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Biomedical subjects

B Shen

Publications and source records attributed to B Shen.

At least 73 records · Page 4Linked to original sources
PubMed2001

[Cloning and identification of deltamethrin-resistance or susceptibility associated genes of Culex pipiens pallens].

OBJECTIVE: To obtain deltamethrin-resistance or susceptibility associated genes of Culex pipiens pallens. METHODS: The differentially expressed genes were obtained by suppression subtractive hybridization (SSH), and identified by cDNA microarray and reverse Northern blotting. RESULTS: 523 and 286 clones were selected respectively in the two directional SSH. 155 and 42 genes were respectively expressed 2-3 and > 3 times higher in the insecticide-resistant strain than in the susceptible strain; 15 and 9 genes were respectively expressed 2-3 and > 3 times higher in the susceptible strain than in the resistant strain. There were 2 genes only expressed in the insecticide-resistant strain. 51 three times differentially expressed clones and 2 specially expressed clones were sequenced. 44 sequences were obtained which belong to 13 new genes. There were 8 over-expressed genes in resistant strain, 7 of which were similar respectively to mitochondrion rRNA gene, 60S ribosomal protein gene, 40S ribosomal protein S4 gene, trypsin gene, chymotrypsin A gene, ospin gene, and 16S ribosomal RNA gene. There were 5 over-expressed genes in susceptible strain, 2 of them being similar with 40S ribosomal protein S29 gene and myosin regulatory light chain 2 gene. In addition, 2 genes specially expressed in resistant strain were similar respectively to glycogen branching enzyme gene and ribosomal protein 46 gene. CONCLUSION: The differentially expressed genes may be associated with deltamethrin-resistance or susceptibility of Culex pipiens pallens.

Animals↗
PubMed2001

[The status and prospects of X-ray photographs marking technology].

This article introduces three different marking technologies used in clinical radiation photography at present and their respective theories, their advantages and disadvantages, analyzes the space-time and technology conditionality of different marking technologies in selecting mark messages. In the future, new marking technology will be fused with the cassette into one and will contain message as much as possible on photograph marks in order to satisfy the need of technicians and doctors.

Radiography↗
PubMed2000

A maize sesquiterpene cyclase gene induced by insect herbivory and volicitin: characterization of wild-type and mutant alleles.

Plants can defend themselves from herbivorous insects by emitting volatile chemical signals that attract natural enemies of the herbivore. For example, maize seedlings attacked by beet armyworm larvae (Spodoptera exigua) produce a mixture of terpenoid and indole volatiles that serve to attract parasitic wasps. A key step in terpenoid biosynthesis is the conversion of acyclic prenyl diphosphates to terpenoid compounds by specific terpenoid synthases (cyclases). We have cloned a maize sesquiterpene cyclase gene, stc1, by transposon tagging and have identified two deletion mutations of the gene. The stc1 gene is located on chromosome 9S and does not seem to have a closely related ortholog in the maize genome. It is induced 15- to 30-fold in maize leaves by beet armyworm larvae feeding or by application of purified volicitin, the insect-derived elicitor, at a mechanically wounded site. stc1 induction is systemic, because undamaged leaves of the same plant show a similar increase in stc1 transcription. Analysis of volatiles from volicitin-treated seedlings revealed that a major naphthalene-based sesquiterpene was present in wild-type seedlings but absent in the Ac-insertion and x-ray-deletion mutants. Therefore, we have identified a maize gene that responds to caterpillar herbivory by producing a chemical defense signal that most likely serves to attract natural enemies of the herbivore.

Alleles↗
PubMed2000

Structural basis for signal transduction by the Toll/interleukin-1 receptor domains.

