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Biomedical subjects

B Shen

Publications and source records attributed to B Shen.

At least 37 records · Page 2Linked to original sources
PubMed2001

Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation.

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.

Animals↗
PubMed2001

The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome.

The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3' untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

Base Sequence↗
PubMed2001

Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication.

DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.

Amino Acid Sequence↗
PubMed2001

Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho < 1.33 g/mL). The top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

Animals↗
PubMed2001

The mouse ocular albinism 1 gene product is an endolysosomal protein.

To gain insight into the role of Oa1, the mouse homolog of the human X-linked ocular albinism 1 protein, its properties and subcellular localization were investigated. Antiserum raised against an expressed segment of the Oa1 protein recognized a band of approximately 48 kDa in immunoblots of extracts of cultured mouse melan-a melanocytes, but not of cells of non-melanocyte origin. When melanocyte extracts were treated with glycopeptidase F, a approximately 44 kDa band appeared. Like the melanogenic enzyme tyrosinase, expression of Oa1 was stimulated by alpha-melanocyte stimulating hormone and inhibited by agouti signal protein. Upon density gradient centrifugation of organelles of melan-a cells, Oa1 protein colocalized with the late endosomal/lysosomal marker Lamp1, but only partial overlap was observed with melanosomal proteins in the high density region of the gradient. Immunofluorescence staining revealed that neither endogenous Oa1 nor an Oa1-green fluorescent protein fusion product colocalized with the melanosomal protein tyrosinase related protein-1 in the cell periphery. In contrast, colocalization of Oa1 and Oa1-green fluorescent protein fusion product with Lamp1 was extensive throughout the cell. These results indicate that Oa1 is a melanocyte-specific integral membrane glycoprotein localized to late endosomes/lysosomes but not mature melanosomes. Considering the microscopic findings in patients with X-linked ocular albinism 1, we speculate that Oa1 may play a role in the trafficking of vesicles to developing melanosomes.

Albinism, Ocular↗
PubMed2001

Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules.

The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examining the biochemical and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a critical role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products.

Amino Acid Sequence↗
PubMed2001

Medical management of postoperative complications of inflammatory bowel disease: pouchitis and Crohn's disease recurrence.

Surgical intervention is often required for patients with inflammatory bowel disease. Total proctocolectomy with ileal pouch-anal anastomosis is the surgical treatment of choice for patients with ulcerative colitis. The main long-term complication of this surgery is pouchitis, with 10-year cumulative incidence rates between 24% and 46%. For patients with Crohn's disease, postoperative recurrence is a significant problem, with clinical recurrence rates as high as 55% at 5 years and 76% at 15 years. Increasing evidence suggests that postoperative medical therapy has the potential to decrease the risk of postoperative Crohn's disease recurrence.

Crohn Disease↗
PubMed2001

A review of diabetic gastropathy.

Diabetes mellitus affects various organs, including the gastrointestinal tract. The stomach is commonly affected, and symptoms related to the upper GI tract are frequently reported. Management of diabetic gastropathy involves dietary modifications, pharmacological agents, and occasionally, alternative feeding methods.

Antiemetics↗
PubMed2001

A novel fluorescent coronenyl-phospholipid analogue for investigations of submicrosecond lipid fluctuations.

A fluorescent phospholipid derivative, the 2'-(4-coronenylbutyric) ester of lyso-egg phosphatidylcholine, has been synthesized for use in studies of submicrosecond lipid dynamics. Synthesis of the phospholipid derivative involves Friedel-Crafts acylation of free coronene, followed by a Huang-Minlon reduction to yield the fatty-acyl derivative, 4-coronenylbutyric acid. Esterification of the carboxylic acid with lyso-phosphatidylcholine is achieved through a mixed anhydride intermediate. The resultant coronenyl-phospholipid adduct (Cor-PC) has been incorporated into sonicated unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) for dynamic lipid studies. Fluorescence quenching studies using potassium iodide, together with steady-state emission anisotropy (EA) measurements, confirm that the coronene moiety of the phospholipid adduct resides towards the head group interfacial region of the lipid bilayer. Unique properties of this new fluorescent phospholipid adduct are its long mean fluorescence lifetime (tau av approximately 112 ns at 14 degrees C), the planar symmetry of the fluorophore and its defined bilayer location. As a consequence, depolarizing motions of the coronene moiety target submicrosecond 'gel-fluid' lipid dynamics arising from a relatively narrow bilayer distribution. Our data suggest that the sensitivity of this new long-lived fluorescent phospholipid analogue to localized transverse submicrosecond lipid dynamics can provide important biological insights into varied processes including lipid-peptide interactions, bilayer fluidity gradients and passive ion transport.

Fluorescent Dyes↗
PubMed2001

Attempt to adsorb N-nitrosamines in solution by use of zeolites.

The strong adsorption of zeolite for N-nitrosamines in solution was first revealed by use of adsorption, and temperature programmed surface reaction (TPSR) techniques. N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) as well as N-nitrosohexamethyleneimine (NHMI) can be adsorbed on zeolite Y, ZSM-5 and A in the solution of methylene chloride or water, which will be helpful for removal of the N-nitrosamines pollution in environmental protection. The equilibrium data were fitted to Freundlich-type isotherms, but the adsorption capacity of zeolites mainly depended on their pore size, surface area and acid-basic properties. Molecular size of adsorbate and solute-solvent interaction also strongly affected the adsorption of N-nitrosamines on zeolite in solution. The extraordinary adsorption properties of NaA zeolite for N-nitrosamines in aqueous solution is first reported and discussed.

