WorldWideScience

Sample records for normal proteolytic processing

  1. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We...... hypothesized that proteolytic processing of gammaENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and m......Ab4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6...

  2. Differential production of proteolytic enzymes by normal human fibroblasts and their counterparts transformed by treatment with 60Co gamma rays

    International Nuclear Information System (INIS)

    Nishitani, Koji; Namba, Masayoshi; Ohkubo, Shigeki; Kimoto, Tetsuo

    1985-01-01

    Production of proteolytic enzymes by human fibroblasts in the process of transformation was investigated. The cells used were normal human fibroblasts (KSM-6) and their in vitro counterparts transformed by treatment with 60 Co gamma rays (KMST-6). Cells seeded by treatment with EDTA were cultured in a serum free medium. Proteolytic enzymes in the culture medium of cells were assayed using a synthetic substrate, N-α-(p-tosyl)-L-arginine ( 3 H) methyl ester hydrochloride. The transformed cells (KMST-6) produced a larger amount of enzymes than normal cells (KMS-6). The enzyme production in both cell lines was high in the exponential growth stage and then decreased as the cells reached confluency. The proteolytic enzymes produced by these cells were trypsin- and thrombin-like enzymes. Cell growth of KMST-6 or KMS-6 was not inhibited by the addition of protease inhibitors to the culture medium. (author)

  3. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

    Directory of Open Access Journals (Sweden)

    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  4. Proteolytic Processing of the Human Immunodeficiency Virus Envelope Glycoprotein Precursor Decreases Conformational Flexibility

    OpenAIRE

    Haim, Hillel; Salas, Ignacio; Sodroski, Joseph

    2013-01-01

    The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentiall...

  5. Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms*

    Science.gov (United States)

    Ehlen, Harald W. A.; Sengle, Gerhard; Klatt, Andreas R.; Talke, Anja; Müller, Stefan; Paulsson, Mats; Wagener, Raimund

    2009-01-01

    The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo. PMID:19531486

  6. Proteolytic events in the processing of secreted proteins in fungi.

    Science.gov (United States)

    Calmels, T P; Martin, F; Durand, H; Tiraby, G

    1991-01-01

    Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

  7. Aberrant proteolytic processing and therapeutic strategies in Alzheimer disease.

    Science.gov (United States)

    Tomita, Taisuke

    2017-05-01

    Amyloid-β peptide (Aβ) and tau are major components of senile plaques and neurofibrillary tangles, respectively, deposited in the brains of Alzheimer disease (AD) patients. Aβ is derived from amyloid-β precursor protein that is sequentially cleaved by two aspartate proteases, β- and γ-secretases. Secreted Aβ is then catabolized by several proteases. Several lines of evidence suggest that accumulation of Aβ by increased production or decreased degradation induces the tau-mediated neuronal toxicity and symptomatic manifestations of AD. Thus, the dynamics of cerebral Aβ, called as "Aβ economy", would be the mechanistic basis of AD pathogenesis. Partial loss of γ-secretase activity leads to the increased generation of toxic Aβ isoforms, indicating that activation of γ-secretase would provide a beneficial effect for AD. After extensive discovery and development efforts, BACE1, which is a β-secretase enzyme, has emerged as a prime drug target for lowering brain Aβ levels. Recent studies revealed the decreased clearance of Aβ in sporadic AD patients, suggesting the importance of the catabolic mechanism in the pathogenesis of AD. I will discuss with these proteolytic mechanisms involved in the regulation of Aβ economy, and development of effective treatment and diagnostics for AD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Normal modified stable processes

    DEFF Research Database (Denmark)

    Barndorff-Nielsen, Ole Eiler; Shephard, N.

    2002-01-01

    This paper discusses two classes of distributions, and stochastic processes derived from them: modified stable (MS) laws and normal modified stable (NMS) laws. This extends corresponding results for the generalised inverse Gaussian (GIG) and generalised hyperbolic (GH) or normal generalised inverse...... Gaussian (NGIG) laws. The wider framework thus established provides, in particular, for added flexibility in the modelling of the dynamics of financial time series, of importance especially as regards OU based stochastic volatility models for equities. In the special case of the tempered stable OU process...

  9. Biogenesis and proteolytic processing of lysosomal DNase II.

    Directory of Open Access Journals (Sweden)

    Susumu Ohkouchi

    Full Text Available Deoxyribonuclease II (DNase II is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected--a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.

  10. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

    2002-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

  11. PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

    Directory of Open Access Journals (Sweden)

    Stefano Lancellotti

    2013-09-01

    Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

  12. Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Meulendyke, Kelly Ann; Wurth, Mark Allen; McCann, Richard O.; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004)...

  13. The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes.

    Directory of Open Access Journals (Sweden)

    Brooke A LaFlamme

    2012-01-01

    Full Text Available Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs. Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.

  14. Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing

    Directory of Open Access Journals (Sweden)

    Heidi Hofer

    2017-06-01

    Full Text Available Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

  15. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells.

    Science.gov (United States)

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian; Schelhaas, Mario

    2015-07-01

    The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that

  16. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

    Directory of Open Access Journals (Sweden)

    Martin Steuble

    2012-06-01

    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  17. Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Pager, Cara Theresia; Wurth, Mark Allen; Dutch, Rebecca Ellis

    2004-01-01

    Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero ...

  18. Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin β.

    Science.gov (United States)

    Martin, Barry Lee; Conley, Sabena Michelle; Harris, Regine Simone; Stanley, Corshe Devon; Niyitegeka, Jean-Marie Vianney; Ongeri, Elimelda Moige

    2016-01-01

    Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin β. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αβ knockout mice (αβKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αβKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αβKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin β cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin β interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury.

  19. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing.

    Directory of Open Access Journals (Sweden)

    Zhitao Wan

    Full Text Available The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline. Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.

  20. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  1. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  2. Combined high pressure and thermal processing on inactivation of type A and proteolytic type B spores of Clostridium botulinum.

    Science.gov (United States)

    Reddy, N Rukma; Marshall, Kristin M; Morrissey, Travis R; Loeza, Viviana; Patazca, Eduardo; Skinner, Guy E; Krishnamurthy, Kathiravan; Larkin, John W

    2013-08-01

    The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A . 7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when

  3. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein.

    Science.gov (United States)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-11-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackievirus B3 (CVB3), substitution N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but its possible benefit has not been investigated yet. In this study, we set out to investigate the possible role of 2C-N2D in the resistance to PI4KB and OSBP inhibition. We show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the poliovirus polyprotein. Therefore, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Lipidperoxidative and proteolytic processes in fungi under the influence of xenobiotica. [Botrytis cinerea; Phytophora infestans

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, H.M.; Edlich, W.; Lyr, H.

    1986-01-01

    As a model system for the investigation of peroxidative effects in the phytopathogenic fungi Botrytis cinerea in submers culture was used. The influence was investigated of hydrogenperoxide with biochemical and cytological methods. The hyphae showed significant changes of the ultrastructure: swelling and vacuolization of mitochondria, loss of ribosomes, vesiculation of endoplasmic reticulum, degradation and lysis of the cell ribosomes, and deposition of lipid droplets on the membranes and in the cytoplasm, dependent on the concentration of the peroxide. Same effects with higher injuries of the membrane system are found in Phytophthorea infestans. A parallel investigation on fibroblasts showed similar effects: pathological changes of mitochondria, but also pycnosis in the nucleus. Comparative ultrastructure investigations were performed in cultures of Phytophthora infestans with tetrachlorcarbon and chloroneb, a specific fungicide substance with a new mode of action. Scavengers like ..cap alpha..-tocopherol and piperonyl butoxide inhibit the toxic effect of the investigated substances in various degrees. Results are discussed in regard to the effect of a lipid peroxidative and proteolytic attack in pesticides of unknown mechanism of action.

  5. Soluble CD146 boosts therapeutic effect of endothelial progenitors through proteolytic processing of short CD146 isoform.

    Science.gov (United States)

    Stalin, Jimmy; Harhouri, Karim; Hubert, Lucas; Garrigue, Philippe; Nollet, Marie; Essaadi, Amel; Muller, Alexandre; Foucault-Bertaud, Alexandrine; Bachelier, Richard; Sabatier, Florence; Pisano, Pascale; Peiretti, Franck; Leroyer, Aurélie S; Guillet, Benjamin; Bardin, Nathalie; Dignat-George, Françoise; Blot-Chabaud, Marcel

    2016-08-01

    Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism. We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146. These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  6. The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Burri, Dominique J.; Pasquato, Antonella; Ramos da Palma, Joel [Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne CH-1011 (Switzerland); Igonet, Sebastien; Oldstone, Michael B.A. [Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037 (United States); Kunz, Stefan, E-mail: Stefan.Kunz@chuv.ch [Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne CH-1011 (Switzerland)

    2013-02-05

    Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

  7. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

  8. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  9. Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome.

    Science.gov (United States)

    Lázaro, Silvia; Gamarra, David; Del Val, Margarita

    2015-12-01

    Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

    Directory of Open Access Journals (Sweden)

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

  11. Dendritic Cells and Their Role in Allergy: Uptake, Proteolytic Processing and Presentation of Allergens

    Directory of Open Access Journals (Sweden)

    Piotr Humeniuk

    2017-07-01

    Full Text Available Dendritic cells (DCs are the most important antigen presenting cells to activate naïve T cells, which results in the case of Type 1 allergies in a Type 2 helper T cell (Th2-driven specific immune response towards allergens. So far, a number of different subsets of specialized DCs in different organs have been identified. In the recent past methods to study the interaction of DCs with allergenic proteins, their different uptake and processing mechanisms followed by the presentation to T cells were developed. The following review aims to summarize the most important characteristics of DC subsets in the context of allergic diseases, and highlights the recent findings. These detailed studies can contribute to a better understanding of the pathomechanisms of allergic diseases and contribute to the identification of key factors to be addressed for therapeutic interventions.

  12. A Conserved Biogenesis Pathway for Nucleoporins: Proteolytic Processing of a 186-Kilodalton Precursor Generates Nup98 and the Novel Nucleoporin, Nup96

    Science.gov (United States)

    Fontoura, Beatriz M.A.; Blobel, Günter; Matunis, Michael J.

    1999-01-01

    The mammalian nuclear pore complex (NPC) is comprised of ∼50 unique proteins, collectively known as nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals. PMID:10087256

  13. Proteolytic activity of protease produced by Pseudomonas fluorescens IB 2312 in skimmed milk subject to the process of high pressure homogenization

    Directory of Open Access Journals (Sweden)

    Claudia Regina Gonçalves Pinho

    2014-08-01

    Full Text Available The presence of thermoresistant proteases produced by psychrotrophic microorganisms have been identified as a limiting factor of the UHT milk shelflife, causing undesirable changes in milk products. High pressure homogenization (HPH processing is a non-thermal method of food preservation, able to promotes the microbiological safety and inactivation of some enzymes. Thus, this work assessed the proteolytic activity of protease produced by Pseudomonas fluorescens in skim milk subjected to high pressure homogenization process. The milk samples were added by the protease enzymatic extract (10% v/v and subjected to pressures up to 300 MPa. The assays showed that pressures on the order of 300 MPa caused a 72.5% reduction in proteolytic activity. Therefore, the process at high pressures resulted in significant inactivation of this thermoresistent enzyme, which possibly favors the shelf-life extension of the UHT milk and also limits the yield and quality loss of cheeses due to undesirable sensory changes in flavor and texture caused by this enzyme.

  14. Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum.

    Science.gov (United States)

    Brown, Janelle L; Tran-Dinh, Nai; Chapman, Belinda

    2012-04-01

    The putrefactive anaerobe Clostridium sporogenes PA 3679 has been widely used as a nontoxigenic surrogate for proteolytic Clostridium botulinum in the validation of thermal processes for low-acid shelf-stable foods, as a target organism in the derivation of thermal processes that reduce the risk of spoilage of such foods to an acceptable level, and as a research model for proteolytic strains of C. botulinum. Despite the importance of this organism, our knowledge of it has remained fragmented. In this article we draw together the literature associated with PA 3679 and discuss the identity of this organism, the phylogenetic relationships that exist between PA 3679 and various strains of C. sporogenes and proteolytic C. botulinum, the heat resistance characteristics of PA 3679, the advantages and limitations associated with its use in the derivation of thermal processing schedules, and the knowledge gaps and opportunities that exist with regard to its use as a research model for proteolytic C. botulinum. Phylogenetic analysis reviewed here suggests that PA 3679 is more closely related to various strains of proteolytic C. botulinum than to selected strains, including the type strain, of C. sporogenes. Even though PA 3679 is demonstrably nontoxigenic, the genetic basis of this nontoxigenic status remains to be elucidated, and the genetic sequence of this microorganism appears to be the key knowledge gap remaining to be filled. Our comprehensive review of comparative heat resistance data gathered for PA 3679 and proteolytic strains of C. botulinum over the past 100 years supports the practice of using PA 3679 as a (typically fail-safe) thermal processing surrogate for proteolytic C. botulinum.

  15. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    Science.gov (United States)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  16. The influence of x-radiation and hypoxia on the proteolytic activity of rat spleen in normal conditions and during neoplasm growth

    International Nuclear Information System (INIS)

    Likholat, E.A.; Reva, A.D.; Sokolov, I.I.; Chernaya, V.I.

    1991-01-01

    The influence of X-radiation on activity of lysosomic enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e. g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent

  17. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    International Nuclear Information System (INIS)

    Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

    1989-01-01

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

  18. Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

    Science.gov (United States)

    Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2015-03-01

    Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. © 2014 John Wiley & Sons Ltd.

  19. Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation.

    Science.gov (United States)

    Dzyuba, Viktoriya; Słowińska, Mariola; Cosson, Jacky; Ciereszko, Andrzej; Boryshpolets, Sergii; Štĕrba, Ján; Rodina, Marek; Linhart, Otomar; Dzyuba, Borys

    2016-12-01

    In sturgeon, the acquisition of the potential for motility activation called spermatozoon maturation takes place outside testes. This process can be accomplished in vitro by pre-incubation of immature testicular spermatozoa in seminal fluid collected from fully mature Wolffian duct sperm. Addition of trypsin inhibitor to the pre-incubation medium disrupts spermatozoon maturation. There are no available data for the role of proteolysis regulators in fish spermatozoon maturation, while their role is recognized in mammalian sperm maturation. The present study evaluated the involvement of seminal fluid proteases and anti-proteolytic activity in the sterlet spermatozoon maturation process. Casein and gelatin zymography and quantification of amidase and anti-proteolytic activity were conducted in sturgeon seminal fluid from Wolffian duct sperm and seminal fluid from testicular sperm, along with spermatozoon extracts from Wolffian duct spermatozoa, testicular spermatozoa, and testicular spermatozoa after in vitro maturation. We did not find significant differences in proteolytic profiles of seminal fluids from Wolffian duct sperm and ones from testicular sperm. Zymography revealed differences in spermatozoon extracts: Wolffian duct spermatozoon extracts were characterized by the presence of a broad proteolytic band ranging from 48 to 41 kDa, while testicular spermatozoon extracts did not show such activity until after in vitro maturation. The differences in amidase activity coincided with these results. It may not be the levels of proteolytic and anti-proteolytic activity per se, but the alterations in their interactions triggering a cascade of signaling events, that is crucial to the maturation process.

  20. Comparative Studies Of The Proteolytic And The Milk Clotting ...

    African Journals Online (AJOL)

    The use of enzymes as catalysts for industrial processes is becoming increasingly widespread, particularly in the food industry. The proteolytic and milk clotting activities of the crude latex of Calotropis procera, Carica papaya, and Musa paradisiaca were examined. The proteolytic activities of the crude latex were determined ...

  1. Defective Proteolytic Processing of Fibrillar Procollagens and Prodecorin Due to Biallelic BMP1 Mutations Results in a Severe, Progressive Form of Osteogenesis Imperfecta.

    Science.gov (United States)

    Syx, Delfien; Guillemyn, Brecht; Symoens, Sofie; Sousa, Ana Berta; Medeira, Ana; Whiteford, Margo; Hermanns-Lê, Trinh; Coucke, Paul J; De Paepe, Anne; Malfait, Fransiska

    2015-08-01

    Whereas the vast majority of osteogenesis imperfecta (OI) is caused by autosomal dominant defects in the genes encoding type I procollagen, mutations in a myriad of genes affecting type I procollagen biosynthesis or bone formation and homeostasis have now been associated with rare autosomal recessive OI forms. Recently, homozygous or compound heterozygous mutations in BMP1, encoding the metalloproteases bone morphogenetic protein-1 (BMP1) and its longer isoform mammalian Tolloid (mTLD), were identified in 5 children with a severe autosomal recessive form of OI and in 4 individuals with mild to moderate bone fragility. BMP1/mTLD functions as the procollagen carboxy-(C)-proteinase for types I to III procollagen but was also suggested to participate in amino-(N)-propeptide cleavage of types V and XI procollagens and in proteolytic trimming of other extracellular matrix (ECM) substrates. We report the phenotypic characteristics and natural history of 4 adults with severe, progressive OI characterized by numerous fractures, short stature with rhizomelic shortening, and deformity of the limbs and variable kyphoscoliosis, in whom we identified novel biallelic missense and frameshift mutations in BMP1. We show that BMP1/mTLD-deficiency in humans not only results in delayed cleavage of the type I procollagen C-propeptide but also hampers the processing of the small leucine-rich proteoglycan prodecorin, a regulator of collagen fibrillogenesis. Immunofluorescent staining of types I and V collagen and transmission electron microscopy of the dermis show impaired assembly of heterotypic type I/V collagen fibrils in the ECM. Our study thus highlights the severe and progressive nature of BMP1-associated OI in adults and broadens insights into the functional consequences of BMP1/mTLD-deficiency on ECM organization. © 2015 American Society for Bone and Mineral Research.

  2. Proteolytic activities in yeast.

    Science.gov (United States)

    Saheki, T; Holzer, H

    1975-03-28

    Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.

  3. Normalized Temperature Contrast Processing in Flash Infrared Thermography

    Science.gov (United States)

    Koshti, Ajay M.

    2016-01-01

    The paper presents further development in normalized contrast processing of flash infrared thermography method by the author given in US 8,577,120 B1. The method of computing normalized image or pixel intensity contrast, and normalized temperature contrast are provided, including converting one from the other. Methods of assessing emissivity of the object, afterglow heat flux, reflection temperature change and temperature video imaging during flash thermography are provided. Temperature imaging and normalized temperature contrast imaging provide certain advantages over pixel intensity normalized contrast processing by reducing effect of reflected energy in images and measurements, providing better quantitative data. The subject matter for this paper mostly comes from US 9,066,028 B1 by the author. Examples of normalized image processing video images and normalized temperature processing video images are provided. Examples of surface temperature video images, surface temperature rise video images and simple contrast video images area also provided. Temperature video imaging in flash infrared thermography allows better comparison with flash thermography simulation using commercial software which provides temperature video as the output. Temperature imaging also allows easy comparison of surface temperature change to camera temperature sensitivity or noise equivalent temperature difference (NETD) to assess probability of detecting (POD) anomalies.

  4. Normalized Temperature Contrast Processing in Infrared Flash Thermography

    Science.gov (United States)

    Koshti, Ajay M.

    2016-01-01

    The paper presents further development in normalized contrast processing used in flash infrared thermography method. Method of computing normalized image or pixel intensity contrast, and normalized temperature contrast are provided. Methods of converting image contrast to temperature contrast and vice versa are provided. Normalized contrast processing in flash thermography is useful in quantitative analysis of flash thermography data including flaw characterization and comparison of experimental results with simulation. Computation of normalized temperature contrast involves use of flash thermography data acquisition set-up with high reflectivity foil and high emissivity tape such that the foil, tape and test object are imaged simultaneously. Methods of assessing other quantitative parameters such as emissivity of object, afterglow heat flux, reflection temperature change and surface temperature during flash thermography are also provided. Temperature imaging and normalized temperature contrast processing provide certain advantages over normalized image contrast processing by reducing effect of reflected energy in images and measurements, therefore providing better quantitative data. Examples of incorporating afterglow heat-flux and reflection temperature evolution in flash thermography simulation are also discussed.

