins from the 8Fe clostridial-type ferredoxins, one of the two motifs present changed to a CysXXCysXXXXCys motif, resulting in the inability to form a 4Fe cluster and the appearance of a 3Fe cluster in that position. In a previous study, we were unsuccessful in using structure as a guide in designing a 4Fe cluster in the 3Fe cluster position of AvFdI. In this study, we have reversed part of the evolutionary process by deleting two residues between the second and third cysteines. UV/Vis, CD, and EPR spectroscopies and direct electrochemical studies of the purified protein reveal that this DeltaT14/DeltaD15 FdI variant is an 8Fe protein containing two [4Fe-4S]2+/+ clusters with reduction potentials of -466 and -612 mV versus SHE. Whole-cell EPR shows that the protein is present as an 8Fe protein in vivo. These data strongly suggest that it is the sequence motif rather than the exact sequence or the structure that is critical for the assembly of a 4Fe cluster in that region of the protein. The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster. In known ferredoxins and enzymes that contain reversibly interconvertible [4Fe-4S]2+/+ and [3Fe-4S]+/0 clusters, the 3Fe form always has a reduction potential ca. 200 mV more positive than the 4Fe cluster in the same position. In contrast, for DeltaT14/DeltaD15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.
Amino Acid Sequence↗