Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography

In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation.

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: Electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host, Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host ( Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of similar to14 and similar to17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coil cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mu m to 0.3 mu m as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or in passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (C) 1997 John Wiley & Sons, Inc.

Characterization of baboon (Papio hamadryas) milk proteins

The major proteins of baboon milk were identified as beta -lactoglobulin (beta LG), alpha -lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)(+) mRNA purified from lactating mammary gland. Baboon beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart

Objective: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. Methods: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 muM) to desensitize PKC, the PKC inhibitor chelerythrine (10 muM), 10 muM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 muM), or the Rho kinase inhibitor Y-27632 (0.1 - 10 muM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. Results: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8 +/- 0.4-fold, P < 0.05, n = 6) and PKCalpha/betaII (1.4 +/- 0.2-fold compared to non-stimulated controls, P < 0.05, n = 7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA. GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. Conclusions: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Analysis of the role of pglI in pilin glycosylation of Neisseria meningitidis

Pilin is the major subunit of the essential virulence factor pili and is glycosylated at Ser63. In this study we investigated the gene pglI to determine whether it is involved in the biosynthesis of the pilin-linked glycan of Neisseria meningitidis strain C311#3. A N. meningitidis C311#3pglI mutant resulted in a change of apparent molecular weight in SDS-PAGE and altered binding of antisera, consistent with a role in the biosynthesis of the pilin-linked glycan. These data, in conjunction with homology with well-characterised acyltransferases suggests a specific role for pglI in the biosynthesis of the basal 2,4-diacetamido-2,4,6-trideoxyhexose residue of the pilin-linked glycan. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Identification of the causal agent of pistachio dieback in Australia

Symptoms associated with pistachio dieback in Australia include decline (little or no current season growth), xylem staining in shoots two or more years old, trunk mu and limb lesions (often covered by black, superficial fungal growth), excessive exudation of resin, dieback and death of the tree. Bacteria belonging to the genus Xanthomonas have been suggested as the causal agent. To confirm the constant association between these bacteria and the disease syndrome, the absence of other pathogens and the identity of the pathogen, we performed a series of isolations and pathogenicity tests. The only microorganism consistently isolated from diseased tissue was a bacterium that produced yellow, mucoid colonies and displayed morphological and cultural characteristics typical of the genus Xanthomonas. Database comparisons of the fatty acid and whole-cell protein profiles of five representative pistachio isolates indicated that they all belonged to X. translucens, but it was not possible to allocate the isolates to pathovar. Pathogenicity tests on cereals and grasses supported this identification. However, Koch's postulates have been only partially fulfilled because not all symptoms associated with pistachio dieback were reproduced on inoculated two-year-old pistachio trees. While discolouration was observed, dieback, excessive resinous exudate and trunk and limb lesions were not produced; expression of these symptoms may be delayed, and long-term monitoring of a small number of inoculated trees is in progress.

Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.

Microarray and proteomic analysis of the human alcoholic brain

The superior frontal cortex (SFC) is selectively damaged in chronic alcohol abuse, with localized neuronal loss and tissue atrophy. Regions such as motor cortex show little neuronal loss except in severe co-morbidity (liver cirrhosis or WKS). Altered gene expression was found in microarray comparisons of alcoholic and control SFC samples [1]. We used Western blots and proteomic analysis to identify the proteins that also show differential expression. Tissue was obtained at autopsy under informed, written consent from uncomplicated alcoholics and age- and sex-matched controls. Alcoholics had consumed 80 g ethanol/day chronically (often, 200 g/day for 20 y). Controls either abstained or were social drinkers ( 20 g/day). All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were homogenized in water and clarified by brief centrifugation (1000g, 3 min) before storage at –80°C. For proteomics the thawed suspensions were centrifuged (15000g, 50 min) to prepare soluble fractions. Aliquots were pooled from SFC samples from the 5 chronic alcoholics and 5 matched controls used in the previous microarray study [1]. 2-Dimensional electrophoresis was performed in triplicate using 18 cm format pH 4–7 and pH 6–11 immobilized pH gradients for firstdimension isoelectric focusing. Following second-dimension SDS-PAGE the proteins were fluorescently stained and the images collected by densitometry. 182 proteins differed by 2-fold between cases and controls. 141 showed lower expression in alcoholics, 33 higher, and 8 were new or had disappeared. To date 63 proteins have been identified using MALDI-MS and MS-MS. Western blots were performed on uncentrifuged individual samples from 76 subjects (controls, uncomplicated alcoholics and cirrhotic alcoholics). A common standard was run on every gel. After transfer, immunolabeling, and densitometry, the intensities of the unknown bands were compared to those of the standards. We focused on proteins from transcripts that showed clear differences in a series of microarray studies, classified into common sets including Regulators of G-protein Signaling and Myelin-associated proteins. The preponderantly lower level of differentially expressed proteins in alcoholics parallels the microarray mRNA analysis in the same samples. We found that mRNA and protein expression do not frequently correspond; this may help identify pathogenic processes acting at the level of transcription, translation, or post-translationally.