  5. Elastinolytic and proteolytic enzymes.

    Science.gov (United States)

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  6. Processing of Superconductor-Normal-Superconductor Josephson Edge Junctions

    Science.gov (United States)

    Kleinsasser, A. W.; Barner, J. B.

    1997-01-01

    The electrical behavior of epitaxial superconductor-normal-superconductor (SNS) Josephson edge junctions is strongly affected by processing conditions. Ex-situ processes, utilizing photoresist and polyimide/photoresist mask layers, are employed for ion milling edges for junctions with Yttrium-Barium-Copper-Oxide (YBCO) electrodes and primarily Co-doped YBCO interlayers.

  7. Does Normal Processing Provide Evidence of Specialised Semantic Subsystems?

    Science.gov (United States)

    Shapiro, Laura R.; Olson, Andrew C.

    2005-01-01

    Category-specific disorders are frequently explained by suggesting that living and non-living things are processed in separate subsystems (e.g. Caramazza & Shelton, 1998). If subsystems exist, there should be benefits for normal processing, beyond the influence of structural similarity. However, no previous study has separated the relative…

  8. MHC molecules protect T cell epitopes against proteolytic destruction

    DEFF Research Database (Denmark)

    Mouritsen, S; Meldal, M; Werdelin, O

    1992-01-01

    There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...... that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC...

  9. The Role of Prison Libraries in Normalization Process

    Directory of Open Access Journals (Sweden)

    Güler Demir

    2013-11-01

    Full Text Available In the modern sense of punishment, notpunishing but restoring should be at the forefront. Thus, the aim is to help theprisoner overcome theperiod of incarceration with minimum damage and to transform this process into a constructive one as far as possible. At this point, the “normalization principle” is funda- mental which is adopted and emphasized by developed countries. The normalization principle requires the setting of conditions of the institution so as to resemble the regular circumstances in the society and the ways of life, to which the incarcerated are accustomed, as closely as possible. The hypothesis of this study is that a qulifiedprison library service will improve the normalization process in prison. Alt- hough prison libraries are negatively affected by the problems that prisons face and although security consideration creates barriers to the normalization process, prison libraries are the places where the incarceratedfeel free and closest to the outside world. In the world, especially in the USA, UK, Canada and Scandinavian countries, an effort to develop library services in prisons has been made since these are believed to contribute to the normalization process. Finally, the study indicates that prison libraries in Turkey don ’t receive enough support and that there are many deficiencies in this field. Hence the study concludes that many issues remain to be resolved in this regard.

  10. Research Applications of Proteolytic Enzymes in Molecular Biology

    OpenAIRE

    Mótyán, János András; Tóth, Ferenc; Tőzsér, József

    2013-01-01

    Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications ...

  11. Effect of normal processes on thermal conductivity of germanium ...

    Indian Academy of Sciences (India)

    Abstract. The effect of normal scattering processes is considered to redistribute the phonon momentum in (a) the same phonon branch – KK-S model and (b) between differ- ent phonon branches – KK-H model. Simplified thermal conductivity relations are used to estimate the thermal conductivity of germanium, silicon and ...

  12. Effect of normal processes on thermal conductivity of germanium ...

    Indian Academy of Sciences (India)

    The effect of normal scattering processes is considered to redistribute the phonon momentum in (a) the same phonon branch – KK-S model and (b) between different phonon branches – KK-H model. Simplified thermal conductivity relations are used to estimate the thermal conductivity of germanium, silicon and diamond ...

  13. Effect of normal processes on thermal conductivity of germanium ...

    Indian Academy of Sciences (India)

    Phonon dispersion; phonon Boltzmann equation; lattice thermal conductivity; normal process; relaxation time; redistribution of phonon momentum. ... Department of Physics, Icfai Tech., ICFAI University, P.O. Kamalghat, Agartala 799 210, India; Department of Physics and Meteorology, Indian Institute of Technology, ...

  14. Research Applications of Proteolytic Enzymes in Molecular Biology

    Directory of Open Access Journals (Sweden)

    József Tőzsér

    2013-11-01

    Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

  15. Proteolytic antibodies in the sera of pregnant women: a case control study

    Directory of Open Access Journals (Sweden)

    Rahimzadeh jahromi M, Mirshahi M, Shamsipour F, Mohamadi M

    2008-07-01

    Full Text Available "n Normal 0 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} Background: The induction of catalytic antibodies (abzymes was first postulated by Pauling in 1948. Various catalytic antibodies have been detected recently in the sera of patients with several autoimmune pathologies such as systemic lupus erythematosus and rheumatoid arthritis. In addition, antibodies with DNase and RNase activity have been discovered in the milk and sera of healthy human mothers, which shows the physiologic role of these antibodies. In this study, we examined the proteolytic activity of antibodies in the sera of pregnant women. "n"nMethods: IgG antibody fractions were isolated from the sera of 30 healthy pregnant women in the first trimester of pregnancy and 10 control samples (men and nonpregnant women by subsequent steps of chromatographic purification on Protein G sepharose and sephacryl S-300. All patients were in their first pregnancy and aged 25-35 years. The conditions for proteolytic activity, such as type of buffer, pH and temperature, were optimized. The proteolytic activity of these antibodies was demonstrated by in-gel assay with gelatin as the substrate. "n"nResults: Antibody treatments at the optimum temperature showed that some samples from pregnant women contain proteolytic abzymes, as demonstrated by in-gel assays. Western blot results confirmed that the proteolytic activity is an intrinsic property of the antibodies. "n"nConclusions: During pregnancy and immediately after delivery women very often experience autoimmune processes similar to

  16. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  17. Normalization.

    Science.gov (United States)

    Cuevas, Eduardo J.

    1997-01-01

    Discusses cornerstone of Montessori theory, normalization, which asserts that if a child is placed in an optimum prepared environment where inner impulses match external opportunities, the undeviated self emerges, a being totally in harmony with its surroundings. Makes distinctions regarding normalization, normalized, and normality, indicating how…

  18. Understanding the work of telehealth implementation using Normalization Process Theory

    OpenAIRE

    Morrison, Janet Gwyneth

    2014-01-01

    This dissertation uses the theoretical constructs of Normalization Process Theory (NPT) to examine the successful implementation of an innovative telehealth service that delivers occupational health nursing services to a large healthcare employee population over a wide geographic area. Telehealth services have come to be regarded as a possible means to improve access to health care services, clinical efficiency, and cost effectiveness in an era where there are shrinking resources and growing ...

  19. Lactobacillus helveticus: the proteolytic system

    Directory of Open Access Journals (Sweden)

    Mansel W Griffiths

    2013-03-01

    Full Text Available Lactobacillus helveticus is one of the species of lactic acid bacteria most commonly used in the production of fermented milk beverages and some types of hard cheese. The versatile nature of this bacterium is based on its highly efficient proteolytic system consisting of cell-envelope proteinases, transport system and intracellular peptidases. Besides the traditional use of L. helveticus in the manufacture of hard cheeses, the production of fermented milk preparations with health promoting properties has become an important industrial application. Studies have shown that fermented dairy products are able to decrease blood pressure, stimulate the immune system, promote calcium absorption, and exert an anti-virulent effect against pathogens. These beneficial effects are produced by a variety of peptides released during the hydrolysis of milk proteins by the proteolytic system of L. helveticus, which provides the bacterium with its nutritional requirements for growth. In recent years, studies have focused on understanding the factors that affect the kinetics of milk protein hydrolysis by specific strains and have concentrated on the effect of pH, temperature, growth phase, and matrix composition on the bacterial enzymatic system. This review focuses on the role of the proteolytic system of L. helveticus in the production of bioactive compounds formed during fermentation of dairy products. Taking advantage of the powerful proteolytic system of this bacterium opens up future opportunities to search for novel food-derived compounds with potential health promoting properties

  20. Factors affecting proteolytic action of Lactococcus lactis in cheese

    NARCIS (Netherlands)

    Youssef, Y.B.

    1992-01-01

    Model cheeses were developed to study the behaviour of proteolytic agents involved in cheese maturation under conditions that closely resemble those in normal cheese. The models were applied to study protein breakdown by Lactococcus lactis ssp. cremoris HP , as a

  1. Characterization of diffusion processes: Normal and anomalous regimes

    Science.gov (United States)

    Alves, Samuel B.; de Oliveira, Gilson F.; de Oliveira, Luimar C.; Passerat de Silans, Thierry; Chevrollier, Martine; Oriá, Marcos; de S. Cavalcante, Hugo L. D.

    2016-04-01

    Many man-made and natural processes involve the diffusion of microscopic particles subject to random or chaotic, random-like movements. Besides the normal diffusion characterized by a Gaussian probability density function, whose variance increases linearly in time, so-called anomalous-diffusion regimes can also take place. They are characterized by a variance growing slower (subdiffusive) or faster (superdiffusive) than normal. In fact, many different underlying processes can lead to anomalous diffusion, with qualitative differences between mechanisms producing subdiffusion and mechanisms resulting in superdiffusion. Thus, a general description, encompassing all three regimes and where the specific mechanisms of each system are not explicit, is desirable. Here, our goal is to present a simple method of data analysis that enables one to characterize a model-less diffusion process from data observation, by observing the temporal evolution of the particle spread. To generate diffusive processes in different regimes, we use a Monte-Carlo routine in which both the step-size and the time-delay of the diffusing particles follow Pareto (inverse-power law) distributions, with either finite or diverging statistical momenta. We discuss on the application of this method to real systems.

  2. Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Olivia R. Buonarati

    2017-07-01

    Full Text Available Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014. Methods: To better determine the apparent molecular masses (MRof the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014 is at best minimal in brain tissue.

  3. Researches Concerning to Minimize Vibrations when Processing Normal Lathe

    Directory of Open Access Journals (Sweden)

    Lenuța Cîndea

    2015-09-01

    Full Text Available In the cutting process, vibration is inevitable appearance, and in situations where the amplitude exceeds the limits of precision dimensional and shape of the surfaces generated vibrator phenomenon is detrimental.Field vibration is an issue of increasingly developed, so the futures will a better understanding of them and their use even in other sectors.The paper developed experimental measurement of vibrations at the lathe machining normal. The scheme described kinematical machine tool, cutting tool, cutting conditions, presenting experimental facility for measuring vibration occurring at turning. Experimental results have followed measurement of amplitude, which occurs during interior turning the knife without silencer incorporated. The tests were performed continuously for different speed, feed and depth of cut.

  4. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    Science.gov (United States)

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  5. Working Memory Processing In Normal Subjects and Subjects with Dyslexia

    Science.gov (United States)

    Bowyer, S. M.; Lajiness-O'Neill, R.; Weiland, B. J.; Mason, K.; Tepley, N.

    2004-10-01

    Magnetoencephalography (MEG) was used to determine the neuroanatomical location of working memory (WM) processes. Differences between subjects with dyslexia (SD; n=5) and normal readers (NR; n=5) were studied during two WM tasks. A spatial WM task (SMW) consisted of blocks visually presented in one of 12 positions for 2 s each. Subjects were to determine if the current position matched the position presented 2 slides earlier (N-Back Test). The verbal task (VMW) consisted of presentation of a single letter. The location of cortical activity during SWM in NR (determined with MR-FOCUSS analysis) was in the right superior temporal gyrus (STG) and right angular gyrus (AG). Similar activation was seen in SD with a slight delay of approximately 20 ms. During VWM activity was seen in LEFT STG and LEFT AG in NR. In contrast for SD, activation was in the RIGHT STG and RIGHT AG. This study demonstrates the possibility to differentiate WM processing in subjects with and without learning disorders.

  6. Improving the normalization of complex interventions: measure development based on normalization process theory (NoMAD): study protocol

    OpenAIRE

    Finch, Tracy L; Rapley, Tim; Girling, Melissa; Mair, Frances S; Murray, Elizabeth; Treweek, Shaun; McColl, Elaine; Steen, Ian Nicholas; May, Carl R

    2013-01-01

    Background Understanding implementation processes is key to ensuring that complex interventions in healthcare are taken up in practice and thus maximize intended benefits for service provision and (ultimately) care to patients. Normalization Process Theory (NPT) provides a framework for understanding how a new intervention becomes part of normal practice. This study aims to develop and validate simple generic tools derived from NPT, to be used to improve the implementation of complex healthca...

  7. A qualitative systematic review of studies using the normalization process theory to research implementation processes

    OpenAIRE

    McEvoy, Rachel; Ballini, Luciana; Maltoni, Susanna; O’Donnell, Catherine A; Mair, Frances S; MacFarlane, Anne

    2014-01-01

    Background There is a well-recognized need for greater use of theory to address research translational gaps. Normalization Process Theory (NPT) provides a set of sociological tools to understand and explain the social processes through which new or modified practices of thinking, enacting, and organizing work are implemented, embedded, and integrated in healthcare and other organizational settings. This review of NPT offers readers the opportunity to observe how, and in what areas, a particul...

  8. The proteolytic system of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmundus Richardus Stephanus

    1997-01-01

    The bacterium Lactococcus lactis usues an extencive proteolytic system to utilize milk proteins (caseins) in orde to meet its need for amino acids. The genetic and biochemical properties of the putative components of the proteolytic pathway are well-described. However, little is known about the role

  9. Proteolytic enzymes of lactic acid bacteria

    NARCIS (Netherlands)

    Law, J; Haandrikman, A

    The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the

  10. Investigations of plant latices with regard to proteolytic activity and the influence on the Interleukin-6 secretion in monocytic cells

    OpenAIRE

    Domsalla, André

    2012-01-01

    Proteolytic enzymes have important functions in physiological and pathophysiological processes, and are frequently used in industrial processes. The search for new proteases with different specificities is always on. In the present work 311 plants from 91 genera from 11 plant families were examined for the presence of proteolytic activity in the latex. 75% of the studied plants were tested positive for the presence of proteolytic activity. Regarding the strength of the activity the families o...

  11. New radionuclide method for determination of gastric content proteolytic activity. (Experiments)

    International Nuclear Information System (INIS)

    Sergienko, V.B.; Popova, L.V.; Khodarev, N.N.; Astashenkova, K.Yu.

    1988-01-01

    A possibility of the use of a radionuclide tubeless rapid method for measuring gastric content proteolytic activity (GCPA) with the help of a protein (gelatin) RP containing capsule was demonstrated in experiments in vitro. There was correlation between the time of dissolution of RP containing capsules and GCPA determined after Mett's method. Reference time intervals were established for normal, raised and lowered proteolytic activity. The method was shown to be physiological, simple and timesaving

  12. Measurements of Normal and Friction Forces in a Rolling Process

    DEFF Research Database (Denmark)

    Henningsen, Poul; Arentoft, Mogens; Wanheim, Tarras

    2004-01-01

    To improve the quality of frictional data and to validate the simulations in rolling, a load transducer for measuring normal and friction stresses in the deformation zone has been developed. The transducer consists of a strain gauge equipped insert embedded in the surface of the roll. The length...... of the insert exceeds the contact length. By analyzing the output from the insert, the friction stress and normal pressure in the contact zone can be determined. The new concept differs from existing pin designs by a lower disturbance of lubricant film and material flow and limited penetration of material...

  13. Measurements of normal and frictional forces in a rolling process

    DEFF Research Database (Denmark)

    Henningsen, Poul; Arentoft, Mogens; Wanheim, Tarras

    2006-01-01

    of the insert exceeds the contact length. By analysing the output from the insert, the frictional stress and normal pressure in the contact zone can be determined. The new concept differs from existing pin designs by less disturbance of lubricant film and material flow and limited penetration of material......To improve the quality of frictional data and to validate the simulations in rolling, a load transducer for measuring normal and frictional stresses in the deformation zone has been developed. The transducer consists of a strain-gauge-equipped insert embedded in the surface of the roll. The length...

  14. The quality of low lactose milk is affected by the side proteolytic activity of the lactase used in the production process

    NARCIS (Netherlands)

    Troise, Antonio Dario; Bandini, Enrica; Donno, De Roberta; Meijer, Geert; Trezzi, Marco; Fogliano, Vincenzo

    2016-01-01

    Lactose intolerance syndrome can be efficiently tackled consuming low lactose products. Lactase is the key tool to manufacture low lactose milk (LLM): its addition during milk processing can be done in batch, i.e. before thermal treatment, or directly in pack after sterilization. In this paper

  15. Measurement of Normal and Friction Forces in a Rolling Process

    DEFF Research Database (Denmark)

    Henningsen, Poul; Arentoft, Mogens; Wanheim, Tarras

    2004-01-01

    by the fric-tion conditions. To achieve this important informa-tion, measurements of the normal pressure and friction stresses in the deformation zone are re-quested. The direction of the friction stresses is changing during the rolling gap. At the entrance of the de-formation zone, the peripherical velocity...... of the roll is higher than for the incoming material, which causes frictional stresses at the material acting in the rolling direction. At the outlet of the rolling gap, the velocity of the deformed material exceeds the velocity of the roll, generating frictional stresses contrary to the direction of rolling...

  16. NORMAL PRESSURE AND FRICTION STRESS MEASUREMENT IN ROLLING PROCESSES

    DEFF Research Database (Denmark)

    Henningsen, Poul; Arentoft, Mogens; Lagergren, Jonas

    2005-01-01

    the output from the transducer, the friction stress and normal pressure in the contact zone can be determined. The new concept differs from existing pin designs by a lower disturbance of lubricant film and material flow and limited penetration of material between transducer and roll. Aluminum, cupper...... and steel strips with a width of 40 mm was rolled with reduction varying from 2.7% to 29%, in a pilot mill. For evaluating the transducer, the measured contact forces are compared with external measurements of roll separating forces and torque. The determined friction coefficients are compared with values...

  17. Analysis of Proteolytic and Fibrinolytic Activity of the Blood Plasma in Patients with Bronchial Asthma Combined with Obesity

    Directory of Open Access Journals (Sweden)

    Fediv A.I. Fediv A.I.

    2016-09-01

    Full Text Available Aim of the study — to conduct the analysis of the performance of proteolytic and fibrinolytic activity of the blood plasma in patients with bronchial asthma (BA combined with obesity. Materials and methods. The study included 40 patients divided into groups. The basic group consisted of 20 patients with BA associated with obesity (ІI group, two comparison groups — of 10 patients with BA and normal body weight (I group and 10 patients with obesity and without pathology of the bronchopulmonary system (ІІІ group. Control group included 10 apparently healthy persons. In patients with BA, there were investigated the indexes of proteolytic and fibrinolytic activity of the blood plasma. Results. Analysis of the obtained data in patients with BA showed an increase of activity of plasma coagulation factors: total fibrinolytic activity increased in comparison to the control group, and in patients with BA it was 1.46 ± 0.13 Е440/ml/h, while in apparently healthy persons — 1.23 ± 0.16 Е440/ml/h (р < 0.05. In addition, an increase was marked and in the group of patients with the isolated obesity (by 38.5 % as compared to the group of apparently healthy persons, that can be explained by the growth of activity of inflammatory process due to the immune changes related to biological activity of fatty tissue as an additional source of proinflammatory cytokines. Conclusions. In combination of bronchial asthma and obesity, there is an activation of fibrinolytic and proteolytic systems of the blood that leads to microcirculatory and hemostatic disorders, enhancement of inflammatory process. In obesity as the state of systemic inflammatory response, the activation of fibrinolytic and proteolytic blood systems is more significant, than in bronchial asthma.