Toll-like receptors (TLRs) and the interleukin-1 receptor superfamily (IL-1Rs) are integral to both innate and adaptive immunity for host defence. These receptors share a conserved cytoplasmic domain, known as the TIR domain. A single-point mutation in the TIR domain of murine TLR4 (Pro712His, the Lps(d) mutation) abolishes the host immune response to lipopolysaccharide (LPS), and mutation of the equivalent residue in TLR2, Pro681His, disrupts signal transduction in response to stimulation by yeast and gram-positive bacteria. Here we report the crystal structures of the TIR domains of human TLR1 and TLR2 and of the Pro681His mutant of TLR2. The structures have a large conserved surface patch that also contains the site of the Lps(d) mutation. Mutagenesis and functional studies confirm that residues in this surface patch are crucial for receptor signalling. The Lps(d) mutation does not disturb the structure of the TIR domain itself. Instead, structural and functional studies indicate that the conserved surface patch may mediate interactions with the down-stream MyD88 adapter molecule, and that the Lps(d) mutation may abolish receptor signalling by disrupting this recruitment.

Amino Acid Sequence↗
PubMed2000

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair.

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.

Cell Cycle↗
PubMed2000

Triple hydroxylation of tetracenomycin A2 to tetracenomycin C involving two molecules of O(2) and one molecule of H(2)O.

The TcmG or ElmG oxygenase-catalyzed triple hydroxylation of tetracenomycin (Tcm) A2 to Tcm C proceeds via a novel monooxygenase-dioxygenase mechanism, deriving the 4- and 12a-OH groups of Tcm C from two molecules of O(2) and the 4a-OH group of Tcm C from a molecule of H(2)O. These results suggest a mechanistic analogy among TcmG, ElmG, and the bacterial and fungal hydroquinone epoxidizing dioxygenases, as well as the mammalian vitamin K-dependent gamma-glutamyl carboxylase.

Anthracyclines↗
PubMed2000

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR.

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.

DNA Mutational Analysis↗
PubMed2000

An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003.

We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003. We report here the identification and characterization of an oxidation domain in BlmIII. The oxidation domain shows local homology to a family of oxidoreductases and is present in all thiazole-forming non-ribosomal peptide synthetase modules known to date. Both the blmIII-Ox domain and blmIII gene were expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity. The oxidation domain contains one molar equivalent of non-covalently bound FMN as a prosthetic group. These results provide experimental evidence for an oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in bleomycin biosynthesis.

Amino Acid Sequence↗
PubMed2000

Evidence for TeV Emission from GRB 970417a.

Milagrito, a detector sensitive to very high energy gamma rays, monitored the northern sky from 1997 February through 1998 May. With a large field of view and a high duty cycle, this instrument was well suited to perform a search for TeV gamma-ray bursts (GRBs). We report on a search made for TeV counterparts to GRBs observed by BATSE. BATSE detected 54 GRBs within the field of view of Milagrito during this period. An excess of events coincident in time and space with one of these bursts, GRB 970417a, was observed by Milagrito. The excess has a chance probability of 2.8x10-5 of being a fluctuation of the background. The probability for observing an excess at least this large from any of the 54 bursts is 1.5x10-3. No significant correlations were detected from the other bursts.

Journal Article↗
PubMed2000

Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer.

Vascular endothelial growth factor (VEGF) is a potent angiogenic polypeptide that activates 2 distinct high-affinity tyrosine kinase receptors, flk-1/KDR and flt-1. In the present study, we characterized the expression of VEGF and its receptors flk-1/KDR and flt-1 in the normal human pancreas and in human pancreatic cancer tissues and cell lines. VEGF, flk-1/KDR and flt-1 mRNA levels were elevated in cancer tissues compared with normal pancreas. By immuno-histochemistry, VEGF, flk-1/KDR and flt-1 immunoreactivity co-localized in many of the cancer cells within the tumor mass. Three (AsPC-1, Capan-1 and MIAPaCa-2) of 6 pancreatic cancer cell lines expressed flk-1/KDR mRNA and protein, and 4 cell lines (AsPC-1, Capan-1, T3M4 and PANC-1) expressed flt-1 mRNA transcripts. Binding studies with (125)I-labeled VEGF165 indicated that only Capan-1 cells exhibited high levels of specific binding. Furthermore, VEGF enhanced the growth of Capan-1 cells but was without effect in the other cell lines. VEGF also enhanced mitogen-activated protein kinase (MAPK) phosphorylation and c-fos induction in Capan-1 cells, whereas the MAPK kinase inhibitor PD98059 abolished the growth-stimulatory effect of VEGF. These data indicate that human pancreatic cancers have the capacity to over-express VEGF and its receptors and suggest that in some instances VEGF may directly promote pancreatic cancer growth via the MAPK pathway.

