Isolation and structural elucidation of AC326-alpha, a new member of the moenomycin group.
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Actinomycetales↗
Biomedical subjects
B Shen
Publications and source records attributed to B Shen.
[Signal transduction and biological function of IL-6 in a human myeloma cell line-XG-7].
OBJECTIVE: To investigate the relationship between IL-6 signal transduction and its biological function on a human IL-6-dependent myeloma cell line-XG-7. METHODS: Electrophoretic mobility shift (EMSA) and immunopeipitation were used respectively to detect the activation of transcription factors (STAT3 and NF-IL-6) and protein kinases (JAK1 and ERK) in the two IL-6 signal transduction pathways by IL-6 in XG-7 cells. Then the cells were transfected with the sense or anti-sense expression plasmids of the transcription factors, which could up- or down-regulate the activation of the signal transduction pathways specially, the changes of the biological function of IL-6 on XG-7 cells was shown by cell number counting and MTT. RESULTS: IL-6 can promote the proliferation of XG-7 cells and activate one of the IL-6 signal transduction pathway-Ras/NF-IL-6; but Jak/STAT signal transduction pathway was not activated at the same conditions. When the activation of Ras/NF-IL-6 pathway was up- or down-regulated, the growth effect of IL-6 on XG-7 cells was strongthened or weakened. CONCLUSIONS: The proliferation of XG-7 cells in the presence of IL-6 is mediated by the activation of Ras/NF-IL-6 signal transduction pathway.
Cell Division↗
[Study of IL-6 signal transduction pathway in a human multiple myeloma cell line--Sko-007].
OBJECTIVE: To investigate the IL-6 signal transduction pathway in a human multiple myeloma (MM) cell line--Sko-007. METHODS: The electrophoretic mobility shift assay (EMSA) and immunoprecipitation were used to detect the activation of IL-6-related transcription factors(TFs)--STAT3, NF-IL-6, NF-kB and protein kinase ERK, Jak1 in Sko-007 cells stimulated by IL-6. RESULTS: Both the transcription factors-STAT3, NF-IL-6 and protein kinases-Jak1, ERK were activated by IL-6 in Sko-007 cells. CONCLUSION: The biological function of IL-6 on Sko-007 cells is mediated by the activation of Jak/STAT and Ras/NF-IL-6 signal transduction pathways.
Humans↗
[A preliminary study on placenta hematopoiesis].
OBJECTIVE: To study the role of placenta on fetal hematopoiesis during embryo ontogeny, so as to find a new source of hematopoietic stem/progenitor cells (HSC/HPC) for clinical transplantation. METHODS: Cord blood was collected separately from cord vein and artery. CD(34)(+) cells were detected by FACS, CFU-GM semisolid agar culture, and hematopoietic growth factors (HGFs) by ELISA. The results of HE stain and CD(34) McAb immunohistochemistry stain of placenta chorionic villi were investigated. RESULTS: The CFU-GM yields and the percentage of CD(34)(+) and CD(34)(+)/CD(38)(minus sign) cells in cord vein were higher than that in artery. The cord vein contained larger amount of hematopoietic stimulating factors and similar level of hematopoietic inhibitory factors as compared with that in artery. Blood island, reticular cells and scattered CD(34)(+) cells were found in the mesenchyme of placenta chorionic villi. CONCLUSION: The placenta might be an another hematopoietic organ in ontogeny. It could be applied to clinical hematopoietic cell transplantation.
Antigens, CD34↗
[Construction of membrane-anchored interleukin-6 receptor beta subunit mutant and its effect on IL-6 signal transduction].
OBJECTIVE: To investigate whether the membrane-anchored gp130 mutant containing the second and the third fibronectin type III modules of the extracellular region could still transduce IL-6 signal. METHOD: The membrane-anchored gp130 mutant cDNA was amplified by the overlap extension PCR and inserted into the mammalian expression vector pRc/RSV. The mutant was proved to be effectively expressed in SKO-007 cells and Jurkat cells by dot blot hybridization. The mutant's effects on IL-6 signaling in these two kinds of cells were detected by electrophoresis mobility shift assay (EMSA). RESULTS: In SKO-007 cells, the mutant could enhance the activated APRF, but the effect was not as strong as that of wide-type gp130. IL-6 could not activate nuclear factors in Jurkat cells. After the expression vectors pRc/RSVgp130 and pRc/RSV were transfected into Jurkat cells, IL-6 could activate APRF, but could not do NF-IL6. Similar to the results in SKO-007 cells, the effects of the mutant was also weaker than that of wild-type gp130. CONCLUSION: Although the mutant could still transduce IL-6 signal, it is not as efficiently as the wild-type gp130. It suggested that the Ig-like domain of gp130 also played an important role in IL-6 signal transduction.