  18. Emotion processes in normal and abnormal development and preventive intervention.

    Science.gov (United States)

    Izard, Carroll E; Fine, Sarah; Mostow, Allison; Trentacosta, Christopher; Campbell, Jan

    2002-01-01

    We present an analysis of the role of emotions in normal and abnormal development and preventive intervention. The conceptual framework stems from three tenets of differential emotions theory (DET). These principles concern the constructs of emotion utilization; intersystem connections among modular emotion systems, cognition, and action; and the organizational and motivational functions of discrete emotions. Particular emotions and patterns of emotions function differentially in different periods of development and in influencing the cognition and behavior associated with different forms of psychopathology. Established prevention programs have not emphasized the concept of emotion as motivation. It is even more critical that they have generally neglected the idea of modulating emotions, not simply to achieve self-regulation, but also to utilize their inherently adaptive functions as a means of facilitating the development of social competence and preventing psychopathology. The paper includes a brief description of a theory-based prevention program and suggestions for complementary targeted interventions to address specific externalizing and internalizing problems. In the final section, we describe ways in which emotion-centered preventions can provide excellent opportunities for research on the development of normal and abnormal behavior.

  19. Effect of normal processes on thermal conductivity of germanium ...

    Indian Academy of Sciences (India)

    and other expressions as given in table 1. Here, the effective relaxation time τα has the second term taking care of the. N-processes, which can result either from intra (Simon mechanism) or inter (Her- ring mechanism) branch redistribution of phonons. For Simon mechanism eq. (1) reduces to Callaway's thermal conductivity ...

  20. Inhibition of proteolytic ferments by taurin in radiation sickness

    International Nuclear Information System (INIS)

    Dokshina, G.A.; Klimova, A.D.; Yartsev, E.I.; Yakovlev, V.G.

    1975-01-01

    The application of taurin potassium phosphate (TKPh) as radioprotective remedy resulted in a considerable influence on the activity of the proteolytic ferments in the irradiated organism. White male rats with a weight of 160 to 180 g being kept in cages without any food for 12 hours before this experiment were irradiated with a gamma unit GUM-Co-60 with a dose of 700 rad (LDsub(70/30)). A 4% solution of the preparation was injected in an amount of 40 mg per rat 1, 3, 5, 7 days after irradiation. Under the effect of ionizing radiation there was a progredient increase of proteolytic ferment activity of liver and spleen which was detected already 30 min after irradiation with maximum rate of proteolysis on the 21st day. After injection of the preparation a two-phase reaction developed: on the 7th to 12th day an increased activity of cathepsins in the tissue and in the following time up to the 30th day of observation an inhibition of ferment activity was demonstrated. Simultaneously it was found that the radiation-induced corticosteroid level was prevented by the preparation. A similar effect was also shown by TKPh inhibiting proteolytic ferment activity in experiments with rats with preceding application of hydrocortisone in high doses. The obtained results permit the assumption that the radioprotective activity of taurin comes about by its effect in the direction of structural integrity of the cell membranes leading to a normalization of the hormonal and fermentative events

  1. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Directory of Open Access Journals (Sweden)

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  2. PROCESS CAPABILITY ESTIMATION FOR NON-NORMALLY DISTRIBUTED DATA USING ROBUST METHODS - A COMPARATIVE STUDY

    Directory of Open Access Journals (Sweden)

    Yerriswamy Wooluru

    2016-06-01

    Full Text Available Process capability indices are very important process quality assessment tools in automotive industries. The common process capability indices (PCIs Cp, Cpk, Cpm are widely used in practice. The use of these PCIs based on the assumption that process is in control and its output is normally distributed. In practice, normality is not always fulfilled. Indices developed based on normality assumption are very sensitive to non- normal processes. When distribution of a product quality characteristic is non-normal, Cp and Cpk indices calculated using conventional methods often lead to erroneous interpretation of process capability. In the literature, various methods have been proposed for surrogate process capability indices under non normality but few literature sources offer their comprehensive evaluation and comparison of their ability to capture true capability in non-normal situation. In this paper, five methods have been reviewed and capability evaluation is carried out for the data pertaining to resistivity of silicon wafer. The final results revealed that the Burr based percentile method is better than Clements method. Modelling of non-normal data and Box-Cox transformation method using statistical software (Minitab 14 provides reasonably good result as they are very promising methods for non - normal and moderately skewed data (Skewness <= 1.5.

  3. Imaging proteolytic activity in live cells and animal models.

    Directory of Open Access Journals (Sweden)

    Stefanie Galbán

    Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

  4. Improving the normalization of complex interventions: measure development based on normalization process theory (NoMAD): study protocol

    Science.gov (United States)

    2013-01-01

    Background Understanding implementation processes is key to ensuring that complex interventions in healthcare are taken up in practice and thus maximize intended benefits for service provision and (ultimately) care to patients. Normalization Process Theory (NPT) provides a framework for understanding how a new intervention becomes part of normal practice. This study aims to develop and validate simple generic tools derived from NPT, to be used to improve the implementation of complex healthcare interventions. Objectives The objectives of this study are to: develop a set of NPT-based measures and formatively evaluate their use for identifying implementation problems and monitoring progress; conduct preliminary evaluation of these measures across a range of interventions and contexts, and identify factors that affect this process; explore the utility of these measures for predicting outcomes; and develop an online users’ manual for the measures. Methods A combination of qualitative (workshops, item development, user feedback, cognitive interviews) and quantitative (survey) methods will be used to develop NPT measures, and test the utility of the measures in six healthcare intervention settings. Discussion The measures developed in the study will be available for use by those involved in planning, implementing, and evaluating complex interventions in healthcare and have the potential to enhance the chances of their implementation, leading to sustained changes in working practices. PMID:23578304

  5. TAT Peptide and Its Conjugates: Proteolytic Stability

    OpenAIRE

    Grunwald, Jacob; Rejtar, Tomas; Sawant, Rupa; Wang, Zhouxi; Torchilin, Vladimir P.

    2009-01-01

    The proteolytic cleavage of TATp, TATp-PEG1000-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG5000-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of hu...

  6. Evaluation of auditory processing and phonemic discrimination in children with normal and disordered phonological development.

    Science.gov (United States)

    Attoni, Tiago Mendonça; Quintas, Victor Gandra; Mota, Helena Bolli

    2010-01-01

    Auditory processing and phonemic discrimination are essential for communication. Retrospective. To evaluate auditory processing and phonemic discrimination in children with normal and disordered phonological development. An evaluation of 46 children was carried out: 22 had phonological disorders and 24 had normally developing speech. Diotic , monotic and dichotic tests were applied to assess auditory processing and a test to evaluate phonemic discrimination abilities. Cross-sectional, contemporary. The values of normally-developing children were within the normal range in all auditory processing tests; these children attained maximum phonemic discrimination test scores. Children with phonological disorders performed worse in the latter, and presented disordered auditory processing. Auditory processing and phonemic discrimination in children with phonological disorders are altered.

  7. A qualitative systematic review of studies using the normalization process theory to research implementation processes

    Science.gov (United States)

    2014-01-01

    Background There is a well-recognized need for greater use of theory to address research translational gaps. Normalization Process Theory (NPT) provides a set of sociological tools to understand and explain the social processes through which new or modified practices of thinking, enacting, and organizing work are implemented, embedded, and integrated in healthcare and other organizational settings. This review of NPT offers readers the opportunity to observe how, and in what areas, a particular theoretical approach to implementation is being used. In this article we review the literature on NPT in order to understand what interventions NPT is being used to analyze, how NPT is being operationalized, and the reported benefits, if any, of using NPT. Methods Using a framework analysis approach, we conducted a qualitative systematic review of peer-reviewed literature using NPT. We searched 12 electronic databases and all citations linked to six key NPT development papers. Grey literature/unpublished studies were not sought. Limitations of English language, healthcare setting and year of publication 2006 to June 2012 were set. Results Twenty-nine articles met the inclusion criteria; in the main, NPT is being applied to qualitatively analyze a diverse range of complex interventions, many beyond its original field of e-health and telehealth. The NPT constructs have high stability across settings and, notwithstanding challenges in applying NPT in terms of managing overlaps between constructs, there is evidence that it is a beneficial heuristic device to explain and guide implementation processes. Conclusions NPT offers a generalizable framework that can be applied across contexts with opportunities for incremental knowledge gain over time and an explicit framework for analysis, which can explain and potentially shape implementation processes. This is the first review of NPT in use and it generates an impetus for further and extended use of NPT. We recommend that in future

  8. Detection of proteolytic signatures for Parkinson's disease

    DEFF Research Database (Denmark)

    Jordal, Peter Lüttge; Dyrlund, Thomas F.; Winge, Kristian

    2016-01-01

    Aim: To investigate if idiopathic Parkinson's disease (IPD) is associated with distinct proteolytic signatures relative to non-neurodegenerative controls (NND) and patients with multiple system atrophy (MSA). Materials & methods: A subtiligase-based N-terminomics screening method was exploited...

  9. The proteolytic systems of lactic acid bacteria

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Mierau, Igor; Hagting, Anja; Poolman, Bert; Konings, Wil N.

    1996-01-01

    Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The

  10. Process convergence of self-normalized sums of i.i.d. random ...

    Indian Academy of Sciences (India)

    Keywords. Domain of attraction; process convergence; self-normalized sums; stable distributions. 1. Introduction ... (1) EX = 0 and X is in the domain of attraction of the normal law. (2) S[nt0]/Vn,2 → N(0, t0) for t0 ∈ (0, ...... [9] Feller W, An Introduction to Probability Theory and its Applications, vol. II (1966). (New York: Wiley).

  11. Normal and language-impaired children's use of reference: syntactic versus pragmatic processing.

    Science.gov (United States)

    Schelletter, Christina; Leinonen, Eeva

    2003-01-01

    The present study investigates children's syntactic and pragmatic processing when specifying referents presented in short video clips. Within Relevance theory, the assumption of 'optimal relevance' implies that utterances are intended to involve the least processing effort on the part of the listener. In the present context, lexically specified NPs are assumed to be more in line with optimal relevance than pronouns. Subjects were 48 normally developing children aged 3;4-8;10 and 30 SLI children aged 5;1-8;9, divided into a low and a normal MLU group. Children's responses were coded according to levels of pragmatic processing and syntactic positions. Normally developing children's referent specifications were found to be increasingly relevant with increasing age. Differences between SLI and normal children were only found for the low MLU group with SLI who used fewer pronouns than the younger children, thereby showing that syntactic limitations alone cannot account for children's specification of referents.

  12. Activation and proteolytic activity of the Treponema pallidum metalloprotease, pallilysin.

    Science.gov (United States)

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹⁹⁸A], HAXXH [E¹⁹⁹A], and HEXXA [H²⁰²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro

  13. Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin

    Science.gov (United States)

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E.

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain

  14. Cytochrome P450 ubiquitination: branding for the proteolytic slaughter?

    Science.gov (United States)

    Correia, Maria Almira; Sadeghi, Sheila; Mundo-Paredes, Eduardo

    2005-01-01

    The hepatic cytochromes P450 (P450s) are monotopic endoplasmic reticulum (ER)-anchored hemoproteins engaged in the enzymatic oxidation of a wide variety of endo- and xenobiotics. In the course of these reactions, the enzymes generate reactive O(2) species and/or reactive metabolic products that can attack the P450 heme and/or protein moiety and structurally and functionally damage the enzyme. The in vivo conformational unraveling of such a structurally damaged P450 signals its rapid removal via the cellular sanitation system responsible for the proteolytic disposal of structurally aberrant, abnormal, and/or otherwise malformed proteins. A key player in this process is the ubiquitin (Ub)-dependent 26S proteasome system. Accordingly, the structurally deformed P450 protein is first branded for recognition and proteolytic removal by the 26S proteasome with an enzymatically incorporated polyUb tag. P450s of the 3A subfamily such as the major human liver enzyme CYP3A4 are notorious targets for this process, and they represent excellent prototypes for the understanding of integral ER protein ubiquitination. Not all the participants in hepatic CYP3A ubiquitination and subsequent proteolytic degradation have been identified. The following discussion thus addresses the various known and plausible events and/or cellular participants involved in this multienzymatic P450 ubiquitination cascade, on the basis of our current knowledge of other eukaryotic models. In addition, because the detection of ubiquitinated P450s is technically challenging, the critical importance of appropriate methodology is also discussed.

  15. Overcoming the Problem of Embedding Change in Educational Organizations: A Perspective from Normalization Process Theory

    Science.gov (United States)

    Wood, Phil

    2017-01-01

    In this article, I begin by outlining some of the barriers which constrain sustainable organizational change in schools and universities. I then go on to introduce a theory which has already started to help explain complex change and innovation processes in health and care contexts, Normalization Process Theory. Finally, I consider what this…

  16. Log-normal distribution from a process that is not multiplicative but is additive.

    Science.gov (United States)

    Mouri, Hideaki

    2013-10-01

    The central limit theorem ensures that a sum of random variables tends to a Gaussian distribution as their total number tends to infinity. However, for a class of positive random variables, we find that the sum tends faster to a log-normal distribution. Although the sum tends eventually to a Gaussian distribution, the distribution of the sum is always close to a log-normal distribution rather than to any Gaussian distribution if the summands are numerous enough. This is in contrast to the current consensus that any log-normal distribution is due to a product of random variables, i.e., a multiplicative process, or equivalently to nonlinearity of the system. In fact, the log-normal distribution is also observable for a sum, i.e., an additive process that is typical of linear systems. We show conditions for such a sum, an analytical example, and an application to random scalar fields such as those of turbulence.

  17. The proteolytic system of lactic acid bacteria.

    Science.gov (United States)

    Mayo, B

    1993-12-01

    Lactic acid bacteria are widely used throughout the world, empirically or deliberately, in the manufacturing of several food and feed stuffs, including milk products (such as cheese, butter, yoghurt, buttermilk, etc.), fermented vegetables (pickles, olives and sauerkraut), sausages, sourdough bread and silage, due to their ability to convert sugars into lactic acid. Of these, dairy products are of outstanding economic importance. Starter cultures used in the dairy industry are mixtures of carefully selected lactic acid bacteria which are added to the milk to fulfil the desired fermentation. Dairy starter cultures must reach high densities in milk in order to produce lactic acid at the required rates for manufacturing. Under these conditions, amino acids supply becomes limitant due to their scarce concentration in milk and to the auxotrophies shown by many starter bacteria. This implies the necessity of a proteolytic system, able to degrade the most abundant protein in milk, casein, into assimilable amino acids and peptides. Casein degradation and utilization require the concerted action of proteinases, peptidases and amino acid and peptide uptake systems. This whole set of enzymes constitutes the proteolytic system. In this article an overview of the recent biochemical and genetic data on the proteolytic system of lactic acid bacteria will be presented.

  18. Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

    Science.gov (United States)

    Biziulevicius, Gediminas A

    2006-01-01

    Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

  19. MSPrep--summarization, normalization and diagnostics for processing of mass spectrometry-based metabolomic data.

    Science.gov (United States)

    Hughes, Grant; Cruickshank-Quinn, Charmion; Reisdorph, Richard; Lutz, Sharon; Petrache, Irina; Reisdorph, Nichole; Bowler, Russell; Kechris, Katerina

    2014-01-01

    Although R packages exist for the pre-processing of metabolomic data, they currently do not incorporate additional analysis steps of summarization, filtering and normalization of aligned data. We developed the MSPrep R package to complement other packages by providing these additional steps, implementing a selection of popular normalization algorithms and generating diagnostics to help guide investigators in their analyses. http://www.sourceforge.net/projects/msprep

  20. Effect of care management program structure on implementation: a normalization process theory analysis

    OpenAIRE

    Holtrop, Jodi Summers; Potworowski, Georges; Fitzpatrick, Laurie; Kowalk, Amy; Green, Lee A.

    2016-01-01

    Background Care management in primary care can be effective in helping patients with chronic disease improve their health status, however, primary care practices are often challenged with implementation. Further, there are different ways to structure care management that may make implementation more or less successful. Normalization process theory (NPT) provides a means of understanding how a new complex intervention can become routine (normalized) in practice. In this study, we used NPT to u...

  1. Proteolytic activity during senescence of plants

    Science.gov (United States)

    Huffaker, R. C.

    1990-01-01

    Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

  2. Process evaluation of discharge planning implementation in healthcare using normalization process theory

    OpenAIRE

    Nordmark, Sofi; Zingmark, Karin; Lindberg, Inger

    2016-01-01

    Background Discharge planning is a care process that aims to secure the transfer of care for the patient at transition from home to the hospital and back home. Information exchange and collaboration between care providers are essential, but deficits are common. A wide range of initiatives to improve the discharge planning process have been developed and implemented for the past three decades. However, there are still high rates of reported medical errors and adverse events related to failures...

  3. SOME LIMIT THEORY FOR THE SELF-NORMALIZED PERIODOGRAM OF STABLE PROCESSES

    NARCIS (Netherlands)

    KLUPPELBERG, C; MIKOSCH, T

    1994-01-01

    Let X(t) = Sigma(j)(infinity) = (-infinity) psi(j)Z(t-j) be a discrete moving average process based on i.i.d. random variables (Z(t),)(t epsilon Z) with common distribution function from the domain of normal attraction of a p-stable law (0

  4. Using natural language processing to improve biomedical concept normalization and relation mining

    NARCIS (Netherlands)

    N. Kang (Ning)

    2013-01-01

    textabstractThis thesis concerns the use of natural language processing for improving biomedical concept normalization and relation mining. We begin with introducing the background of biomedical text mining, and subsequently we will continue by describing a typical text mining pipeline, some key

  5. Process convergence of self-normalized sums of i.i.d. random ...

    Indian Academy of Sciences (India)

    Yn,2(·), i.e., for p = 2, holds iff α = 2 and identified the limiting process as a standard. Brownian motion in sup norm. Keywords. Domain of ... sample standard deviation in the Student's t statistic). The normalizing factor is random ... index of stability α which equals 2 (for definition, see §2). This paper is organized as follows.

  6. Development of a theory of implementation and integration: Normalization Process Theory.

    NARCIS (Netherlands)

    May, C.R.; Mair, F.; Finch, T.; Macfarlane, A.; Dowrick, C.; Treweek, S.; Rapley, T.; Ballini, L.; Ong, B.N.; Rogers, A.; Murray, E.; Elwyn, G.; Legare, F.; Gunn, J.; Montori, V.M.

    2009-01-01

    ABSTRACT: BACKGROUND: Theories are important tools in the social and natural sciences. The methods by which they are derived are rarely described and discussed. Normalization Process Theory explains how new technologies, ways of acting, and ways of working become routinely embedded in everyday

  7. Understanding the implementation of complex interventions in health care: the normalization process model

    Directory of Open Access Journals (Sweden)

    Rogers Anne

    2007-09-01

    Full Text Available Abstract Background The Normalization Process Model is a theoretical model that assists in explaining the processes by which complex interventions become routinely embedded in health care practice. It offers a framework for process evaluation and also for comparative studies of complex interventions. It focuses on the factors that promote or inhibit the routine embedding of complex interventions in health care practice. Methods A formal theory structure is used to define the model, and its internal causal relations and mechanisms. The model is broken down to show that it is consistent and adequate in generating accurate description, systematic explanation, and the production of rational knowledge claims about the workability and integration of complex interventions. Results The model explains the normalization of complex interventions by reference to four factors demonstrated to promote or inhibit the operationalization and embedding of complex interventions (interactional workability, relational integration, skill-set workability, and contextual integration. Conclusion The model is consistent and adequate. Repeated calls for theoretically sound process evaluations in randomized controlled trials of complex interventions, and policy-makers who call for a proper understanding of implementation processes, emphasize the value of conceptual tools like the Normalization Process Model.

