Description of 165 episodes of fungemia: A multicenter study.

The aim of this article is to describe the main features of fungemia belonged to the Multicenter Study Sepsis Data realised in 34 spanish hospitals. The hospitals were been given a protocol and a software specially designed for this study. 165 episodes of fungemia has been recovered (total: 5,000 episodes of bacteremia). The main results are showed as follows. Sex: male 100 (60.6%); female 65 (39.4%). Areas: medical 64 (38.8%); intensive care unit 48 (29.1%); surgical 39 (23.6%); paediatric 14 (8.5%). Underlying disease: neoplasia 43 (26.1%); HIV infection 28 (16.9%); chronic obtructive pulmonary disease 18 (10.9%); diabetes mellitus and parenteral drug abuse 15 (9.1%) every one. Nosocomial fungemia: 119 (72.1%). Community-acquired fungemia: 42 (25.5%). Sources: primary 41 (24.9%); catheter 40 (24.2%); respiratory 23 (13.9%); urinary 17 (10.3%); abdominal 8 (4.9%); skin/soft tissues 4 (4.4%); surgical wound 6 (3.6%). Fungi most often isolated: Candida albicans, 73 isolates (44.2%); Candida parapsilosis, 20 (12.1%); Cryptococcus spp., 12 (7.3%) and Candida glabrata, 6 (3.6%). Polymicrobial fungemia: 19 (11.5%). Fluconazole (54.4%) and amphotericin B (41.9%) were the antifungal agents most often used. Mortality: 33.3%.     

Palaeontology: the 165-million-year itch

New flea-like fossils from China provide a rare, tantalizing glimpse of bizarre insects in the Cretaceous and Jurassic. Possibly the oldest flea-like animals known, they provide a challenge to the functional morphologist to infer which animals they may have targeted.     

165 Protein domains: a thermodynamic definition

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earlier crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous thermodynamically based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, domains are equated to chain segments that retain full cooperativity when excised from their parent structures. Implementing this approach computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property, grounded in solution thermodynamics.     

pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.     

Vascular endothelial growth factor 165 (VEGF(165)) activities are inhibited by carboxymethyl benzylamide dextran that competes for heparin binding to VEGF(165) and VEGF(165).KDR Complexes

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.     

Monoclonal antibodies against vascular endothelial growth factor 165 (VEGF165): neutralization of biological activity and recognition of the epitope

Five antibodies against vascular endothelial growth factor 165 (VEGF165) were obtained. These antibodies, Ab-2, Ab-20, Ab-153, Ab-309, Ab-342, were able to recognize not only native VEGF165, but also reducing VEGF165. Three of the antibodies, Ab-153, Ab-309, Ab-342, were identified as VEGF165 neutralizing antibody on the basis of its ability to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) induced by VEGF165 and the binding of [125I]-VEGF165 to receptors on HUVEC. The fragments of E1 (VEGF120-135) and E2 (VEGF46-60) recognized by neutralizing antibodies may be related the receptor binding domain of VEGF.     

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.     

Characterization and optimization of vascular endothelial growth factor165 (rhVEGF165) expression in Escherichia coli

Vascular endothelial growth factors₁₆₅ (VEGF₁₆₅) is the most potent and widely used pro-angiogenic factor. Here we determined optimal culture condition of recombinant human VEGF₁₆₅ (rhVEGF₁₆₅) in Escherichia coli (E. coli). rhVEGF₁₆₅ expression was the highest in 0.25% of l-arabinose induction concentration, at 20 °C induction temperature, and for 5 h induction time under the control of araBAD promoter using pBADHisA vector. In biological activity test, rhVEGF₁₆₅ significantly increased the proliferative activity of CPAE cells (p < 0.001) and upregulated the expressions of endothelial cell growth-related genes, such as platelet endothelial cell adhesion molecule (PECAM-1), endothelial-specific receptor tyrosine kinase (TEK), kinase insert domain protein receptor (KDR), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1) in calf pulmonary artery endothelial (CPAE) cells.     

VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding

Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR.     

Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys¹⁶⁵, a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys¹⁶⁵ modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys¹⁶⁵ was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys¹⁶⁵ is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys¹⁶⁵. The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.     

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.     

Simple model for the effect of Glu165----Asp165 mutation on the rate of catalysis in triose phosphate isomerase

We present an ab-initio self-consistent field calculation with a 4-31G basis set on a simple model for proton abstraction from hydroxyacetone (a model for dihydroxyacetone phosphate; DHAP) by formate, which is a model for Glu165 in triose phosphate isomerase. Earlier, we showed that the electrophilic groups on the enzyme (the NH3+ of Lys13 and the NH of His95) were essential to efficient catalysis by triose phosphate isomerase. These groups stabilized the enediolate formed by proton abstraction from the DHAP model so that proton transfer from this molecule to Glu165 became likely. In this study, we carry this analysis one step further. First, we re-examine the energy profile for proton transfer, using the fact that our earlier calculations showed that the combined effect of His95 and Lys13 on the reactant DHAP and intermediate enediolate was to make them equal in energy. Then, we analyze the likely effect of changing Glu165 to Asp165 and relate this to experiments on the kinetics of enzyme catalysis by the Glu165----Asp165 mutant.     

Metabolic products of microorganisms, 165th communication

Summary Isolation of siderochromes from culture filtrates by extraction with a mixture of chloroform and phenol was replaced by adsorption chromatography with Amberlite XAD-2. For the antibiotic albomycin, the purification was possible by ion-exchange chromatography with SP-Sephadex, and for the sideramine ferricrocin by exclusion chromatography with Bio-Gel P-2. Quantitation of albomycin was done by an agar plate diffusion test, and of ferricrocin by high-pressure liquid chromatography and photometric determination. Thinlayer and high-pressure liquid chromatographic systems were developed for checking homogeneity.Metabolic products of microorganisms. 164. W.A. König, C. Krauss, H. Zähner: 6-Chlor-Genistein und 6,3-Dichlor-Genistein. Helv. Chim. Acta in press     

A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia

In Arabidopsis leaf primordia, the expression of HD‐Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD‐Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD‐Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial‐biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell‐to‐cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis‐acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial‐biased activity of miR165 in the leaf primordia. We also found that the abaxial‐determining genes YABBYs are trans‐acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial–abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial‐biased patterning of miR165 activity is confined.     

枯草杆菌蛋白酶E的156和165位突变

应用定点突变方法,在M222A突变的枯草杆菌蛋白酶E基因上进行E156S和V165I定点突变. 将突变基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DB104中进行表达,得到突变种(M222A,E156S)和(M222A,E156S,V165I)蛋白酶E. 性质测定表明,E156S突变使蛋白酶比活力增加90%,并不影响酶的热稳定性和抗氧化性. 而V165I突变使蛋白酶比活力降低.