Antigens, CD↗
[Detection of antidepressant and antipsychotic drugs in human hair and evaluation of the results].
The presence of therapeutic drugs in the hair of psychiatric patients was investigated, and the method for simultaneous determination of antidepressant and antipsychotic drugs by GC/NPD and GC/MS was also established. Eight different psychotroic drugs were detected from the hair of 32 subjects, and chlorpromazine and clozapine concentrations in the hair were found to be dependent on the dosage and the correlation coefficients were 0.8047 (P < 0.001, n = 16) and 0.7097 (P < 0.001, n = 16) respectively. Segmental analysis demonstrated that there was a correlation between the subject's drug exposure history and the distribution of drug along the hair shaft.
Adult↗
[Expression of recombinant human stem cell factor in insect cells].
The cDNA of human stem cell factor(hSCF) containing signal sequence was cloned into the transfer vector pVL941 of AcNPV to construct a recombinant transfer vector pVL941-SCF. Sf9 cells were cotransfected with wild type viral DNA and pVL914-SCF to produce the recombinant virus AcNPV-SCF by homologous recombination in cell. Southern-hybridization analysis suggested that the recombinant viral DNA contained hSCF cDNA fragment. The Sf9 cells infected with the recombinant baculovirus AcNPV-SCF expressed biologically active rhSCF which was secreted into the cell culture. The synergistic activities of SCF in conjunction with human interleukin-3(hIL-3) was measured by MTT colorimetric method and TF-1 cell line. The expression level of Sf9 cells reached its highest at about 1970 units/ml in the 3rd day after the infection with AcNPV-SCF. Three SCF bands with molecular masses of 18 x 10(3), 20 x 10(3) and 22 x 10(3) were detected by immunoblotting.
Animals↗
TeV Observations of Markarian 501 with the Milagrito Water Cerenkov Detector.
The Milagrito water Cerenkov detector near Los Alamos, New Mexico, was operated as a sky monitor at energies of a few TeV between 1997 February and 1998 May, including the period of the strong, long-lasting 1997 flare of Markarian 501. Milagrito served as a test run for the full Milagro detector. An event excess with a significance of 3.7 sigma from Markarian 501 was observed, in agreement with expectations.
Journal Article↗
Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance.
Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
Cell Division↗
Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway.
Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.
Base Sequence↗
Conformational flexibility of a ubiquitin conjugation enzyme (E2).
Ubiquitination plays important roles in a variety of biological processes, such as DNA repair, cell cycle regulation, and p53-dependent processes. Despite intensive studies in ubiquitination, the mechanism of substrate recognition is still not well understood. Each E2 has its own substrate specificity, yet substrate proteins recognized by each E2 are highly diverse. To better understand how E2 proteins confer both substrate specificity and diversity, we have studied conformational flexibility of an E2, UBC9, using nuclear magnetic resonance 15N relaxation and hydrogen-deuterium exchange measurements. Two regions in human UBC9 show higher mobility over a wide range of time scales. Combined with previous biochemical studies, both regions are likely to be important for protein-protein recognition in the ubiquitin pathway. The region near the N-terminus may be important for interactions with the E1-UBL1 conjugate. The region near the C-terminus, which undergoes conformational exchange may be important for substrate binding and catalytic activity. Since E2 enzymes share high homology in primary sequences and three-dimensional structures, the conformational flexibility of UBC9 may represent a general feature of E2 enzymes. This study provides a new perspective for further studies of protein-protein recognition in ubiquitination.
Amides↗
Structural changes measured by X-ray scattering from human flap endonuclease-1 complexed with Mg2+ and flap DNA substrate.
Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5'-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1. 34-mer DNA flap complex, and the D181A.34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+ binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of approximately 26 A, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5'-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolated Io scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1.34-mer DNA flap complex has an Rg value of approximately 34 A, whereas the D181A.34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1.34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.
DNA↗
Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.
The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.
3T3 Cells↗
N-methyl-D-aspartate receptor antagonists are less effective in blocking long-term potentiation at apical than basal dendrites in hippocampal CA1 of awake rats.