  8. Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation

    Czech Academy of Sciences Publication Activity Database

    Dzyuba, V.; Słowińska, M.; Cosson, J.; Ciereszko, A.; Boryshpolets, S.; Štěrba, Ján; Rodina, M.; Linhart, O.; Dzyuba, B.

    2016-01-01

    Roč. 42, č. 6 (2016), s. 1755-1766 ISSN 0920-1742 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : amidase activity * anti-proteolytic activity * casein and gelatin zymography * siberian sturgeon sperm * sperm atozoon maturation * sterlet sperm Subject RIV: ED - Physiology Impact factor: 1.647, year: 2016

  9. Comparing Sensory Information Processing and Alexithymia between People with Substance Dependency and Normal.

    Science.gov (United States)

    Bashapoor, Sajjad; Hosseini-Kiasari, Seyyedeh Tayebeh; Daneshvar, Somayeh; Kazemi-Taskooh, Zeinab

    2015-01-01

    Sensory information processing and alexithymia are two important factors in determining behavioral reactions. Some studies explain the effect of the sensitivity of sensory processing and alexithymia in the tendency to substance abuse. Giving that, the aim of the current study was to compare the styles of sensory information processing and alexithymia between substance-dependent people and normal ones. The research method was cross-sectional and the statistical population of the current study comprised of all substance-dependent men who are present in substance quitting camps of Masal, Iran, in October 2013 (n = 78). 36 persons were selected randomly by simple randomly sampling method from this population as the study group, and 36 persons were also selected among the normal population in the same way as the comparison group. Both groups was evaluated by using Toronto alexithymia scale (TAS) and adult sensory profile, and the multivariate analysis of variance (MANOVA) test was applied to analyze data. The results showed that there are significance differences between two groups in low registration (P processing and difficulty in describing emotions (P process sensory information in a different way than normal people and show more alexithymia features than them.

  10. TAT peptide and its conjugates: proteolytic stability.

    Science.gov (United States)

    Grunwald, Jacob; Rejtar, Tomas; Sawant, Rupa; Wang, Zhouxi; Torchilin, Vladimir P

    2009-08-19

    The proteolytic cleavage of TATp, TATp-PEG(1000)-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG(5000)-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t(1/2) ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t(1/2) ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t(1/2) ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional "smart" drug delivery systems with detachable PEG.

  11. PMA: Protein Microarray Analyser, a user-friendly tool for data processing and normalization.

    Science.gov (United States)

    Da Gama Duarte, Jessica; Goosen, Ryan W; Lawry, Peter J; Blackburn, Jonathan M

    2018-02-27

    Protein microarrays provide a high-throughput platform to measure protein interactions and associated functions, and can aid in the discovery of cancer biomarkers. The resulting protein microarray data can however be subject to systematic bias and noise, thus requiring a robust data processing, normalization and analysis pipeline to ensure high quality and robust results. To date, a comprehensive data processing pipeline is yet to be developed. Furthermore, a lack of analysis consistency is evident amongst different research groups, thereby impeding collaborative data consolidation and comparison. Thus, we sought to develop an accessible data processing tool using methods that are generalizable to the protein microarray field and which can be adapted to individual array layouts with minimal software engineering expertise. We developed an improved version of a previously developed pipeline of protein microarray data processing and implemented it as an open source software tool, with particular focus on widening its use and applicability. The Protein Microarray Analyser software presented here includes the following tools: (1) neighbourhood background correction, (2) net intensity correction, (3) user-defined noise threshold, (4) user-defined CV threshold amongst replicates and (5) assay controls, (6) composite 'pin-to-pin' normalization amongst sub-arrays, and (7) 'array-to-array' normalization amongst whole arrays.

  12. Normal processes of phonon-phonon scattering and thermal conductivity of germanium crystals with isotopic disorder

    CERN Document Server

    Kuleev, I G

    2001-01-01

    The effect of normal processes of the phonon-phonon scattering on the thermal conductivity of the germanium crystals with various isotopic disorder degrees is considered. The phonon pulse redistribution in the normal scattering processes both inside each oscillatory branch (the Simons mechanism) and between various phonon oscillatory branches (the Herring mechanism) is accounted for. The contributions of the longitudinal and cross-sectional phonons drift motion into the thermal conductivity are analyzed. It is shown that the pulse redistribution in the Herring relaxation mechanism leads to essential suppression of the longitudinal phonons drift motion in the isotopically pure germanium crystals. The calculations results of thermal conductivity for the Herring relaxation mechanism agree well with experimental data on the germanium crystals with various isotopic disorder degrees

  13. Adaptive Convergence Rates of a Dirichlet Process Mixture of Multivariate Normals

    OpenAIRE

    Tokdar, Surya T.

    2011-01-01

    It is shown that a simple Dirichlet process mixture of multivariate normals offers Bayesian density estimation with adaptive posterior convergence rates. Toward this, a novel sieve for non-parametric mixture densities is explored, and its rate adaptability to various smoothness classes of densities in arbitrary dimension is demonstrated. This sieve construction is expected to offer a substantial technical advancement in studying Bayesian non-parametric mixture models based on stick-breaking p...

  14. Accident and Off-Normal Response and Recovery from Multi-Canister Overpack (MCO) Processing Events

    International Nuclear Information System (INIS)

    ALDERMAN, C.A.

    2000-01-01

    In the process of removing spent nuclear fuel (SNF) from the K Basins through its subsequent packaging, drymg, transportation and storage steps, the SNF Project must be able to respond to all anticipated or foreseeable off-normal and accident events that may occur. Response procedures and recovery plans need to be in place, personnel training established and implemented to ensure the project will be capable of appropriate actions. To establish suitable project planning, these events must first be identified and analyzed for their expected impact to the project. This document assesses all off-normal and accident events for their potential cross-facility or Multi-Canister Overpack (MCO) process reversal impact. Table 1 provides the methodology for establishing the event planning level and these events are provided in Table 2 along with the general response and recovery planning. Accidents and off-normal events of the SNF Project have been evaluated and are identified in the appropriate facility Safety Analysis Report (SAR) or in the transportation Safety Analysis Report for Packaging (SARP). Hazards and accidents are summarized from these safety analyses and listed in separate tables for each facility and the transportation system in Appendix A, along with identified off-normal events. The tables identify the general response time required to ensure a stable state after the event, governing response documents, and the events with potential cross-facility or SNF process reversal impacts. The event closure is predicated on stable state response time, impact to operations and the mitigated annual occurrence frequency of the event as developed in the hazard analysis process

  15. Normalization and gene p-value estimation: issues in microarray data processing.

    Science.gov (United States)

    Fundel, Katrin; Küffner, Robert; Aigner, Thomas; Zimmer, Ralf

    2008-05-28

    Numerous methods exist for basic processing, e.g. normalization, of microarray gene expression data. These methods have an important effect on the final analysis outcome. Therefore, it is crucial to select methods appropriate for a given dataset in order to assure the validity and reliability of expression data analysis. Furthermore, biological interpretation requires expression values for genes, which are often represented by several spots or probe sets on a microarray. How to best integrate spot/probe set values into gene values has so far been a somewhat neglected problem. We present a case study comparing different between-array normalization methods with respect to the identification of differentially expressed genes. Our results show that it is feasible and necessary to use prior knowledge on gene expression measurements to select an adequate normalization method for the given data. Furthermore, we provide evidence that combining spot/probe set p-values into gene p-values for detecting differentially expressed genes has advantages compared to combining expression values for spots/probe sets into gene expression values. The comparison of different methods suggests to use Stouffer's method for this purpose. The study has been conducted on gene expression experiments investigating human joint cartilage samples of osteoarthritis related groups: a cDNA microarray (83 samples, four groups) and an Affymetrix (26 samples, two groups) data set. The apparently straight forward steps of gene expression data analysis, e.g. between-array normalization and detection of differentially regulated genes, can be accomplished by numerous different methods. We analyzed multiple methods and the possible effects and thereby demonstrate the importance of the single decisions taken during data processing. We give guidelines for evaluating normalization outcomes. An overview of these effects via appropriate measures and plots compared to prior knowledge is essential for the biological

  16. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    Science.gov (United States)

    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

  17. Development of a theory of implementation and integration: Normalization Process Theory

    Directory of Open Access Journals (Sweden)

    May Carl R

    2009-05-01

    Full Text Available Abstract Background Theories are important tools in the social and natural sciences. The methods by which they are derived are rarely described and discussed. Normalization Process Theory explains how new technologies, ways of acting, and ways of working become routinely embedded in everyday practice, and has applications in the study of implementation processes. This paper describes the process by which it was built. Methods Between 1998 and 2008, we developed a theory. We derived a set of empirical generalizations from analysis of data collected in qualitative studies of healthcare work and organization. We developed an applied theoretical model through analysis of empirical generalizations. Finally, we built a formal theory through a process of extension and implication analysis of the applied theoretical model. Results Each phase of theory development showed that the constructs of the theory did not conflict with each other, had explanatory power, and possessed sufficient robustness for formal testing. As the theory developed, its scope expanded from a set of observed regularities in data with procedural explanations, to an applied theoretical model, to a formal middle-range theory. Conclusion Normalization Process Theory has been developed through procedures that were properly sceptical and critical, and which were opened to review at each stage of development. The theory has been shown to merit formal testing.

  18. Optical Imaging of Matrix Metalloproteinase-7 Activity in Vivo Using a Proteolytic Nanobeacon

    Directory of Open Access Journals (Sweden)

    Randy L. Scherer

    2008-05-01

    Full Text Available Matrix metalloproteinases (MMPs are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor and AF750 as an internal reference to monitor relative substrate concentration (reference. In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APCMin mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APCMin mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APCMin adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

  19. Milk contamination in different points of the dairy process. ii mesophilic, psychrotrophic and proteolytic microorganisms/ Contaminação do leite em diferentes pontos da produção leiteira: ii microrganismos mesófilos, psicrotróficos e proteolíticos

    Directory of Open Access Journals (Sweden)

    Viviane Vieira Gusmão

    2004-05-01

    Full Text Available Psychrotrophics microorganisms are able to multiply under refrigeration temperatures. They can produce heat-resistant extracellular enzymes that cause organoleptic and structural changes in milk and dairy products. With the objective of determining the frequency of microorganisms in the dairy process, four farms were evaluated in Londrina city – Brazil and different points were assessed. Mesophilic (48hs at 35oC, psychrotrophic (25hs at 21oC and proteolytic (72hs at 22oC microorganisms were researched, and the proteolytic predominant microflora were determined by using the Gram stain technique. The main psychrotrophic contamination points were the residual water on milk cans, on bulk tank and bad cleaned teats, and milk cans and teats were the most important sources of proteolytics. Gram negative bacilli represented 69% of the microorganisms in the refrigerated milk that means 45% of the total proteolytics microorganisms in the dairy process. Gram negative bacilli is most frequent on bad cleaned teats, teatcups, residual water in milk cans and bulk tank. The psychrotroph count in the refrigerated milk was more than the mesophilic count, showing that that psychrotrophics are the best indicatator for refrigerated milk contamination. The psycrotrophics count at all points assessed were greater than the ideal limit (10% of the mesophilic count. Thus as refrigeration at 4oC is not sufficient to control the multiplication of the psycrotrophics group, good practices should be associated to avoid or control milk contamination by psycrotrophics.Os psicrotróficos são microrganismos que têm capacidade de multiplicação em temperaturas de refrigeração. Produzem enzimas termorresistentes como proteases e lipases que promovem alterações organolépticas no leite e comprometem a produção de derivados. Com o objetivo de determinar a freqüência desses microrganismos a produção do leite, foram estudadas 04 propriedades da região de Londrina, PR

  20. Classification of normal and pathological aging processes based on brain MRI morphology measures

    Science.gov (United States)

    Perez-Gonzalez, J. L.; Yanez-Suarez, O.; Medina-Bañuelos, V.

    2014-03-01

    Reported studies describing normal and abnormal aging based on anatomical MRI analysis do not consider morphological brain changes, but only volumetric measures to distinguish among these processes. This work presents a classification scheme, based both on size and shape features extracted from brain volumes, to determine different aging stages: healthy control (HC) adults, mild cognitive impairment (MCI), and Alzheimer's disease (AD). Three support vector machines were optimized and validated for the pair-wise separation of these three classes, using selected features from a set of 3D discrete compactness measures and normalized volumes of several global and local anatomical structures. Our analysis show classification rates of up to 98.3% between HC and AD; of 85% between HC and MCI and of 93.3% for MCI and AD separation. These results outperform those reported in the literature and demonstrate the viability of the proposed morphological indexes to classify different aging stages.

  1. Exploring the process of inference generation in sarcasm: a review of normal and clinical studies.

    Science.gov (United States)

    McDonald, S

    1999-07-01

    This article evaluates two theoretical accounts of how sarcasm is understood; the traditional model, which asserts that listeners derive a counterfactual inference from the sarcastic comment, and relevance theory, which asserts that listeners recognize sarcasm as a scornful echo of a previous assertion. Evidence from normal speakers provides only partial support for both theories. Evidence from brain-injured populations suggests that aspects of the pragmatic process can be arrested in ways not predicted by either theory. It is concluded that sarcasm is more effortful to process than nonsarcastic comments and that inferences about the facts of the situation and the mental state of the speaker (e.g., attitudes, knowledge, and intentions) are important to comprehending sarcasm. It is questioned whether inferences about mental state are relatively more difficult for brain-injured subjects and, if so, whether this is a continuum of difficulty or reflects reliance upon different cognitive processes. Copyright 1999 Academic Press.

  2. Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Yu. Ya. Kit

    2014-10-01

    Full Text Available The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP, bovine serum albumin (BSA, and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  3. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    International Nuclear Information System (INIS)

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-01-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated

  4. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    Science.gov (United States)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

  5. IBUPROFEN AS A MEDICATION FOR A CORRECTION OF SYMPTOMS OF NORMAL VACCINAL PROCESS IN CHILDREN

    Directory of Open Access Journals (Sweden)

    T.A. Chebotareva

    2008-01-01

    Full Text Available The pathogenetic approach to treatment of symptoms of normal vaccinal process in children after standard vaccination, based on the results of application of anti9inflammatory medications — ibuprofen (nurofen for children and paracetamol is presented in this article. Clinical activity of ibuprofen was established on the basis of clinica catamnestic observation of 856 vaccinated children aged from 3 months to 3 years. recommendations for application of these medications as a treatment for a correction of vaccinal reactions are given.Key words: children, ibuprofen, paracetamol, vaccination.

  6. Effect of care management program structure on implementation: a normalization process theory analysis.

    Science.gov (United States)

    Holtrop, Jodi Summers; Potworowski, Georges; Fitzpatrick, Laurie; Kowalk, Amy; Green, Lee A

    2016-08-15

    Care management in primary care can be effective in helping patients with chronic disease improve their health status, however, primary care practices are often challenged with implementation. Further, there are different ways to structure care management that may make implementation more or less successful. Normalization process theory (NPT) provides a means of understanding how a new complex intervention can become routine (normalized) in practice. In this study, we used NPT to understand how care management structure affected how well care management became routine in practice. Data collection involved semi-structured interviews and observations conducted at 25 practices in five physician organizations in Michigan, USA. Practices were selected to reflect variation in physician organizations, type of care management program, and degree of normalization. Data were transcribed, qualitatively coded and analyzed, initially using an editing approach and then a template approach with NPT as a guiding framework. Seventy interviews and 25 observations were completed. Two key structures for care management organization emerged: practice-based care management where the care managers were embedded in the practice as part of the practice team; and centralized care management where the care managers worked independently of the practice work flow and was located outside the practice. There were differences in normalization of care management across practices. Practice-based care management was generally better normalized as compared to centralized care management. Differences in normalization were well explained by the NPT, and in particular the collective action construct. When care managers had multiple and flexible opportunities for communication (interactional workability), had the requisite knowledge, skills, and personal characteristics (skill set workability), and the organizational support and resources (contextual integration), a trusting professional relationship

  7. In situ end labelling: effect of proteolytic enzyme pretreatment and hydrochloric acid.

    Science.gov (United States)

    Carr, N. J.; Talbot, I. C.

    1997-01-01

    AIM: To determine the effect of dilute hydrochloric acid on the in situ end labelling (ISEL) reaction, with and without a variety of different proteolytic enzymes. METHODS: Sections of tissue fixed in buffered formalin were pretreated with trypsin, protease XIV (at two different concentrations), or protease XXIV (for two different incubation times), with and without subsequent 1 M hydrochloric acid treatment. The results were compared with those obtained using hydrochloric acid alone, with proteinase K, and pepsin pretreatment, and with no pretreatment. RESULTS: When hydrochloric acid was added to the sections in addition to trypsin, protease XIV, and protease XXIV, there was a significant increase in ISEL reactivity in both apoptotic nuclei and morphologically normal nuclei. Hydrochloric acid alone had no significant effect. CONCLUSIONS: Hydrochloric acid has a distinctive effect on the ISEL reaction that is dependent on prior proteolytic digestion. Images PMID:9292152

  8. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Directory of Open Access Journals (Sweden)

    Monica Salamone

    Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  9. Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk.

    Science.gov (United States)

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-11-02

    We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o -phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli , Staphylococcus aureus , Salmonella cholere enteridis , Listeria monocytogenes , Listeria innocua and Enterobacter aerogenes . The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus , which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes . The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

  10. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  11. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

  12. Temporal and speech processing skills in normal hearing individuals exposed to occupational noise

    Directory of Open Access Journals (Sweden)

    U Ajith Kumar

    2012-01-01

    Full Text Available Prolonged exposure to high levels of occupational noise can cause damage to hair cells in the cochlea and result in permanent noise-induced cochlear hearing loss. Consequences of cochlear hearing loss on speech perception and psychophysical abilities have been well documented. Primary goal of this research was to explore temporal processing and speech perception Skills in individuals who are exposed to occupational noise of more than 80 dBA and not yet incurred clinically significant threshold shifts. Contribution of temporal processing skills to speech perception in adverse listening situation was also evaluated. A total of 118 participants took part in this research. Participants comprised three groups of train drivers in the age range of 30-40 (n= 13, 41 50 ( = 13, 41-50 (n = 9, and 51-60 (n = 6 years and their non-noise-exposed counterparts (n = 30 in each age group. Participants of all the groups including the train drivers had hearing sensitivity within 25 dB HL in the octave frequencies between 250 and 8 kHz. Temporal processing was evaluated using gap detection, modulation detection, and duration pattern tests. Speech recognition was tested in presence multi-talker babble at -5dB SNR. Differences between experimental and control groups were analyzed using ANOVA and independent sample t-tests. Results showed a trend of reduced temporal processing skills in individuals with noise exposure. These deficits were observed despite normal peripheral hearing sensitivity. Speech recognition scores in the presence of noise were also significantly poor in noise-exposed group. Furthermore, poor temporal processing skills partially accounted for the speech recognition difficulties exhibited by the noise-exposed individuals. These results suggest that noise can cause significant distortions in the processing of suprathreshold temporal cues which may add to difficulties in hearing in adverse listening conditions.