Adenocarcinoma↗
PubMed2000

Construction and analysis of three-dimensional graphic model of single-chain Fv derived from an anti-human placental acidic isoferritin monoclonal antibody by computer.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the inker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

Amino Acid Sequence↗
PubMed2000

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase.

BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.

Amino Acid Sequence↗
PubMed2000

Crystallization and preliminary X-ray analysis of tetracenomycin A2 oxygenase: a flavoprotein hydroxylase involved in polyketide biosynthesis.

The tcm operon in Streptomyces glaucescens encodes a group of enzymes involved in the synthesis of the polyketide tetracenomycin (Tcm) C that exhibits both antitumor and antibiotic activities. Here, the crystallization and preliminary data characterization of the tcmG gene product, Tcm A2 oxygenase, which catalyzes the triple hydroxylation of Tcm A2 to form Tcm C, are reported. Tcm A2 oxygenase crystallizes in two different space groups, both with six monomers per asymmetric unit, resulting in large unit-cell parameters. Synchrotron data have been collected from both the hexagonal and tetragonal crystal forms to 4.5 and 4.2 A, respectively. The self-rotation function searches in both space groups suggest the monomers assemble into a complex with D(3) symmetry.

Anthracyclines↗
PubMed2000

Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the cagA gene that encodes the C-1027 apoprotein.

C-1027, the most potent member of the enediyne antitumor antibiotic family, is produced by Streptomyces globisporus C-1027 and consists of an apoprotein (encoded by the cagA gene) and a nonpeptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazolinate, a deoxyamino hexose, a beta-amino acid, and an enediyne core. By adopting a strategy for cloning of the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to cagA, we have localized 75 kb of contiguous DNA from S. globisporus. DNA sequence analysis of two regions of the cloned gene cluster revealed two genes, sgcA and sgcB, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the cagA gene resides approximately 14 kb upstream of the sgcAB locus. The involvement of the cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production. These results represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis and provide a starting point for future genetic and biochemical investigations of C-1027 biosynthesis.

Amino Acid Sequence↗
PubMed2000

Genetic localization and molecular characterization of the nonS gene required for macrotetrolide biosynthesis in Streptomyces griseus DSM40695.

The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (+/-)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (+/-)-NA suggests that NonS catalyzes the formation of (-)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

Bacterial Proteins↗
PubMed2000

Tegumental changes in adult Schistosoma mansoni harbored in mice treated with artemether.

A detailed temporal examination was made of alterations induced by artemether in the tegument of adult Schistosoma mansoni worms using scanning electron microscopy (SEM). Mice infected with S. mansoni cercariae 42 days previously were treated intragastrically with artemether at a single dose of 400 mg/kg. Groups of 3 mice were killed at 24 hr, 72 hr, and 7 days after treatment; the worms were collected by perfusion and examined by SEM. Twenty-four hours after artemether treatment, focal damage to the tubercles on the tegumental surface of male worms was seen. In both male and female worms, there was focal swelling and fusion of tegumental ridges, and sometimes peeling. After 72 hr, the damage to the tegument had increased, especially in female worms, with extensive swelling, fusion, and peeling of the tegumental ridges. In the most severely damaged worms, host leukocytes were seen to be adhered to the damaged tegument. Damage to the oral sucker was also occasionally seen in both male and female worms. Seven days after treatment, the appearance of the tegument had returned to normal in some male and female worms, whereas others still showed apparent damage. The results demonstrate that artemether damages the tegument of adult S. mansoni, and the intensity of damage is more severe in female worms than in males.

Animals↗
PubMed2000

Diabetic gastropathy: a practical approach to a vexing problem.

The clinical presentation of diabetic gastropathy varies, and a diagnosis usually must be confirmed with tests that evaluate the structure and function of the upper gut. Although glucose control, dietary changes, and drug therapy are the current mainstays of treatment, they may not be effective. Gastric pacing, a new technique that stimulates gastric motility, may give physicians another management tool.

Anti-Bacterial Agents↗