Incorporation of nitric oxide-releasing crosslinked polyethyleneimine microspheres into vascular grafts

Over the years, many attempts have been made to increase the patency of small- to medium-sized prosthetic vascular grafts. However, none of them has greatly affected long-term rates. Recently, nitric oxide (NO) has been shown to inhibit thrombus formation in such grafts, suggesting that local delivery of NO may help to increase graft patency. This study describes the site-specific delivery of NO by entrapping NO-releasing microspheres in the pores of a vascular graft. NO-releasing polyethyleneimine microspheres (PEIX) were developed using a novel water-in-oil emulsion technique involving chemical crosslinking with a bis-epoxide. The PEIX microspheres were then derivatized with NO forming the [N(O)NO]- moiety of the diazeniumdiolates formerly known as NONOates. These polymeric NO-releasing particles were found to spontaneously release 194 nmol NO/mg with a half-life of over 66 h under physiologic conditions. Fluorescein isothiocyanate-labeled microspheres were then embedded into the pores of a 60-micron nonreinforced Gore-tex vascular graft using a simple evacuation technique and evaluated for microsphere placement and NO release. Scanning electron microscopic analysis showed the microspheres entrapped in the pores of the vascular graft releasing 10 nmol NO/mg with a half-life of 51 h. The microspheres remained entrapped in the graft even after immersion and NO release, as confirmed by fluorescence of the medium. These results suggest that NO-releasing particles can be incorporated into the pores of a vascular graft to deliver therapeutic amounts of NO for the prevention of thrombosis in small-diameter prosthetic grafts.     

Effects of ion irradiation on fatigue of Fe-12Cr-20Mn stainless steel for fusion reactor applications

To minimize waste disposal problems associated with the residual radioactivity of the first wall material of a fusion reactor, fast induced radioactive decay (FIRD) alloys based on the Fe-Cr-Mn system are being investigated. The objective of this research was to evaluate the effects of irradiation on cyclic strain localization and fatigue crack initiation in a FIRD Fe-12Cr-20Mn alloy and to compare the response to commercially available 316 stainless steel. The alloys were irradiated with 200 keV Fe ions to a dose of 1 x 10 ions/cm² and 15.5 keV He ions to a dose of 7 x 10¹⁵ ions/cm² to simulate the irradiation-induced defect structure and helium concentration that would be produced in a fusion reactor. Irradiated specimens were fatigued in a cantilever beam fatigue testing machine with the deflection set to produce a fully reversed total strain amplitude of 0.25% on the surface of the specimen. Acetate replicas were obtained during the fatigue tests to provide a record of surface fatigue damage. Transmission electron microscopy (TEM) analyses were performed to characterize the microstructural changes resulting from the irradiations and interactions between fatigue-induced glide dislocations and the irradiation-induced defects. Results indicate that the irradiated Fe-Cr-Mn alloy exhibits fatigue properties similar to 316 stainless steel. Glide dislocations produced by fatigue cycling annihilate irradiation-induced defects. The defect annihilation causes the formation of cleared channels in which the cyclic plastic strain is localized. Subsurface slip bands penetrate the irradiated regions through the cleared channels and serve as fatigue crack initiation sites.     

Structural model of the catalytic domain of an enzyme with cell adhesion activity: human vascular adhesion protein-1 (HVAP-1) D4 domain is an amine oxidase

Human vascular adhesion protein-1 (HVAP-1) is a multifunctional protein having at least two different cellular roles, functioning both as a lymphocyte-endothelial cell adhesion protein and as an enzyme with monoamine oxidase activity. HVAP-1 is a 180 kDa homodimeric glycoprotein consisting of a membrane-spanning domain and three predicted extracellular copper-containing amine oxidase domains. In HVAP-1 the extracellular domains are composed of a large domain D4, containing the active site and forming the interface of the dimer, while the smaller D2 and D3 domains surround the D4 dimer near the entrance to the active site. The structural model of the catalytic D4 domain of HVAP-1 reveals that all components necessary for enzymatic monoamine oxidase activity are indeed present within the HVAP-1 and pinpoints residues that may be key to substrate entry through a channel to the active site and residues likely to be involved in substrate specificity as well as structural features critical to dimer formation. Proper glycosylation is required for the cell adhesion function of HVAP- 1 and the predicted location of the sugar units at the solvent-exposed surface suits this function well.      