  13. Embedding new technologies in practice - a normalization process theory study of point of care testing.

    Science.gov (United States)

    Jones, Caroline H D; Glogowska, Margaret; Locock, Louise; Lasserson, Daniel S

    2016-10-19

    Many point of care diagnostic technologies are available which produce results within minutes, and offer the opportunity to deliver acute care out of hospital settings. Increasing access to diagnostics at the point of care could increase the volume and scope of acute ambulatory care. Yet these technologies are not routinely used in many settings. We aimed to explore how point of care testing is used in a setting where it has become 'normalized' (embedded in everyday practice), in order to inform future adoption and implementation in other settings. We used normalization process theory to guide our case study approach. We used a single case study design, choosing a community based ambulatory care unit where point of care testing is used routinely. A focused ethnographic approach was taken, including non-participant observation of all activities related to point of care testing, and semi-structured interviews, with all clinical staff involved in point of care testing at the unit. Data were analysed thematically, guided by normalization process theory. Fourteen days of observation and six interviews were completed. Staff had a shared understanding of the purpose, value and benefits of point of care testing, believing it to be integral to the running of the unit. They organised themselves as a team to ensure that point of care testing worked effectively; and one key individual led a change in practice to ensure more consistency and trust in procedures. Staff assessed point of care testing as worthwhile for the unit, their patients, and themselves in terms of job satisfaction and knowledge. Potential barriers to adoption of point of care testing were evident (including lack of trust in the accuracy of some results compared to laboratory testing; and lack of ease of use of some aspects of the equipment); but these did not prevent point of care testing from becoming embedded, because the importance and value attributed to it were so strong. This case study offers insights

  14. Activation of human monocytes by proteolytic fragments of gliadin

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Lenka; Tučková, Ludmila; Cinová, Jana; Cimburek, Zdeněk; Tlaskalová, Helena

    2003-01-01

    Roč. 87, č. 1 (2003), s. 08 ISSN 0165-2478 Institutional research plan: CEZ:AV0Z5020903 Keywords : proteolytic * fragments * gliadin Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003

  15. ISOLATION OF PROTEOLYTIC PSYCHROTROPHIC YEASTS FROM FRESH RAW SEAFOODS

    NARCIS (Netherlands)

    KOBATAKE, M; KREGERVANRIJ, NJW; PLACIDO, MTLC; VANUDEN, N

    A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces. Rhodotorula, Sterigmatomyces and Trichosporon, and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic

  16. Proteolytic activity of alkaliphilic, salt-tolerant actinomycetes from ...

    African Journals Online (AJOL)

    tolerant alkaliphilic. All the isolates need to be further studied for the ability of their potential protease enzyme production. Key words: Alkaliphilic actinomycetes, salt tolerant actinomycetes, desert soil, isolation, proteolytic activity.

  17. [Inhibitors of proteolytic enzymes under abiotic stresses in plants (review)].

    Science.gov (United States)

    Mosolov, V V; Valueva, T A

    2011-01-01

    Data on the role of proteolytic enzyme inhibitors in plant adaptation to various unfavorable environmental abiotic factors--water deficiency, salinization of soil, extreme temperatures, etc.--and also probable functions of proteinases inhibitors in natural plant senescense are considered.

  18. Nonepileptic seizures under levetiracetam therapy: a case report of forced normalization process

    Directory of Open Access Journals (Sweden)

    Anzellotti F

    2014-05-01

    Full Text Available Francesca Anzellotti, Raffaella Franciotti, Holta Zhuzhuni, Aurelio D'Amico, Astrid Thomas, Marco Onofrj Department of Neuroscience and Imaging, Aging Research Centre, Gabriele d'Annunzio University Foundation, Gabriele d'Annunzio University, Chieti, Italy Abstract: Nonepileptic seizures (NES apparently look like epileptic seizures, but are not associated with ictal electrical discharges in the brain. NES constitute one of the most important differential diagnoses of epilepsy. They have been recognized as a distinctive clinical phenomenon for centuries, and video/electroencephalogram monitoring has allowed clinicians to make near-certain diagnoses. NES are supposedly unrelated to organic brain lesions, and despite the preponderance of a psychiatric/psychological context, they may have an iatrogenic origin. We report a patient with NES precipitated by levetiracetam therapy; in this case, NES was observed during the disappearance of epileptiform discharges from the routine video/electroencephalogram. We discuss the possible mechanisms underlying NES with regard to alternative psychoses associated with the phenomenon of the forced normalization process. Keywords: nonepileptic seizures, forced normalization, levetiracetam, behavioral side effects

  19. Nonepileptic seizures under levetiracetam therapy: a case report of forced normalization process.

    Science.gov (United States)

    Anzellotti, Francesca; Franciotti, Raffaella; Zhuzhuni, Holta; D'Amico, Aurelio; Thomas, Astrid; Onofrj, Marco

    2014-01-01

    Nonepileptic seizures (NES) apparently look like epileptic seizures, but are not associated with ictal electrical discharges in the brain. NES constitute one of the most important differential diagnoses of epilepsy. They have been recognized as a distinctive clinical phenomenon for centuries, and video/electroencephalogram monitoring has allowed clinicians to make near-certain diagnoses. NES are supposedly unrelated to organic brain lesions, and despite the preponderance of a psychiatric/psychological context, they may have an iatrogenic origin. We report a patient with NES precipitated by levetiracetam therapy; in this case, NES was observed during the disappearance of epileptiform discharges from the routine video/electroencephalogram. We discuss the possible mechanisms underlying NES with regard to alternative psychoses associated with the phenomenon of the forced normalization process.

  20. Fisher informations and local asymptotic normality for continuous-time quantum Markov processes

    Science.gov (United States)

    Catana, Catalin; Bouten, Luc; Guţă, Mădălin

    2015-09-01

    We consider the problem of estimating an arbitrary dynamical parameter of an open quantum system in the input-output formalism. For irreducible Markov processes, we show that in the limit of large times the system-output state can be approximated by a quantum Gaussian state whose mean is proportional to the unknown parameter. This approximation holds locally in a neighbourhood of size {t}-1/2 in the parameter space, and provides an explicit expression of the asymptotic quantum Fisher information in terms of the Markov generator. Furthermore we show that additive statistics of the counting and homodyne measurements also satisfy local asymptotic normality and we compute the corresponding classical Fisher informations. The general theory is illustrated with the examples of a two-level system and the atom maser. Our results contribute towards a better understanding of the statistical and probabilistic properties of the output process, with relevance for quantum control engineering, and the theory of non-equilibrium quantum open systems.

  1. The effect of state worry and trait anxiety on working memory processes in a normal sample.

    Science.gov (United States)

    Walkenhorst, Elizabeth; Crowe, Simon F

    2009-03-01

    This study investigated the effects of trait anxiety and state worry on working memory performance in a normal sample. Phase one investigated the effects of trait anxiety and state worry on the capacity of specific working memory components. Phase two investigated the validity of Eysenck and Calvo's (1992) Processing Efficiency Theory of worry. Sixty adult participants (40 females and 20 males with a mean age of 26 years) were assigned to a 2 (trait anxiety: Low vs. high)x2 (state worry: Low vs. high) between-subjects design. Contrary to prediction, worry did not lead to a decrement in performance on verbal working memory tasks but unexpectedly enhanced performance on visual tasks in participants with low trait anxiety (LTA). The results were also in opposition to expectations for Phase two. Individuals in the conditions of high trait anxiety and/or high state worry (LTA/HW, HTA/LW, and HTA/HW) displayed shorter response latencies than individuals in the LTA and low state worry (LTA/LW) condition on both verbal and spatial working memory (i.e., N-back) tasks. Although non-pathological worry is predominantly a verbal-linguistic activity, it may also be complemented by the processing of visual imagery which facilitates problem-solving and adaptive functions.

  2. STUDY ON ENERGY EXCHANGE PROCESSES IN NORMAL OPERATION OF METRO ROLLING STOCK WITH REGENERATIVE BRAKING SYSTEMS

    Directory of Open Access Journals (Sweden)

    A. O. Sulym

    2017-10-01

    Full Text Available Purpose. The analysis of the existing studies showed that the increasing of energy efficiency of metro rolling stock becomes especially important and requires timely solutions. It is known that the implementation of regenerative braking systems on rolling stock will allow significantly solving this problem. It was proved that one of the key issues regarding the introduction of the above-mentioned systems is research on efficient use of electric energy of regenerative braking. The purpose of the work is to evaluate the amount of excessive electric power of regenerative braking under normal operation conditions of the rolling stock with regenerative braking systems for the analysis of the energy saving reserves. Methodology. Quantifiable values of electrical energy consumed for traction, returned to the contact line and dissipated in braking resistors (excessive energy are determined using results of experimental studies of energy exchange processes under normal operating conditions of metro rolling stock with regenerative systems. Statistical methods of data processing were applied as well. Findings. Results of the studies analysis of metro rolling stock operation under specified conditions in Sviatoshinsko-Brovarskaia line of KP «Kyiv Metro system» stipulate the following: 1 introduction of regenerative braking systems into the rolling stock allows to return about 17.9-23.2% of electrical energy consumed for traction to the contact line; 2 there are reserves for improving of energy efficiency of rolling stock with regenerative systems at the level of 20.2–29.9 % of electrical energy consumed for traction. Originality. For the first time, it is proved that the most significant factor that influences the quantifiable values of the electrical energy regeneration is a track profile. It is suggested to use coefficients which indicate the amount and reserves of unused (excessive electrical energy for quantitative evaluation. Studies on

  3. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis.

    Science.gov (United States)

    Saita, Shotaro; Nolte, Hendrik; Fiedler, Kai Uwe; Kashkar, Hamid; Venne, A Saskia; Zahedi, René P; Krüger, Marcus; Langer, Thomas

    2017-04-01

    Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria.

  4. Physical-chemical and biocatalytic properties of a proteolytic complex of the preparation "Protepsin"

    Directory of Open Access Journals (Sweden)

    L. V. Antipova

    2016-01-01

    Full Text Available Enzymatic technologies were included strongly into practical activities of the person, the volume of the world market constantly grows and is updated. However the domestic production of enzymatic preparations very lags behind world level that is in many respects connected with insufficient scientific and technical base for a wide circulation of technologies in large-scale production. At the same time there were Russian producers of enzymatic preparations from animal fabrics and bodies for processing of raw materials of an animal origin, according to forecasts, of interest in rational use of resources of an animal origin. In article data on research of properties of the enzymatic preparation "Protepsin" and an assessment of prospects of application are provided in processing of raw materials of an animal origin. The enzymatic preparation "Protepsin" made in the conditions of JSC Plant of Endocrine Enzymes (Rzhavki, Moscow region activity at action on proteins of meat shows, including the strengthened structure, has milk-clotting effect, is active in the field of pH 4,0-6,0 and temperature 20-45zs. The proteinaceous complex includes 4 fractions, 2 from which possess the general proteolytic activity. One of them shows the general proteolytic and milk-clotting activity. Enzymes differ in an amino-acid set and molecular weight. The method of a disk electrophoresis determined molecular-mass structure of "Protepsin". The preparation inactivation conditions guaranteeing its safety in the production technology of foodstuff as active proteolytic enzymes in the course of digestion can cause violations of integrity of fabrics and corresponding diseases are shown. Thus, conditions of use of a perspective proteolytic preparation in technology of a wide range of food of an animal origin are in a complex proved and picked up.

  5. Information processing in micro and meso-scale neural circuits during normal and disease states

    Science.gov (United States)

    Luongo, Francisco

    Neural computation can occur at multiple spatial and temporal timescales. The sum total of all of these processes is to guide optimal behaviors within the context of the constraints imposed by the physical world. How the circuits of the brain achieves this goal represents a central question in systems neuroscience. Here I explore the many ways in which the circuits of the brain can process information at both the micro and meso scale. Understanding the way information is represented and processed in the brain could shed light on the neuropathology underlying complex neuropsychiatric diseases such as autism and schizophrenia. Chapter 2 establishes an experimental paradigm for assaying patterns of microcircuit activity and examines the role of dopaminergic modulation on prefrontal microcircuits. We find that dopamine type 2 (D2) receptor activation results in an increase in spontaneous activity while dopamine type 1 (D1) activation does not. Chapter 3 of this dissertation presents a study that illustrates how cholingergic activation normally produces what has been suggested as a neural substrate of attention; pairwise decorrelation in microcircuit activity. This study also shows that in two etiologicall distinct mouse models of autism, FMR1 knockout mice and Valproic Acid exposed mice, this ability to decorrelate in the presence of cholinergic activation is lost. This represents a putative microcircuit level biomarker of autism. Chapter 4 examines the structure/function relationship within the prefrontal microcircuit. Spontaneous activity in prefrontal microcircuits is shown to be organized according to a small world architecture. Interestingly, this architecture is important for one concrete function of neuronal microcircuits; the ability to produce temporally stereotyped patterns of activation. In the final chapter, we identify subnetworks in chronic intracranial electrocorticographic (ECoG) recordings using pairwise electrode coherence and dimensionality reduction

  6. Large-scale grain growth in the solid-state process: From "Abnormal" to "Normal"

    Science.gov (United States)

    Jiang, Minhong; Han, Shengnan; Zhang, Jingwei; Song, Jiageng; Hao, Chongyan; Deng, Manjiao; Ge, Lingjing; Gu, Zhengfei; Liu, Xinyu

    2018-02-01

    Abnormal grain growth (AGG) has been a common phenomenon during the ceramic or metallurgy processing since prehistoric times. However, usually it had been very difficult to grow big single crystal (centimeter scale over) by using the AGG method due to its so-called occasionality. Based on the AGG, a solid-state crystal growth (SSCG) method was developed. The greatest advantages of the SSCG technology are the simplicity and cost-effectiveness of the technique. But the traditional SSCG technology is still uncontrollable. This article first summarizes the history and current status of AGG, and then reports recent technical developments from AGG to SSCG, and further introduces a new seed-free, solid-state crystal growth (SFSSCG) technology. This SFSSCG method allows us to repeatedly and controllably fabricate large-scale single crystals with appreciable high quality and relatively stable chemical composition at a relatively low temperature, at least in (K0.5Na0.5)NbO3(KNN) and Cu-Al-Mn systems. In this sense, the exaggerated grain growth is no longer 'Abnormal' but 'Normal' since it is able to be artificially controllable and repeated now. This article also provides a crystal growth model to qualitatively explain the mechanism of SFSSCG for KNN system. Compared with the traditional melt and high temperature solution growth methods, the SFSSCG method has the advantages of low energy consumption, low investment, simple technique, composition homogeneity overcoming the issues with incongruent melting and high volatility. This SFSSCG could be helpful for improving the mechanical and physical properties of single crystals, which should be promising for industrial applications.

  7. the use of proteolytic enzymes (chymoral) in sporting injuries

    African Journals Online (AJOL)

    The results lined showed a statistically significant improvement he patients taking the active drug, reducing the time. :esolution of inflammatory oedema by 50%. 10nstein3 was able to show considerable improvement boxers who used proteolytic enzymes both prophy-. icaUy and therapeutically. In this trial the double-blind.

  8. Milk clotting and proteolytic activity of enzyme preparation from ...

    African Journals Online (AJOL)

    Some microorganisms have the ability to produce enzymes that could clot milk and used as a substitute for calf rennet. Strains of lactic acid bacteria (LAB) could produce proteolytic enzymes that may have the potential to be used as a source of milk clotting enzyme (MCE). In the present study, LAB isolated from shrimp paste ...

  9. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... Jeonju 561-756, South Korea. *Corresponding author (Fax, +82-063-270-2605; Email, inho.hwang@jbnu.ac.kr). The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube ...

  10. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    Directory of Open Access Journals (Sweden)

    Belém Sampaio-Marques

    2015-05-01

    Full Text Available Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies.

  11. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA expression of myogenic ...

  12. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA ...

  13. Genetics of the proteolytic system of lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1990-01-01

    The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have

  14. Proteolytic enzymes in seawater: contribution of prokaryotes and protists

    Science.gov (United States)

    Obayashi, Y.; Suzuki, S.

    2016-02-01

    Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

  15. Endurance Exercise: Normal Physiology and Limitations Imposed by Pathological Processes (Part 1).

    Science.gov (United States)

    Frontera, Walter R.; Adams, Richard P.

    1986-01-01

    The physiologic and metabolic adjustments of the body to a single endurance exercise session are analyzed in terms of the respiratory system, the cardiovascular system, and oxygen delivery to the muscles. Patients with cardiorespiratory and neuromuscular diseases are compared to normal individuals. (Author/MT)

  16. Attentional Processing and Teacher Ratings in Hyperactive, Learning Disabled and Normal Boys.

    Science.gov (United States)

    Brown, Ronald T.; Wynne, Martha Ellen

    Sustained attention and inhibitory control of 15 nonhyperactive, learning disabled (LD) boys, 15 hyperactive but not LD boys, and 15 normal boys (11-12 years old) were studied, on teacher ratings of impulse control in the classroom and testing results. Coming to attention, decision making, sustained attention, and attention-concentration were…

  17. Neural processing of healthy foods in normal-weight and overweight children and adults

    NARCIS (Netherlands)

    van Meer, Anne-Floor

    2017-01-01

    Childhood obesity is a growing problem almost everywhere in the world. The chance for an overweight child to become an overweight adult is much larger than for a normal-weight child, which emphasizes the importance of prevention. Weight gain, and thus overweight and obesity, is largely caused by

  18. Does Silent Reading Speed in Normal Adult Readers Depend on Early Visual Processes? Evidence from Event-Related Brain Potentials

    Science.gov (United States)

    Korinth, Sebastian Peter; Sommer, Werner; Breznitz, Zvia

    2012-01-01

    Little is known about the relationship of reading speed and early visual processes in normal readers. Here we examined the association of the early P1, N170 and late N1 component in visual event-related potentials (ERPs) with silent reading speed and a number of additional cognitive skills in a sample of 52 adult German readers utilizing a Lexical…

  19. A non-permselective membrane reactor for chemical processes normally requiring strict stoichiometric feed rates of reactants

    NARCIS (Netherlands)

    Sloot, H.J.; Versteeg, Geert; van Swaaij, Willibrordus Petrus Maria

    1990-01-01

    A novel type of membrane reactor with separated feeding of the reactants is presented for chemical processes normally requiring strict stoichiometric feed rates of premixed reactants. The reactants are fed in the reactor to the different sides of a porous membrane which is impregnated with a

  20. Arrays of surface-normal electroabsorption modulators for the generation and signal processing of microwave photonics signals

    NARCIS (Netherlands)

    Noharet, Bertrand; Wang, Qin; Platt, Duncan; Junique, Stéphane; Marpaung, D.A.I.; Roeloffzen, C.G.H.

    2011-01-01

    The development of an array of 16 surface-normal electroabsorption modulators operating at 1550nm is presented. The modulator array is dedicated to the generation and processing of microwave photonics signals, targeting a modulation bandwidth in excess of 5GHz. The hybrid integration of the

  1. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  2. Embedding new technologies in practice ? a normalization process theory study of point of care testing

    OpenAIRE

    Jones, Caroline H. D.; Glogowska, Margaret; Locock, Louise; Lasserson, Daniel S.

    2016-01-01

    Background Many point of care diagnostic technologies are available which produce results within minutes, and offer the opportunity to deliver acute care out of hospital settings. Increasing access to diagnostics at the point of care could increase the volume and scope of acute ambulatory care. Yet these technologies are not routinely used in many settings. We aimed to explore how point of care testing is used in a setting where it has become ?normalized? (embedded in everyday practice), in o...

  3. Recruitment of Bone Marrow-Derived Valve Interstitial Cells is a Normal Homeostatic Process

    Science.gov (United States)

    Hajdu, Zoltan; Romeo, Stephen J.; Fleming, Paul A.; Markwald, Roger R.; Visconti, Richard P.; Drake, Christopher J.