Adsorption↗
PubMed2001

STAT3 exerts two-way regulation in the biological effects of IL-6 in M1 leukemia cells.

The signal transducer and activator of transcription (STAT) proteins have been implicated in cytokine-regulated proliferation, differentiation and cell survival. Interleukin-6 (IL-6), a pleiotropic cytokine, induces a robust and sustained activation of STAT3 in M1 acute myeloid leukemia cells, which in turn undergo growth arrest, terminal differentiation and apoptosis in response to IL-6. The roles of STAT3 activation in IL-6-mediated responses in M1 cells are not fully understood. We introduced STAT3 antisense cDNA into M1 cells. STAT3 antisense cDNA blocked the expression and IL-6-induced tyrosine phosphorylation and DNA binding of STAT3, and resulted in reduction of both IL-6-induced growth arrest at G(0)/G(1) phase and macrophage differentiation in the M1 transformants. This observation is in accordance with previous reports and confirms that STAT3 plays an essential role in IL-6-induced growth arrest and terminal differentiation in M1 leukemia cells. On the other hand, STAT3 antisense cDNA augmented IL-6-induced apoptosis of M1 cells, which was supported by the cell cycle assay, DNA fragmentation assay and detection of the p17 active fragment of Caspase 3. As proliferation inhibition and differentiation induction stands for a negative signal, while survival maintenance stands for a positive signal, we conclude that STAT3 exerts two-way regulation on the biological effects of IL-6 in M1 leukemia cells.

Animals↗
PubMed2001

Independent dose calculations for the PEACOCK System.

An independent dose calculation method has been developed to validate intensity-modulated radiation therapy (IMRT) plans from the NOMOS PEACOCK System. After the plan is generated on the CORVUS planning system, the beam parameters are imported into an independent workstation. The beam parameters consist of intensity maps at each gantry angle and each arc position. In addition, CT scans of the patient are imported into the independent workstation to obtain the external contour of the patient. The coordinate system is defined relative to the alignment point chosen in the CORVUS plan. The independent calculation uses the pencil beam data viz tissue maximum ratio (TMR) and beam profiles for a single 1 x 0.8-cm beamlet formed by the NOMOS multileaf intensity-modulating collimator (MIMiC) leaf. The pencil beam data were measured for the 6-MV photon beam from Siemens PRIMUS linear accelerator using film dosimetry. The dose at a point is calculated using the depth and off-axis distance from a given pencil beam, corrected for its beam intensity. Isodose distributions are generated using the independent dose calculations and compared to the CORVUS plans. Isodose distributions show good agreement with the CORVUS plans for a number of clinical cases. The independent dose calculation algorithm is described in this paper.

Algorithms↗
PubMed2001

Comparative study between IMRT with NOMOS BEAK and linac-based radiosurgery in the treatment of intracranial lesions.

A comparative study was undertaken to examine intracranial irradiation using intensity-modulation radiation therapy (IMRT) and linear accelerator-based radiosurgery. The IMRT was examined using the Peacock system with a BEAK attachment. A clinical case involving a metastatic brain lesion, treated with 3 radiosurgery isocenters, was planned for IMRT. The radiosurgery was planned using the Leibinger planning system. The IMRT was planned using the CORVUS planning system. The CORVUS planning system uses an inverse planning algorithm, a recent development in radiotherapy. Isodose distributions and dose volume histograms were generated and compared. Analysis of the dosimetry shows that the dose conformity and homogeneity within the target using the RTOG guidelines are superior for IMRT. The advantages of IMRT using inverse planning system include the ease of planning and execution of treatment, especially for cases that involve concave targets that require multiple isocenters using radiosurgery.

Algorithms↗
PubMed2001

Independent dose calculations for the corvus MLC IMRT.

Two independent dose calculation methods have been explored to validate MLC-based IMRT plans from the NOMOS CORVUS system. After the plan is generated on the CORVUS planning system, the beam parameters are imported into an independent workstation. The beam parameters consist of intensity maps at each gantry angle. In addition, CT scans of the patient are imported into the independent workstation to obtain the external contour of the patient. The coordinate system is defined relative to the alignment point chosen in the CORVUS plan. The 2 independent calculation methods are based on a pencil beam kernel convolution and a Clarkson-type differential scatter summation, respectively. The pencil beam data for a 1 x 1-cm beam, as formed by the multileaf collimator, were measured for the 6-MV photon beam from a Siemens PRIMUS linear accelerator using film dosimetry. In the pencil beam method, the dose at a point is calculated using the depth and off-axis distance from a given pencil beam, corrected for beam intensity. The scatter summation method used the conversion of measured depth dose data into scatter maximum ratios. In this method, the differential scatter from each pencil beam is corrected for the beam intensity. Isodose distributions were generated using the independent dose calculations and compared to the CORVUS plans. Although isodose distributions from both methods show good agreement with the CORVUS plan, our implementation of the differential scatter summation approach seems more favorable. The 2 independent dose calculation algorithms are described in this paper.

Radiotherapy Dosage↗
PubMed2001

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.

Amino Acid Sequence↗