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is usedas a thermolabile switch in 'hot start' variations of PCR to minimizenon-specific amplification events. Earlier studies have established thatTP7 binds to the polymerase domain of TaqP, competes with primer templatecomplex for binding and is a potent inhibitor of the polymerase activity ofTaqP. We report crystallographic determination of the structure of an Fabfragment of TP7 and computational docking of the structure with the knownthree-dimensional structure of the enzyme. Our observations stronglysuggest that the origin of inhibitory ability of TP7 is its binding toenzyme residues involved in DNA binding and polymerization mechanism.Although criteria unbiased by extant biochemical data have been used inidentification of a putative solution, the resulting complex offers aneminently plausible structural explanation of biochemical observations. Theresults presented are of general significance for interpretation of dockingexperiments and in design of small molecular inhibitors of TaqP, that arenot structurally similar to substrates, for use in PCR. Structural andfunctional similarities noted among DNA polymerases, and the fact thatseveral DNA polymerases are pharmacological targets, make discovery ofnon-substrate based inhibitors of additional importance.     

The greatest co-authorships of finance theory literature (1896–2006): scientometrics based on complex networks

Scientometrics - We use some modern scientometrics tools to detect which articles written in co-authorship are the most influential in the finance theory literature from 1896 to 2006. To develop a...     

Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.     

Testis-like steroidogenesis in the ovotestis of the European mole, Talpa europaea

A gene (AglyA) encoding serine hydroxymethyltransferase of Acinetobacter radioresistens CMC-1 was cloned and sequenced. Nucleotide sequence analysis of AglyA predicted a single open reading frame (ORF) of 1251 bp encoding a 417-amino acid polypeptide. Two putative MetR-like binding sites (5'-TGAAACATGAGCT) and (5'-TGAGCAAAGTTCA), centered at bp -123 and -95 relative to the +1 translation start site were found, which have six out of nine and eight out of nine nucleotides that match to the consensus sequence of Escherichia coli (5'-TGAANNT/ANNTTCA), respectively. The enzyme also showed a high level of homology to other sources of serine hydroxymethyltransferase proteins.     

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.     

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.     

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

OBJECTIVE: To assess the ability of the International Association for the Study of Pain Complex Regional Pain Syndrome (CRPS) diagnostic criteria and associated features to discriminate between CRPS patients and patients with painful diabetic neuropathy. DESIGN: Prospective assessment of signs and symptoms in a series of CRPS and diabetic neuropathy patients. SETTING: University of Washington Multidisciplinary Pain Center. PATIENTS: A consecutive series of 18 CRPS patients and 30 diabetic neuropathy patients. INTERVENTIONS: Patients completed a 10-item patient history questionnaire assessing symptoms of CRPS prior to medical evaluation. The evaluating physician completed a 10-item patient examination questionnaire assessing objective signs of CRPS. OUTCOME MEASURES: The analyses conducted were designed to test the ability of CRPS signs and symptoms and associated features to discriminate between CRPS patients and diabetic neuropathy patients. RESULTS: Data analysis suggested that CRPS decision rules may lead to overdiagnosis of the disorder. Diagnosis based on self-reported symptoms can be diagnostically useful in some circumstances. The addition of trophic tissue changes, range of motion changes, and "burning" quality of pain did not improve diagnostic accuracy, but the addition of motor neglect signs did. Test of a CRPS scoring system resulted in improved accuracy relative to current criteria and decision rules. CONCLUSIONS: Poorly understood disorders lacking prototypical signs/symptoms and diagnostic laboratory testing must rely on the development of reliable diagnostic guidelines. The results of this study should assist in the further refinement of the CRPS diagnostic criteria.