    2011-01-01

    Advances in understanding of the maintenance of the cardiac valves during normal cardiac function and response to injury have lead to several novel findings, including that there is contribution of extra-cardiac cells to the major cellular population of the valve: the valve interstitial cell (VIC). While suggested to occur in human heart studies, we have been able to experimentally demonstrate, using a mouse model, that cells of bone marrow hematopoietic stem cell origin engraft into the valves and synthesize collagen type I. Based on these initial findings, we sought to further characterize this cell population in terms of its similarity to VICs and begin to elucidate its contribution to valve homeostasis. To accomplish this, chimeric mice whose bone marrow was repopulated with enhanced green fluorescent protein (EGFP) expressing total nucleated bone marrow cells were used to establish a profile of EGFP+ valve cells in terms of their expression of hematopoietic antigens, progenitor markers, fibroblast- and myofibroblast-related molecules, as well as their distribution within the valves. Using this profile, we show that normal (non-irradiated, non-transplanted) mice have BM-derived cell populations that exhibit identical morphology and phenotype to those observed in transplanted mice. Collectively, our findings establish that the engraftment of bone marrow-derived cells occurs as part of normal valve homeostasis. Further, our efforts demonstrate that the use of myeloablative irradiation, which is commonly employed in studies involving bone marrow transplantation, does not elicit changes in the bone marrow-derived VIC phenotype in recipient mice. PMID:21871458

  4. Rates of convergence and asymptotic normality of curve estimators for ergodic diffusion processes

    NARCIS (Netherlands)

    J.H. van Zanten (Harry)

    2000-01-01

    textabstractFor ergodic diffusion processes, we study kernel-type estimators for the invariant density, its derivatives and the drift function. We determine rates of convergence and find the joint asymptotic distribution of the estimators at different points.

  5. Reducing Error Rates for Iris Image using higher Contrast in Normalization process

    Science.gov (United States)

    Aminu Ghali, Abdulrahman; Jamel, Sapiee; Abubakar Pindar, Zahraddeen; Hasssan Disina, Abdulkadir; Mat Daris, Mustafa

    2017-08-01

    Iris recognition system is the most secured, and faster means of identification and authentication. However, iris recognition system suffers a setback from blurring, low contrast and illumination due to low quality image which compromises the accuracy of the system. The acceptance or rejection rates of verified user depend solely on the quality of the image. In many cases, iris recognition system with low image contrast could falsely accept or reject user. Therefore this paper adopts Histogram Equalization Technique to address the problem of False Rejection Rate (FRR) and False Acceptance Rate (FAR) by enhancing the contrast of the iris image. A histogram equalization technique enhances the image quality and neutralizes the low contrast of the image at normalization stage. The experimental result shows that Histogram Equalization Technique has reduced FRR and FAR compared to the existing techniques.

  6. Genetics of the proteolytic system of lactic acid bacteria

    OpenAIRE

    Kok, Jan

    1990-01-01

    The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellullar pe...

  7. Production of extracellular proteolytic enzymes by Beauveria bassiana

    Directory of Open Access Journals (Sweden)

    Józefa Chrzanowska

    2014-08-01

    Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

  8. Estimation of the flare duty cycle of AGNs based on log-normal red-noise processes

    Science.gov (United States)

    Ou, Z.-W.; Biteau, J.

    2017-12-01

    Active Galactic Nuclei (AGN) show variability on time scales ranging from years down to minutes, e.g. in the TeV band, with outbursts often called ‘flares’. We aim at estimating the number of flares observable during a long-term monitoring campaign, depending on their flux and variability time scales. We use backward Fourier transform to construct AGN light curves as realizations of a pseudo-red-noise, log-normal process. Using a simple definition of a flare, we map their duty cycle as a function of threshold-flux and flare-duration values. The flare duty cycle can be entirely defined by two quantities: the slope of the power spectral density and the normalized variance of the process. We also produce visibility windows in order to estimate the effect of sampling on the observable number of flares.

  9. Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility.

    Science.gov (United States)

    Lamothe, Shawn M; Hulbert, Maggie; Guo, Jun; Li, Wentao; Yang, Tonghua; Zhang, Shetuan

    2018-01-05

    The human ether-a-go-go related gene ( hERG)-encoded channel hERG undergoes N-linked glycosylation at position 598, which is located in the unusually long S5-pore linker of the channel. In other work we have demonstrated that hERG is uniquely susceptible to proteolytic cleavage at the S5-pore linker by proteinase K (PK) and calpain (CAPN). The scorpion toxin BeKm-1, which binds to the S5-pore linker of hERG, protects hERG from such cleavage. In the present study, our data revealed that, compared with normal glycosylated hERG channels, nonglycosylated hERG channels were significantly more susceptible to cleavage by extracellular PK. Furthermore, the protective effect of BeKm-1 on hERG from PK-cleavage was lost when glycosylation of hERG was inhibited. The inactivation-deficient mutant hERG channels S620T and S631A were resistant to PK cleavage, and inhibition of glycosylation rendered both mutants susceptible to PK cleavage. Compared with normal glycosylated channels, nonglycosylated hERG channels were also more susceptible to cleavage mediated by CAPN, which was present in the medium of human embryonic kidney cells under normal culture conditions. Inhibition of CAPN resulted in an increase of nonglycosylated hERG current. In summary, our results revealed that N-linked glycosylation protects hERG against protease-mediated degradation and thus contributes to hERG channel stability on the plasma membrane.-Lamothe, S. M., Hulbert, M., Guo, J., Li, W., Yang, T., Zhang, S. Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility.

  10. False memories and normal aging: Links between inhibitory capacities and monitoring processes.

    Science.gov (United States)

    Colombel, Fabienne; Tessoulin, Marine; Gilet, Anne-Laure; Corson, Yves

    2016-05-01

    Empirical evidence suggests an increased production of false memories with advancing age. The activation-monitoring theory proposes that strategic monitoring processes influence the probability of false recall in the DRM paradigm. In the present study, we examined the hypothesis that a low level of inhibition may impair the efficient use of monitoring processes during information retrieval and thus increase the production of false memories in aging. Accordingly, we conducted a study in which older adults with low or high levels of inhibition performed a standard DRM task or an inclusion DRM task that disables monitoring processes. The results indicated that low inhibitory capacities were associated with fewer correct recalls and increased production of critical lures (false memories), suggesting difficulties in using monitoring processes at the time of retrieval. Our findings also showed that the relationship between Age and the production of critical lures in a standard DRM task is mediated by Inhibition. These results are interpreted as suggesting that inhibitory abilities may partly be linked to the impairment of monitoring processes in the elderly. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  11. Implementation of secondary fracture prevention services after hip fracture: a qualitative study using extended Normalization Process Theory

    OpenAIRE

    Drew, Sarah; Judge, Andrew; May, Carl; Farmer, Andrew; Cooper, Cyrus; Javaid, M Kassim; Gooberman-Hill, Rachael

    2015-01-01

    Background National and international guidance emphasizes the need for hospitals to have effective secondary fracture prevention services, to reduce the risk of future fractures in hip fracture patients. Variation exists in how hospitals organize these services, and there remain significant gaps in care. No research has systematically explored reasons for this to understand how to successfully implement these services. The objective of this study was to use extended Normalization Process Theo...

  12. Regular-, irregular-, and pseudo-character processing in Chinese: The regularity effect in normal adult readers

    Directory of Open Access Journals (Sweden)

    Dustin Kai Yan Lau

    2014-03-01

    Full Text Available Background Unlike alphabetic languages, Chinese uses a logographic script. However, the pronunciation of many character’s phonetic radical has the same pronunciation as the character as a whole. These are considered regular characters and can be read through a lexical non-semantic route (Weekes & Chen, 1999. Pseudocharacters are another way to study this non-semantic route. A pseudocharacter is the combination of existing semantic and phonetic radicals in their legal positions resulting in a non-existing character (Ho, Chan, Chung, Lee, & Tsang, 2007. Pseudocharacters can be pronounced by direct derivation from the sound of its phonetic radical. Conversely, if the pronunciation of a character does not follow that of the phonetic radical, it is considered as irregular and can only be correctly read through the lexical-semantic route. The aim of the current investigation was to examine reading aloud in normal adults. We hypothesized that the regularity effect, previously described for alphabetical scripts and acquired dyslexic patients of Chinese (Weekes & Chen, 1999; Wu, Liu, Sun, Chromik, & Zhang, 2014, would also be present in normal adult Chinese readers. Method Participants. Thirty (50% female native Hong Kong Cantonese speakers with a mean age of 19.6 years and a mean education of 12.9 years. Stimuli. Sixty regular-, 60 irregular-, and 60 pseudo-characters (with at least 75% of name agreement in Chinese were matched by initial phoneme, number of strokes and family size. Additionally, regular- and irregular-characters were matched by frequency (low and consistency. Procedure. Each participant was asked to read aloud the stimuli presented on a laptop using the DMDX software. The order of stimuli presentation was randomized. Data analysis. ANOVAs were carried out by participants and items with RTs and errors as dependent variables and type of stimuli (regular-, irregular- and pseudo-character as repeated measures (F1 or between subject

  13. Both left and right posterior parietal activations contribute to compensatory processes in normal aging

    Science.gov (United States)

    Huang, Chih-Mao; Polk, Thad A.; Goh, Joshua O.; Park, Denise C.

    2012-01-01

    Older adults often exhibit greater brain activation in prefrontal cortex compared to younger adults, and there is some evidence that this increased activation compensates for age-related neural degradation that would otherwise adversely affect cognitive performance. Less is known about aging and compensatory recruitment in the parietal cortex. In this event-related functional magnetic resonance imaging study, we presented healthy young and old participants with two Stroop-like tasks (number magnitude and physical size). In young, the number magnitude task activated right parietal cortex and the physical size task activated left parietal cortex. In older adults, we observed contralateral parietal recruitment that depended on the task: in the number magnitude task older participants recruited left posterior parietal cortex (in addition to the right parietal activity observed in young) while in the physical size task they recruited right (in addition to left) posterior parietal cortex. In both cases, the additional parietal activity was associated with better performance suggesting that it played a compensatory role. Older adults also recruited left prefrontal cortex during both tasks and this common activation was also associated with better performance. The results provide evidence for task-specific compensatory recruitment in parietal cortex as well as task-independent compensatory recruitment in prefrontal cortex in normal aging. PMID:22063904

  14. Experimental and bioinformatic investigation of the proteolytic degradation of the C-terminal domain of a fungal tyrosinase.

    Science.gov (United States)

    Faccio, Greta; Arvas, Mikko; Thöny-Meyer, Linda; Saloheimo, Markku

    2013-04-01

    Proteolytic processing is a key step in the production of polyphenol oxidases such as tyrosinases, converting the inactive proenzyme to an active form. In general, the fungal tyrosinase gene codes for a ~60 kDa protein that is, however, isolated as an active enzyme of ~40 kDa, lacking the C-terminal domain. Using the secreted tyrosinase 2 from Trichoderma reesei as a model protein, we performed a mutagenesis study of the residues in proximity of the experimentally determined cleavage site which are possibly involved in the proteolytic process. However, the mutant forms of tyrosinase 2 were not secreted in a full-length form retaining the C-terminal domain, but they were processed to give a ~45 kDa active form. Aiming at explaining this phenomenon, we analysed in silico the properties of the C-terminal domain of tyrosinase 2, of 23 previously retrieved homologous tyrosinase sequences from fungi (C. Gasparetti, G. Faccio, M. Arvas, J. Buchert, M. Saloheimo, K. Kruus, Appl. Microbiol. Biotechnol. 86 (2010) 213-226) and of nine well-characterised polyphenol oxidases. Based on the results of our study, we exclude the key role of specific amino acids at the cleavage site in the proteolytic process and report an overall higher sensitivity to proteolysis of the linker region and of the whole C-terminal domain of fungal tyrosinases. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Process Convergence of Self-Normalized Sums of i.i.d. Random ...

    Indian Academy of Sciences (India)

    ... either of tightness or finite dimensional convergence to a non-degenerate limiting distribution does not hold. This work is an extension of the work by Csörgő et al. who showed Donsker's theorem for Y n , 2 ( ⋅ p ) , i.e., for p = 2 , holds i f f =2 and identified the limiting process as a standard Brownian motion in sup norm.

  16. Methylphenidate Normalizes Frontocingulate Underactivation During Error Processing in Attention-Deficit/Hyperactivity Disorder

    OpenAIRE

    Rubia, Katya; Halari, Rozmin; Mohammad, Abdul-Majeed; Taylor, Eric; Brammer, Michael

    2011-01-01

    Background Children with attention-deficit/hyperactivity disorder (ADHD) have deficits in performance monitoring often improved with the indirect catecholamine agonist methylphenidate (MPH). We used functional magnetic resonance imaging to investigate the effects of single-dose MPH on activation of error processing brain areas in medication-naive boys with ADHD during a stop task that elicits 50% error rates. Methods Twelve medication-naive boys with ADHD were scanned twice, under either a si...

  17. Nitric oxide production and intensity of free radical processes in young men with high normal and hypertensive blood pressure.

    Science.gov (United States)

    Lyamina, Nadezhda P; Dolotovskaya, Polina V; Lyamina, Svetlana V; Malyshev, Igor Y; Manukhina, Eugenia B

    2003-07-01

    Young individuals with high normal blood pressure (HNBP) are at risk for hypertension. The aim of our study was to compare NO production and the intensity of free radical processes in young males with different BP levels. Male subjects aged 18-45 years with normal BP, HNBP, and hypertension underwent physical and cardiological examination. NO production was evaluated by measuring plasma nitrite and nitrate (NOx) and 24-hour urinary excretion of NOx. Lipid peroxidation (LP) intensity and serum antioxidant activity (AOA) were measured using the biochemiluminescence method. HNBP was associated with increased 24-hour systolic BP (SBP), diastolic BP (DBP), SBP and DBP variability, plasma and 24-hour urinary NOx and LP intensity, and decreased total AOA as compared to normotensive controls. We observed a direct nonlinear correlation between plasma NOx and SBP and between 24-hour urinary NOx and DBP, and a close inverse correlation between LP intensity and AOA in patients with HNBP. In the presence of two cardiovascular risk factors (smoking and obesity), patients with HNBP displayed higher LP intensity and lower levels of NOx than in both nonsmokers with normal body weight and control subjects. In hypertensive patients, SBP, DBP and LP intensity inversely correlated with plasma and urinary NOx. Activation of LP processes and depression of AOA proceed in parallel with declining NO production and severity of hypertension. Early correction of the revealed disorders before the appearance of clinical symptoms may be promising in terms of prevention and treatment of cardiovascular disease.

  18. Cue acquisition: A feature of Malawian midwives decision making process to support normality during the first stage of labour.

    Science.gov (United States)

    Chodzaza, Elizabeth; Haycock-Stuart, Elaine; Holloway, Aisha; Mander, Rosemary

    2018-03-01

    to explore Malawian midwives decision making when caring for women during the first stage of labour in the hospital setting. this focused ethnographic study examined the decision making process of 9 nurse-midwives with varying years of clinical experience in the real world setting of an urban and semi urban hospital from October 2013 to May 2014.This was done using 27 participant observations and 27 post-observation in-depth interviews over a period of six months. Qualitative data analysis software, NVivo 10, was used to assist with data management for the analysis. All data was analysed using the principle of theme and category formation. analysis revealed a six-stage process of decision making that include a baseline for labour, deciding to admit a woman to labour ward, ascertaining the normal physiological progress of labour, supporting the normal physiological progress of labour, embracing uncertainty: the midwives' construction of unusual labour as normal, dealing with uncertainty and deciding to intervene in unusual labour. This six-stage process of decision making is conceptualised as the 'role of cue acquisition', illustrating the ways in which midwives utilise their assessment of labouring women to reason and make decisions on how to care for them in labour. Cue acquisition involved the midwives piecing together segments of information they obtained from the women to formulate an understanding of the woman's birthing progress and inform the midwives decision making process. This understanding of cue acquisition by midwives is significant for supporting safe care in the labour setting. When there was uncertainty in a woman's progress of labour, midwives used deductive reasoning, for example, by cross-checking and analysing the information obtained during the span of labour. Supporting normal labour physiological processes was identified as an underlying principle that shaped the midwives clinical judgement and decision making when they cared for women in

  19. TECHNIQUES OF PAIN REDUCTION IN THE NORMAL LABOR PROCESS : SYSTEMATIC REVIEW

    Directory of Open Access Journals (Sweden)

    Wan Anita

    2017-10-01

    Full Text Available Pain during labor is a physiological condition commonly experienced by most maternity mothers. Labor pain is a subjective experience caused by uterine muscle ischemia, withdrawal and traction of uterine ligaments, ovarian traction, fallopian tubes and lower uterine distension, pelvic floor muscles and perineum. The pain in labor arises from psychic responses and physical reflexes. The purpose of this Systematic review is to look at effective methods for reducing pain in the labor process so that it can be used as an alternative method of reducing pain in patients who will give birth. This review systematic review of the published artike through google scholar site with 17 journals reviewed. In an effort to reduce labor pain there are various methods that can be used in providing midwifery care in the process of childbirth. Based on this systematic review it can be concluded that many methods of pain reduction that can be used in reducing labor pain are counter pressure and abdominal lifting, hypnobirthing, religious and murottal music, classical music and local music, relaxation, compress, warm ginger drink, acupressur , TENS, account and aromatherapy.

  20. Use of vaccinia virus vectors to study protein processing in human disease. Normal nerve growth factor processing and secretion in cultured fibroblasts from patients with familial dysautonomia.

    Science.gov (United States)

    Edwards, R H; Rutter, W J

    1988-07-01

    Familial dysautonomia is a hereditary disorder that affects autonomic and sensory neurons. Nerve growth factor (NGF) is required for the normal development of sympathetic and sensory neurons and it has been postulated that an abnormality involving NGF may be responsible for familial dysautonomia. Previous studies have shown that the beta-NGF gene is not linked to the disease. However, NGF appears to be abnormal by immunochemical assays; the putative altered form of NGF could result from a disturbance in the processing pathway. To study the processing of the 35-kD glycosylated NGF precursor and the secretion of NGF in familial dysautonomia, we have employed a recombinant vaccinia virus vector to express high levels of NGF mRNA in primary fibroblast cultures from patients with the disorder; the processing pathway was then studied directly. Cells from several unrelated patients all produce the same 35-kD NGF precursor, process this normally to NGF within the cell, and release NGF into the medium. There are no differences in the ability of cells from patients and from unaffected relatives to process and secrete NGF. The use of similar recombinant vaccinia virus vectors to express proteins at high level in primary cell lines should facilitate the detection of posttranslational processing defects in a variety of human disorders.

  1. The effects of limited bandwidth and noise on verbal processing time and word recall in normal-hearing children.

    Science.gov (United States)

    McCreery, Ryan W; Stelmachowicz, Patricia G

    2013-09-01

    Understanding speech in acoustically degraded environments can place significant cognitive demands on school-age children who are developing the cognitive and linguistic skills needed to support this process. Previous studies suggest the speech understanding, word learning, and academic performance can be negatively impacted by background noise, but the effect of limited audibility on cognitive processes in children has not been directly studied. The aim of the present study was to evaluate the impact of limited audibility on speech understanding and working memory tasks in school-age children with normal hearing. Seventeen children with normal hearing between 6 and 12 years of age participated in the present study. Repetition of nonword consonant-vowel-consonant stimuli was measured under conditions with combinations of two different signal to noise ratios (SNRs; 3 and 9 dB) and two low-pass filter settings (3.2 and 5.6 kHz). Verbal processing time was calculated based on the time from the onset of the stimulus to the onset of the child's response. Monosyllabic word repetition and recall were also measured in conditions with a full bandwidth and 5.6 kHz low-pass cutoff. Nonword repetition scores decreased as audibility decreased. Verbal processing time increased as audibility decreased, consistent with predictions based on increased listening effort. Although monosyllabic word repetition did not vary between the full bandwidth and 5.6 kHz low-pass filter condition, recall was significantly poorer in the condition with limited bandwidth (low pass at 5.6 kHz). Age and expressive language scores predicted performance on word recall tasks, but did not predict nonword repetition accuracy or verbal processing time. Decreased audibility was associated with reduced accuracy for nonword repetition and increased verbal processing time in children with normal hearing. Deficits in free recall were observed even under conditions where word repetition was not affected

  2. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.