Long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) at the apical or basal dendrites of CA1 pyramidal cells was induced by stimulation with a 1-s train of 200-Hz pulses in awake rats, with or without the presence of various doses of an N-methyl-D-aspartate (NMDA) receptor antagonist. Apical LTP was blocked by an intracerebroventricular (i.c.v.) dose of 40 microg D-2-amino-5-phosphonopentanoic acid (D-AP5) or 20 mg/kg i.p. D-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP-40116), whereas basal LTP was blocked by half the dose of D-AP5 or CGP-40116. The noncompetitive antagonist MK-801 (< or =1 mg/kg i.p.) had no significant effect on apical LTP. Apical LTP was not blocked by i.c.v. nifedipine. The effect of an NMDA receptor antagonist alone on apical and basal fEPSPs was also evaluated, to assess the net effect of the NMDA receptor antagonist in blocking LTP. MK-801 (0.5-1 mg/kg i.p.) or CGP-40116 (10-20 mg/kg i.p.) but not D-AP5 suppressed apical fEPSPs for several hours and confounded the expression of apical LTP during this time. We concluded that hippocampal LTP at different synapses has different sensitivity to NMDA receptor antagonists and that a general blockade of hippocampal NMDA receptor functions cannot be inferred by a single hippocampal LTP measure.
2-Amino-5-phosphonovalerate↗
The influence of the p53 gene on the in vitro chemosensitivity of colorectal cancer cells.
PURPOSE: The p53 gene is considered one of the most important in the control of apoptosis, and its mutations have a close relationship with chemosensitivity. The aim of this work was to investigate the role of p53 in the apoptosis of colorectal cancer cells in vitro, induced by 5-fluorouracil (5-FU) and hydroxy-camptothecin (HCPT). METHODS: A total of 39 colorectal cancer samples from patients were treated in vitro with 5-FU (10 microg/ml), 5-FU (10 microg/ml) + leucovorin (5 microg/ml), HCPT (0.1 microg/ml) and HCPT (0.1 microg/ml) + Salvia mitorrhiza (6 microl), using an in situ terminal deoxynucleotidyltransferase assay to detect chemosensitivity. p53 gene mutations from tumor DNA were detected, after amplification by the polymerase chain reaction of exons 5-8, by non-radioactive single-strand conformation polymorphism. RESULTS: p53 gene mutations were observed in 43.6% (17/39) of colorectal carcinomas, when the terminal deoxynucleotidyltransferase assay was used to detect the tumor apoptotic rate. Cells with mutated p53 had lower chemosensitivity than those without (p < 0.01). CONCLUSION: Routine assessment of p53 status may be helpful in selecting patients with the wildtype p53 gene, who have a predictably better response to chemotherapy.
Adult↗
Spectral modulation of cortical connections measured by EEG coherence in humans.
OBJECTIVE: Description of coherence patterns of cortical EEG. METHODS: EEG recordings were collected from 9 subdural electrode grids implanted in 6 patients undergoing EEG monitoring for refractory epilepsy. RESULTS: Coherence decreased with increasing inter-electrode distance and exhibited considerable variability at the same inter-electrode distances. Analysis of variance demonstrated that both spatial and temporal factors contributed significantly (P < 0.01) to this variability. The spatial factor contributed the largest portion (up to 90%) of the variability, and was modulated by frequency and inter-electrode distance. CONCLUSION: The finding that the mean frequency modulation was consistent over time for the same pair of electrodes and was different between different pairs of electrodes indicated a spatial-spectral pattern of cortical synchrony. The connections (pairs of electrodes) could be accordingly predicted from their spatial-spectral pattern, which suggested that the spatial heterogeneity of neuronal synchrony was expressed not only by the degree of synchrony, but also by distinct spectral channels of synchrony. A model based on neuronal connection and activation is proposed to account for the observations.
Analysis of Variance↗
It's time to bring the best and brightest back to gastroenterology.
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Gastroenterology↗
Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003.
BACKGROUND: Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a structurally diverse group of peptides by the multiple-carrier thiotemplate mechanism. All NRPSs known to date are exclusively type I modular enzymes that consist of domains, such as adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains, for individual enzyme activities. Although several A and PCP domains have been demonstrated to function independently, aminoacylation in trans has been successful only between PCPs and their cognate A domains. RESULTS: We have identified within the bleomycin-biosynthesis gene cluster from Streptomyces verticillus ATCC15003 the blmI gene that encodes a discrete PCP protein. We overexpressed the blmI gene in Escherichia coli, purified the BlmI protein, and demonstrated that apo-BlmI can be efficiently modified into holo-BlmI either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases). Unlike the PCP domains in type I NRPSs, BlmI lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS. CONCLUSIONS: BlmI represents the first characterized type II PCP. The BlmI type II PCP, like the PCP domains of type I NRPSs, can be 4'-phospho-pantetheinylated by PCP-specific PPTases but is biochemically distinct in that it can be aminoacylated by an A domain from a completely unrelated type I NRPS. Our results provide for the first time the genetic and biochemical evidence to support the existence of a type II NRPS, which might be useful in the combinatorial manipulation of NRPS proteins to generate novel peptides.
Adenine↗