    Directory of Open Access Journals (Sweden)

    Sonu Kumar

    Full Text Available CleavPredict (http://cleavpredict.sanfordburnham.org is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs. It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP, and posttranslational modification (PTM sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.

  3. In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)

    2015-10-01

    A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.

  4. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw.

    Science.gov (United States)

    Orlandelli, Ravely Casarotti; de Almeida, Tiago Tognolli; Alberto, Raiani Nascimento; Polonio, Julio Cesar; Azevedo, João Lúcio; Pamphile, João Alencar

    2015-06-01

    Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

  5. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

    Directory of Open Access Journals (Sweden)

    Ravely Casarotti Orlandelli

    2015-06-01

    Full Text Available Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

  6. Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples.

    Science.gov (United States)

    Connon, Catherine C; LeFebvre, Aaron K; Benjamin, Robert C

    2016-11-01

    The goal of this project was to develop a "normalized" extraction procedure to be used in conjunction with previously validated 3μL fast PCR reactions (42-51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24-28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch ® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3μL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection - further referred to as new "first pass" procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95-99% and 88-91% pass rates, respectively. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Caudal articular process dysplasia of thoracic vertebrae in neurologically normal French bulldogs, English bulldogs, and Pugs: Prevalence and characteristics.

    Science.gov (United States)

    Bertram, Simon; Ter Haar, Gert; De Decker, Steven

    2018-02-20

    The aims of this study were to evaluate the prevalence and anatomical characteristics of thoracic caudal articular process dysplasia in French bulldogs, English bulldogs and Pugs presenting for problems unrelated to spinal disease. In this retrospective cross-sectional study, computed tomography scans of the thoracic vertebral column of these three breeds were reviewed for the presence and location of caudal articular process hypoplasia and aplasia, and compared between breeds. A total of 271 dogs met the inclusion criteria: 108 French bulldogs, 63 English bulldogs, and 100 Pugs. A total of 70.4% of French bulldogs, 84.1% of English bulldogs, and 97.0% of Pugs showed evidence of caudal articular process dysplasia. Compared to French and English bulldogs, Pugs showed a significantly higher prevalence of caudal articular process aplasia, but also a lower prevalence of caudal articular process hypoplasia, a higher number of affected vertebrae per dog and demonstrated a generalized and bilateral spatial pattern more frequently. Furthermore, Pugs showed a significantly different anatomical distribution of caudal articular process dysplasia along the vertebral column, with a high prevalence of caudal articular process aplasia between T10 and T13. This area was almost completely spared in French and English bulldogs. As previously suggested, caudal articular process dysplasia is a common finding in neurologically normal Pugs but this also seems to apply to French and English bulldogs. The predisposition of clinically relevant caudal articular process dysplasia in Pugs is possibly not only caused by the higher prevalence of caudal articular process dysplasia, but also by breed specific anatomical characteristics. © 2018 American College of Veterinary Radiology.

  8. Development of potent and proteolytically stable human neuromedin U receptor agonists

    DEFF Research Database (Denmark)

    De Prins, An; Martin, Charlotte; Van Wanseele, Yannick

    2018-01-01

    Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the devel......Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe...... residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting...

  9. Coal liquefaction process streams characterization and evaluation: High performance liquid chromatography (HPLC) of coal liquefaction process streams using normal-phase separation with uv diode array detection

    Energy Technology Data Exchange (ETDEWEB)

    Clifford, D.J.; McKinney, D.E.; Hou, Lei; Hatcher, P.G. [Pennsylvania State Univ., University Park, PA (United States). Dept. of Materials Science and Engineering

    1994-01-01

    This study demonstrated the considerable potential of using two-dimensional, high performance liquid chromatography (HPLC) with normal-phase separation and ultraviolet (UV) diode array detection for the examination of filtered process liquids and the 850{degrees}F{sup {minus}} distillate materials derived from direct coal liquefaction process streams. A commercially available HPLC column (Hypersil Green PAH-2) provided excellent separation of the complex mixture of polynuclear aromatic hydrocarbons (PAHs) found in coal-derived process streams process. Some characteristics of the samples delineated by separation could be attributed to processing parameters. Mass recovery of the process derived samples was low (5--50 wt %). Penn State believes, however, that, improved recovery can be achieved. High resolution mass spectrometry and gas chromatography/mass spectrometry (GC/MS) also were used in this study to characterize the samples and the HPLC fractions. The GC/MS technique was used to preliminarily examine the GC-elutable portion of the samples. The GC/MS data were compared with the data from the HPLC technique. The use of an ultraviolet detector in the HPLC work precludes detecting the aliphatic portion of the sample. The GC/MS allowed for identification and quantification of that portion of the samples. Further development of the 2-D HPLC analytical method as a process development tool appears justified based on the results of this project.

  10. Proteolytic Cleavage of ProBDNF into Mature BDNF in the Basolateral Amygdala Is Necessary for Defeat-Induced Social Avoidance

    Science.gov (United States)

    Dulka, Brooke N.; Ford, Ellen C.; Lee, Melissa A.; Donnell, Nathaniel J.; Goode, Travis D.; Prosser, Rebecca; Cooper, Matthew A.

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is essential for memory processes. The present study tested whether proteolytic cleavage of proBDNF into mature BDNF (mBDNF) within the basolateral amygdala (BLA) regulates the consolidation of defeat-related memories. We found that acute social defeat increases the expression of mBDNF, but not proBDNF, in…

  11. Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt

    NARCIS (Netherlands)

    Settachaimongkon, S.; Nout, M.J.R.; Antunes Fernandes, E.C.; Hettinga, K.A.; Vervoort, J.J.M.; Hooijdonk, van A.C.M.; Zwietering, M.H.; Smid, E.J.; Valenberg, van H.J.F.

    2014-01-01

    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L.

  12. The proteolytic activities of chymopapain, papain, and papaya proteinase III.

    Science.gov (United States)

    Zucker, S; Buttle, D J; Nicklin, M J; Barrett, A J

    1985-04-05

    The three proteinases present in papaya latex: papain (EC 3.4.22.2) chymopapain and papaya proteinase III (EC 3.4.22.6), were standardized by active-site titration, and compared in proteolytic activity against azocasein, serum albumin and cartilage proteoglycan. The activities were all of the same order, although there were differences in pH dependence. SDS-polyacrylamide gel electrophoresis of the early products of digestion of albumin and phosphorylase a showed very similar patterns for the three papaya proteinases. Kinetic parameters for hydrolysis of benzyloxycarbonyl-phenylalanyl-arginyl-7(4-methyl)coumarylamide were determined for the three enzymes. Values for kcat/Km varied only within a factor of 2, but the individual constants were much higher for papain than for chymopapain and papaya proteinase III. In contrast to the results obtained with the synthetic substrate, the kinetic parameters for the initial hydrolysis of succinyl-albumin were very similar for the three papaya proteinases. This was consistent with their similar proteolytic activities in other assays.

  13. Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues

    2017-09-01

    Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

  14. Persistence of bacterial proteolytic enzymes in lake ecosystems.

    Science.gov (United States)

    Kiersztyn, Bartosz; Siuda, Waldemar; Chróst, Ryszard J

    2012-04-01

    This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. MHD effects on turbulent dissipation process in channel flows with an imposed wall-normal magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Yoshinobu, E-mail: yamamotoy@yamanashi.ac.jp [Division of Mechanical Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu 400-8511 (Japan); Kunugi, Tomoaki [Department of Nuclear Engineering, Kyoto University, C3-d2S06, Kyoto-Daigaku Katsura, Nishikyo-Ku, Kyoto 615-8540 (Japan)

    2016-11-01

    Highlights: • We succeeded the establishing DNS database for the epsilon transport equation for turbulent channel flows under high-Re and Ha conditions. • Turbulent dissipation process under the MHD effects shows the similarity under the same R condition, where R is the Reynolds number based on the Hartmann layer thickness and the laminar centerline velocity. • Modulation of the non-MHD terms is important from the view point of the turbulence modeling. - Abstract: In this study, we succeeded in establishing DNS database significantly increased the range of Reynolds number and Hartmann numbers for the epsilon transport equation in turbulent channel flows imposed on a wall-normal uniform magnetic field. Maximum friction Reynolds number based on the channel half-height is 1000 with the Hartmann number based on the channel half-height, 0, 24, and 34. The contribution of MHD source term derived from Lorentz force on the turbulent dissipation process is very small. However, modulation of the other terms in this process was remarkably observed with Hartmann number increasing. The turbulent dissipation process under the MHD effects is similar under the same R condition, where R is the Reynolds number based on the Hartmann layer thickness and the laminar centerline velocity.

  16. Normal uptake of 68Ga-DOTA-TOC by the pancreas uncinate process mimicking malignancy at somatostatin receptor PET.

    Science.gov (United States)

    Jacobsson, Hans; Larsson, Patricia; Jonsson, Cathrine; Jussing, Emma; Grybäck, Per

    2012-04-01

    To characterize a commonly occurring increased uptake by the uncinate process of the pancreas at PET/CT using 68Ga-DOTA-d-Phe1-Tyr3-octreotide (68Ga-DOTA-TOC). This tracer has replaced In pentetreotide (OctreoScan®) for somatostatin receptor scintigraphy at our laboratory. Fifty of our first 74 PET/CT examinations with 68Ga-DOTA-TOC could be evaluated in retrospect. None of these patients had surgery or showed any pathology in the pancreas head at the concomitant CT. Thirty-five of the 50 examinations (70%) showed an uptake by the uncinate process sufficiently intense to be interpreted as pathologic and simulating a tumor. Mean SUVmax was 9.2. Mean SUVmean using an isoactivity cut-off of >75% and >50% was 7.8 and 6.0, respectively. Volume calculations of the uncinate process activity using these definitions gave 0.9 mL and 4.2 mL, respectively. There is a frequent physiological uptake of 68Ga-DOTA-TOC by the pancreas uncinate process. This may be caused by an accumulation of pancreatic polypeptide-containing cells expressing somatostatin receptors. If there is a normal finding at concomitant diagnostic CT, this uptake should be regarded as physiological.

  17. Patchy ‘coherence’: using normalization process theory to evaluate a multi-faceted shared decision making implementation program (MAGIC)

    Science.gov (United States)

    2013-01-01

    Background Implementing shared decision making into routine practice is proving difficult, despite considerable interest from policy-makers, and is far more complex than merely making decision support interventions available to patients. Few have reported successful implementation beyond research studies. MAking Good Decisions In Collaboration (MAGIC) is a multi-faceted implementation program, commissioned by The Health Foundation (UK), to examine how best to put shared decision making into routine practice. In this paper, we investigate healthcare professionals’ perspectives on implementing shared decision making during the MAGIC program, to examine the work required to implement shared decision making and to inform future efforts. Methods The MAGIC program approached implementation of shared decision making by initiating a range of interventions including: providing workshops; facilitating development of brief decision support tools (Option Grids); initiating a patient activation campaign (‘Ask 3 Questions’); gathering feedback using Decision Quality Measures; providing clinical leads meetings, learning events, and feedback sessions; and obtaining executive board level support. At 9 and 15 months (May and November 2011), two rounds of semi-structured interviews were conducted with healthcare professionals in three secondary care teams to explore views on the impact of these interventions. Interview data were coded by two reviewers using a framework derived from the Normalization Process Theory. Results A total of 54 interviews were completed with 31 healthcare professionals. Partial implementation of shared decision making could be explained using the four components of the Normalization Process Theory: ‘coherence,’ ‘cognitive participation,’ ‘collective action,’ and ‘reflexive monitoring.’ Shared decision making was integrated into routine practice when clinical teams shared coherent views of role and purpose (‘coherence’). Shared

  18. Increasing viability, numbers, and motility of sperm in men with normal spermatogenesis exposed to saffron extract after freezing- thawing process

    Directory of Open Access Journals (Sweden)

    Y. Khazaei

    2017-04-01

    Full Text Available Background: Sperm freezing method is used frequently in assisted reproductive techniques, on the other hand in different studies negative effect of freezing have been shown on different sperm parameters. Objective: The aim of this study was to determine the effect of saffron extract as an antioxidant, on the different sperm parameters in men with normal spermatogenesis after freezing-thawing process. Methods: In this case-control study, collecting of samples was done in 2015 year from the Infertility Treatment Center, ACECR Branch of Qazvin, Qazvin, Iran. These men had normal spermatogenesis and their spouse had infertility problem. Semen samples was divided in two groups, control without saffron extract, and case with 50 mg/ml saffron extract. Then, samples freezed with snap freezing method. After two weeks, they were thawed and different sperm parameters were assessed. Data were analyzed by two-tail T test. Findings: Our results showed, mean percent of viability (72±0.99, motility (87±0.43, and the number of sperm cells (62.5±3.8 in treaded group was elevated significantly (P0.05. Conclusion: Our results showed that possibly antioxidant agents of saffron extract could scavenge free radicals and thus, optimize different sperm parameters (viability, motility, and number after freezing and thawing.

  19. Self-Growth of Centimeter-Scale Single Crystals by Normal Sintering Process in Modified Potassium Sodium Niobate Ceramics

    Science.gov (United States)

    Ahn, Cheol-Woo; Lee, Ho-Yong; Han, Guifang; Zhang, Shujun; Choi, Si-Young; Choi, Jong-Jin; Kim, Jong-Woo; Yoon, Woon-Ha; Choi, Joon-Hwan; Park, Dong-Soo; Hahn, Byung-Dong; Ryu, Jungho

    2015-01-01

    In this manuscript, an interesting phenomenon is reported. That is the self-growth of single crystals in Pb-free piezoelectric ceramics. These crystals are several centimeters in size. They are grown without any seed addition through a normal sintering process in modified potassium sodium niobate ceramics. It has been achieved by the composition designed to compensate the Na+ loss which occurs during the liquid phase sintering. The composition of the crystals is (K0.4925Na0.4925−xBa0.015+x/2)Nb0.995+xO3 [x is determined by the Na+ loss, due to Na2O volatilization]. These crystals have high piezoelectric voltage coefficients (g33, 131 10−3Vm/N), indicating that they are good candidates for piezoelectric sensors and energy harvesting devices. We hope that this report can offer the opportunity for many researchers to have an interest in these crystals. PMID:26631973

  20. Quantification of diffusion tensor imaging in normal white matter maturation of early childhood using an automated processing pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Loh, K.B.; Ramli, N.; Tan, L.K.; Roziah, M. [University of Malaya, Department of Biomedical Imaging, University Malaya Research Imaging Centre (UMRIC), Faculty of Medicine, Kuala Lumpur (Malaysia); Rahmat, K. [University of Malaya, Department of Biomedical Imaging, University Malaya Research Imaging Centre (UMRIC), Faculty of Medicine, Kuala Lumpur (Malaysia); University Malaya, Biomedical Imaging Department, Kuala Lumpur (Malaysia); Ariffin, H. [University of Malaya, Department of Paediatrics, Faculty of Medicine, Kuala Lumpur (Malaysia)

    2012-07-15

    The degree and status of white matter myelination can be sensitively monitored using diffusion tensor imaging (DTI). This study looks at the measurement of fractional anistropy (FA) and mean diffusivity (MD) using an automated ROI with an existing DTI atlas. Anatomical MRI and structural DTI were performed cross-sectionally on 26 normal children (newborn to 48 months old), using 1.5-T MRI. The automated processing pipeline was implemented to convert diffusion-weighted images into the NIfTI format. DTI-TK software was used to register the processed images to the ICBM DTI-81 atlas, while AFNI software was used for automated atlas-based volumes of interest (VOIs) and statistical value extraction. DTI exhibited consistent grey-white matter contrast. Triphasic temporal variation of the FA and MD values was noted, with FA increasing and MD decreasing rapidly early in the first 12 months. The second phase lasted 12-24 months during which the rate of FA and MD changes was reduced. After 24 months, the FA and MD values plateaued. DTI is a superior technique to conventional MR imaging in depicting WM maturation. The use of the automated processing pipeline provides a reliable environment for quantitative analysis of high-throughput DTI data. (orig.)

  1. On the (unimportance of working memory in speech-in-noise processing for listeners with normal hearing thresholds

    Directory of Open Access Journals (Sweden)

    Christian Füllgrabe

    2016-08-01

    Full Text Available With the advent of cognitive hearing science, increased attention has been given to individual differences in cognitive functioning and their explanatory power in accounting for inter-listener variability in the processing of speech in noise (SiN. The psychological construct that has received much interest in recent years is working memory (WM. Empirical evidence indeed confirms the association between WM capacity (WMC and SiN identification in older hearing-impaired listeners. However, some theoretical models propose that variations in WMC are an important predictor for variations in speech processing abilities in adverse perceptual conditions for all listeners, and this notion has become widely accepted within the field. To assess whether WMC also plays a role when listeners without hearing loss process speech in adverse listening conditions, we surveyed published and unpublished studies in which the Reading-Span test (a widely used measure of WMC was administered in conjunction with a measure of SiN identification, using sentence material routinely used in audiological and hearing research. A meta-analysis revealed that, for young listeners with audiometrically normal hearing, individual variations in WMC are estimated to account for, on average, less than 2% of the variance in SiN identification scores. This result cautions against the (intuitively appealing assumption that individual variations in WMC are predictive of SiN identification independently of the age and hearing status of the listener.

  2. Evaluation of the radiographic process using a experimental monobath solution compared with normal (Kodak) and rapid (RAY) developer solutions

    International Nuclear Information System (INIS)

    Baratieri, N.M.M.

    1985-01-01

    A comparative evaluation of the radiographic image quality of two dental X-ray films (Kodak's EP-21 and Agfa-Gevaert DOS-1) when processed in a normal (Kodak) a rapid (Ray) and a experimental monobath solutions, is presented. These films, processed in those solutions had their time of development, temperature and agitation performances checked by sensitometry; pH and color by routine methods and hipo rests by spectrophotometry. The radiographies were also analysed by able professionals regarding the best development time. The data so obtained allowed the conclusions that the best development time for the monobath was 3 minutes at 20 0 C but 25 or 30 0 C give also acceptable results at shorter times. The agitation of 10 seconds every minute is an important factor concerning image quality. pH and color do alter rapidally but with little influence in the final result. We found a certain amount of residual chemical compounds which were not identified but that are not hipo components, and being important to note that they seem not act upon the emulsion at least during one year after processing. (author) [pt

  3. Quantification of diffusion tensor imaging in normal white matter maturation of early childhood using an automated processing pipeline

    International Nuclear Information System (INIS)

    Loh, K.B.; Ramli, N.; Tan, L.K.; Roziah, M.; Rahmat, K.; Ariffin, H.

    2012-01-01

    The degree and status of white matter myelination can be sensitively monitored using diffusion tensor imaging (DTI). This study looks at the measurement of fractional anistropy (FA) and mean diffusivity (MD) using an automated ROI with an existing DTI atlas. Anatomical MRI and structural DTI were performed cross-sectionally on 26 normal children (newborn to 48 months old), using 1.5-T MRI. The automated processing pipeline was implemented to convert diffusion-weighted images into the NIfTI format. DTI-TK software was used to register the processed images to the ICBM DTI-81 atlas, while AFNI software was used for automated atlas-based volumes of interest (VOIs) and statistical value extraction. DTI exhibited consistent grey-white matter contrast. Triphasic temporal variation of the FA and MD values was noted, with FA increasing and MD decreasing rapidly early in the first 12 months. The second phase lasted 12-24 months during which the rate of FA and MD changes was reduced. After 24 months, the FA and MD values plateaued. DTI is a superior technique to conventional MR imaging in depicting WM maturation. The use of the automated processing pipeline provides a reliable environment for quantitative analysis of high-throughput DTI data. (orig.)

  4. A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts.

    Science.gov (United States)

    Sakashita, Tetsuya; Hamada, Nobuyuki; Kawaguchi, Isao; Hara, Takamitsu; Kobayashi, Yasuhiko; Saito, Kimiaki

    2014-05-01

    A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/µm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log-log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE.

  5. A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts

    International Nuclear Information System (INIS)

    Sakashita, Tetsuya; Kobayashi, Yasuhiko; Hamada, Nobuyuki; Kawaguchi, Isao; Hara, Takamitsu; Saito, Kimiaki

    2014-01-01

    A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/μm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log–log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE. (author)

  6. Conformational quiescence of ADAMTS-13 prevents proteolytic promiscuity.

    Science.gov (United States)

    South, K; Freitas, M O; Lane, D A

    2016-10-01

    Essentials Recently, ADAMTS-13 has been shown to undergo substrate induced conformation activation. Conformational quiescence of ADAMTS-13 may serve to prevent off-target proteolysis in plasma. Conformationally active ADAMTS-13 variants are capable of proteolysing the Aα chain of fibrinogen. This should be considered as ADAMTS-13 variants are developed as potential therapeutic agents. Click to hear Dr Zheng's presentation on structure function and cofactor-dependent regulation of ADAMTS-13 SUMMARY: Background Recent work has revealed that ADAMTS-13 circulates in a 'closed' conformation, only fully interacting with von Willebrand factor (VWF) following a conformational change. We hypothesized that this conformational quiescence also maintains the substrate specificity of ADAMTS-13 and that the 'open' conformation of the protease might facilitate proteolytic promiscuity. Objectives To identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F). Methods Fibrinogen proteolysis was characterized using SDS PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fibrin formation was monitored by turbidity measurements and fibrin structure visualized by confocal microscopy. Results ADAMTS-13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS-13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC-MS/MS to be Aα chain Lys225-Met226. Proteolysis of fibrinogen by GoF ADAMTS-13 impairs fibrin formation in plasma-based assays, alters clot structure and increases clot permeability. Although GoF ADAMTS-13 does not appear to proteolyse preformed cross-linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to tissue-type plasminogen activator (t-PA)-induced lysis by plasmin and increases the

  7. Evaluating Complex Interventions and Health Technologies Using Normalization Process Theory: Development of a Simplified Approach and Web-Enabled Toolkit

    LENUS (Irish Health Repository)

    May, Carl R

    2011-09-30

    Abstract Background Normalization Process Theory (NPT) can be used to explain implementation processes in health care relating to new technologies and complex interventions. This paper describes the processes by which we developed a simplified version of NPT for use by clinicians, managers, and policy makers, and which could be embedded in a web-enabled toolkit and on-line users manual. Methods Between 2006 and 2010 we undertook four tasks. (i) We presented NPT to potential and actual users in multiple workshops, seminars, and presentations. (ii) Using what we discovered from these meetings, we decided to create a simplified set of statements and explanations expressing core constructs of the theory (iii) We circulated these statements to a criterion sample of 60 researchers, clinicians and others, using SurveyMonkey to collect qualitative textual data about their criticisms of the statements. (iv) We then reconstructed the statements and explanations to meet users\\' criticisms, embedded them in a web-enabled toolkit, and beta tested this \\'in the wild\\'. Results On-line data collection was effective: over a four week period 50\\/60 participants responded using SurveyMonkey (40\\/60) or direct phone and email contact (10\\/60). An additional nine responses were received from people who had been sent the SurveyMonkey form by other respondents. Beta testing of the web enabled toolkit produced 13 responses, from 327 visits to http:\\/\\/www.normalizationprocess.org. Qualitative analysis of both sets of responses showed a high level of support for the statements but also showed that some statements poorly expressed their underlying constructs or overlapped with others. These were rewritten to take account of users\\' criticisms and then embedded in a web-enabled toolkit. As a result we were able translate the core constructs into a simplified set of statements that could be utilized by non-experts. Conclusion Normalization Process Theory has been developed through

  8. Evaluating complex interventions and health technologies using normalization process theory: development of a simplified approach and web-enabled toolkit

    Directory of Open Access Journals (Sweden)

    Murray Elizabeth

    2011-09-01

    Full Text Available Abstract Background Normalization Process Theory (NPT can be used to explain implementation processes in health care relating to new technologies and complex interventions. This paper describes the processes by which we developed a simplified version of NPT for use by clinicians, managers, and policy makers, and which could be embedded in a web-enabled toolkit and on-line users manual. Methods Between 2006 and 2010 we undertook four tasks. (i We presented NPT to potential and actual users in multiple workshops, seminars, and presentations. (ii Using what we discovered from these meetings, we decided to create a simplified set of statements and explanations expressing core constructs of the theory (iii We circulated these statements to a criterion sample of 60 researchers, clinicians and others, using SurveyMonkey to collect qualitative textual data about their criticisms of the statements. (iv We then reconstructed the statements and explanations to meet users' criticisms, embedded them in a web-enabled toolkit, and beta tested this 'in the wild'. Results On-line data collection was effective: over a four week period 50/60 participants responded using SurveyMonkey (40/60 or direct phone and email contact (10/60. An additional nine responses were received from people who had been sent the SurveyMonkey form by other respondents. Beta testing of the web enabled toolkit produced 13 responses, from 327 visits to http://www.normalizationprocess.org. Qualitative analysis of both sets of responses showed a high level of support for the statements but also showed that some statements poorly expressed their underlying constructs or overlapped with others. These were rewritten to take account of users' criticisms and then embedded in a web-enabled toolkit. As a result we were able translate the core constructs into a simplified set of statements that could be utilized by non-experts. Conclusion Normalization Process Theory has been developed through

  9. Inability of non-proteolytic Clostridium botulinum to grow in mussels inoculated via immersion and packaged in high oxygen atmospheres.

    Science.gov (United States)

    Newell, Carter R; Doyle, Michael; Ma, Li

    2015-04-01

    A series of botulism challenge studies were conducted to determine if botulinum toxin would be produced in mussels (Mytilus edulis) inoculated with non-proteolytic Clostridium botulinum spores and held under modified atmosphere (MA) packaging conditions at normal (4 °C) and abusive (12 °C) temperatures. Spore mixtures of six strains of non-proteolytic C. botulinum were introduced into live mussels through immersion in a seawater solution with cultured algae. Mussels were packed in a commercial high-oxygen (60-65% O2) MA-package with a buffer, and also packed under a vacuum. Feeding live mussels cultured algae (10(4) cells/ml) with a C. botulinum spore suspension (10(3) spores/ml) in seawater at 4 °C for 6 h resulted in the uptake of spores into mussel tissue (500/g) and the mussel GI tract (100/g). Under all of the experimental conditions evaluated, none of the fresh mussels became toxic, even after spoilage and in the absence of oxygen. However, control samples using tuna or cooked mussel meats became toxic in the absence of oxygen. Botulinum toxin was not produced in fresh mussels packaged under the MA-packaging conditions evaluated, even at an abusive storage temperature (12 °C) for at least 12 days or at normal storage temperate (4 °C) for at least 21 days, which is beyond their shelf life. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.

    Science.gov (United States)

    Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N

    2016-12-01

    Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.

  11. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    International Nuclear Information System (INIS)

    Safarik, I. Ivo; Safarikova, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed

  12. High stability of self-assembled peptide nanowires against thermal, chemical, and proteolytic attacks.

    Science.gov (United States)

    Ryu, Jungki; Park, Chan Beum

    2010-02-01

    Understanding the self-assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio-inspired materials. In order to realize the potential of the peptide-based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self-assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self-assembly of diphenylalanine (Phe-Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150 degrees C led to the formation of PNWs, while its incubation with water vapor at 25 degrees C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier-transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200 degrees C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non-polar). In contrast, PNTs started to disintegrate even at 100 degrees C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant beta-sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self-assembled PNWs are suitable for future nano-scale applications requiring harsh processing conditions. 2009 Wiley Periodicals, Inc.

  13. Regulation of host hemoglobin binding by the Staphylococcus aureus Clp proteolytic system.

    Science.gov (United States)

    Farrand, Allison J; Reniere, Michelle L; Ingmer, Hanne; Frees, Dorte; Skaar, Eric P

    2013-11-01

    Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacterium Staphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery in S. aureus. This study sought to identify the effect of the Clp protease on the iron-regulated surface determinant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical to S. aureus pathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription of isdB is decreased by clpP or clpX inactivation. In contrast, inactivation of clpC enhances isdB transcription and protein abundance. Loss of clpP or clpX impairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition in S. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.

  14. A framework for analysis of abortive colony size distributions using a model of branching processes in irradiated normal human fibroblasts.

    Science.gov (United States)

    Sakashita, Tetsuya; Hamada, Nobuyuki; Kawaguchi, Isao; Ouchi, Noriyuki B; Hara, Takamitsu; Kobayashi, Yasuhiko; Saito, Kimiaki

    2013-01-01

    Clonogenicity gives important information about the cellular reproductive potential following ionizing irradiation, but an abortive colony that fails to continue to grow remains poorly characterized. It was recently reported that the fraction of abortive colonies increases with increasing dose. Thus, we set out to investigate the production kinetics of abortive colonies using a model of branching processes. We firstly plotted the experimentally determined colony size distribution of abortive colonies in irradiated normal human fibroblasts, and found the linear relationship on the log-linear or log-log plot. By applying the simple model of branching processes to the linear relationship, we found the persistent reproductive cell death (RCD) over several generations following irradiation. To verify the estimated probability of RCD, abortive colony size distribution (≤ 15 cells) and the surviving fraction were simulated by the Monte Carlo computational approach for colony expansion. Parameters estimated from the log-log fit demonstrated the good performance in both simulations than those from the log-linear fit. Radiation-induced RCD, i.e. excess probability, lasted over 16 generations and mainly consisted of two components in the early (probability over 5 generations, whereas abortive colony size distribution was robust against it. These results suggest that, whereas short-term RCD is critical to the abortive colony size distribution, long-lasting RCD is important for the dose response of the surviving fraction. Our present model provides a single framework for understanding the behavior of primary cell colonies in culture following irradiation.

  15. Partial digestion of neurophysins with proteolytic enzymes: unusual interactions between bovine neurophysin II and chymotrypsin.

    Science.gov (United States)

    Rabbani, L D; Pagnozzi, M; Chang, P; Breslow, E

    1982-03-02

    Bovine neurophysin II was partially digested by chymotrypsin and by chymotrypsin followed by carboxy-peptidase B to produce large fragments collectively representing deletions of residues 1-5 and 91-95. All such fragments were capable of binding peptides to the principal hormone-binding site of neurophysin with normal or near-normal affinity, indicating that residues 1-5 and 91-95 do not directly participate in binding. In addition, preliminary results with thermolysin-derived fragments suggested that residue 6 does not participate in peptide binding. During the course of chymotrypsin studies, it was demonstrated that bovine neurophysin II behaves as a transient competitive inhibitor of chymotrypsin; for neurophysin-peptide complexes, Ki congruent to 8 x 10(-6) M. This inhibition is dependent on neurophysin conformation and is relieved by the anomalous preferential splitting by chymotrypsin of Arg-Arg and Phe-Pro bonds near the carboxyl terminus of neurophysin II. It is suggested that this phenomenon might reflect the interaction of neurophysin II with a chymotrypsin-related enzyme in the pituitary. One approach used in the study of binding properties of proteolytically modified neurophysin was affinity chromatography; the preparation and properties of a conveniently prepared affinity column for neurophysin are described.

  16. Age-group differences in speech identification despite matched audiometrically normal hearing: contributions from auditory temporal processing and cognition

    Science.gov (United States)

    Füllgrabe, Christian; Moore, Brian C. J.; Stone, Michael A.

    2015-01-01

    Hearing loss with increasing age adversely affects the ability to understand speech, an effect that results partly from reduced audibility. The aims of this study were to establish whether aging reduces speech intelligibility for listeners with normal audiograms, and, if so, to assess the relative contributions of auditory temporal and cognitive processing. Twenty-one older normal-hearing (ONH; 60–79 years) participants with bilateral audiometric thresholds ≤ 20 dB HL at 0.125–6 kHz were matched to nine young (YNH; 18–27 years) participants in terms of mean audiograms, years of education, and performance IQ. Measures included: (1) identification of consonants in quiet and in noise that was unmodulated or modulated at 5 or 80 Hz; (2) identification of sentences in quiet and in co-located or spatially separated two-talker babble; (3) detection of modulation of the temporal envelope (TE) at frequencies 5–180 Hz; (4) monaural and binaural sensitivity to temporal fine structure (TFS); (5) various cognitive tests. Speech identification was worse for ONH than YNH participants in all types of background. This deficit was not reflected in self-ratings of hearing ability. Modulation masking release (the improvement in speech identification obtained by amplitude modulating a noise background) and spatial masking release (the benefit obtained from spatially separating masker and target speech) were not affected by age. Sensitivity to TE and TFS was lower for ONH than YNH participants, and was correlated positively with speech-in-noise (SiN) identification. Many cognitive abilities were lower for ONH than YNH participants, and generally were correlated positively with SiN identification scores. The best predictors of the intelligibility of SiN were composite measures of cognition and TFS sensitivity. These results suggest that declines in speech perception in older persons are partly caused by cognitive and perceptual changes separate from age-related changes in

  17. Implementing nutrition guidelines for older people in residential care homes: a qualitative study using Normalization Process Theory

    Directory of Open Access Journals (Sweden)

    Bamford Claire

    2012-10-01

    Full Text Available Abstract Background Optimizing the dietary intake of older people can prevent nutritional deficiencies and diet-related diseases, thereby improving quality of life. However, there is evidence that the nutritional intake of older people living in care homes is suboptimal, with high levels of saturated fat, salt, and added sugars. The UK Food Standards Agency therefore developed nutrient- and food-based guidance for residential care homes. The acceptability of these guidelines and their feasibility in practice is unknown. This study used the Normalization Process Theory (NPT to understand the barriers and facilitators to implementing the guidelines and inform future implementation. Methods We conducted a process evaluation in five care homes in the north of England using qualitative methods (observation and interviews to explore the views of managers, care staff, catering staff, and domestic staff. Data were analyzed thematically and discussed in data workshops; emerging themes were then mapped to the constructs of NPT. Results Many staff perceived the guidelines as unnecessarily restrictive and irrelevant to older people. In terms of NPT, the guidelines simply did not make sense (coherence, and as a result, relatively few staff invested in the guidelines (cognitive participation. Even where staff supported the guidelines, implementation was hampered by a lack of nutritional knowledge and institutional support (collective action. Finally, the absence of observable benefits to clients confirmed the negative preconceptions of many staff, with limited evidence of reappraisal following implementation (reflexive monitoring. Conclusions The successful implementation of the nutrition guidelines requires that the fundamental issues relating to their perceived value and fit with other priorities and goals be addressed. Specialist support is needed to equip staff with the technical knowledge and skills required for menu analysis and development and to

  18. A framework for analysis of abortive colony size distributions using a model of branching processes in irradiated normal human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Tetsuya Sakashita

    Full Text Available Clonogenicity gives important information about the cellular reproductive potential following ionizing irradiation, but an abortive colony that fails to continue to grow remains poorly characterized. It was recently reported that the fraction of abortive colonies increases with increasing dose. Thus, we set out to investigate the production kinetics of abortive colonies using a model of branching processes.We firstly plotted the experimentally determined colony size distribution of abortive colonies in irradiated normal human fibroblasts, and found the linear relationship on the log-linear or log-log plot. By applying the simple model of branching processes to the linear relationship, we found the persistent reproductive cell death (RCD over several generations following irradiation. To verify the estimated probability of RCD, abortive colony size distribution (≤ 15 cells and the surviving fraction were simulated by the Monte Carlo computational approach for colony expansion. Parameters estimated from the log-log fit demonstrated the good performance in both simulations than those from the log-linear fit. Radiation-induced RCD, i.e. excess probability, lasted over 16 generations and mainly consisted of two components in the early (<3 generations and late phases. Intriguingly, the survival curve was sensitive to the excess probability over 5 generations, whereas abortive colony size distribution was robust against it. These results suggest that, whereas short-term RCD is critical to the abortive colony size distribution, long-lasting RCD is important for the dose response of the surviving fraction.Our present model provides a single framework for understanding the behavior of primary cell colonies in culture following irradiation.

  19. Implementing nutrition guidelines for older people in residential care homes: a qualitative study using Normalization Process Theory

    Science.gov (United States)

    2012-01-01

    Background Optimizing the dietary intake of older people can prevent nutritional deficiencies and diet-related diseases, thereby improving quality of life. However, there is evidence that the nutritional intake of older people living in care homes is suboptimal, with high levels of saturated fat, salt, and added sugars. The UK Food Standards Agency therefore developed nutrient- and food-based guidance for residential care homes. The acceptability of these guidelines and their feasibility in practice is unknown. This study used the Normalization Process Theory (NPT) to understand the barriers and facilitators to implementing the guidelines and inform future implementation. Methods We conducted a process evaluation in five care homes in the north of England using qualitative methods (observation and interviews) to explore the views of managers, care staff, catering staff, and domestic staff. Data were analyzed thematically and discussed in data workshops; emerging themes were then mapped to the constructs of NPT. Results Many staff perceived the guidelines as unnecessarily restrictive and irrelevant to older people. In terms of NPT, the guidelines simply did not make sense (coherence), and as a result, relatively few staff invested in the guidelines (cognitive participation). Even where staff supported the guidelines, implementation was hampered by a lack of nutritional knowledge and institutional support (collective action). Finally, the absence of observable benefits to clients confirmed the negative preconceptions of many staff, with limited evidence of reappraisal following implementation (reflexive monitoring). Conclusions The successful implementation of the nutrition guidelines requires that the fundamental issues relating to their perceived value and fit with other priorities and goals be addressed. Specialist support is needed to equip staff with the technical knowledge and skills required for menu analysis and development and to devise ways of evaluating

  20. Transport and processing of beta-hexosaminidase in normal and mucolipidosis-II cultured fibroblasts. Effect of monensin and nigericin.

    Science.gov (United States)

    Vladutiu, G D

    1984-01-01

    The univalent-cation ionophores monensin (4.0 microM) and nigericin (0.5 microM) inhibited the abnormal excretion of beta-hexosaminidase from mucolipidosis-II cultured fibroblasts by 62 and 76% respectively, with a corresponding intracellular accumulation of the enzyme. As shown by lectin binding, the enzyme which accumulated in monensin-treated cells did not contain galactose residues, whereas the corresponding enzyme from nigericin-treated cells was galactosylated. The results suggest that monensin acts at an early point in the process of hydrolase glycosylation, and nigericin acts later, both presumably within the Golgi region, allowing the accumulation of different glycosylated forms of the enzyme. The intra- and extra-cellular distribution of beta-hexosaminidase in ionophore-treated normal cells was essentially unchanged, whereas concanavalin A precipitability of excreted enzyme was increased and its ability to be taken up by deficient fibroblasts was decreased. The bivalent-cation ionophore A23187 (1 microM) reduced beta-hexosaminidase excretion from mucolipidosis-II cells by 82% and by 96% when used with EGTA (1 mM). However, there was no intracellular accumulation of enzyme, suggesting that the effect of this ionophore was restricted to the inhibition of synthesis. It therefore appears that the actual transport of beta-hexosaminidase in mucolipidosis-II cells is affected by univalent-cation ionophores in a selective manner. These findings suggest that individual ionophores could be used to identify the sites of hydrolase oligosaccharide processing in the Golgi region by causing intermediate glycosylated forms of the transported hydrolase to accumulate in a specific Golgi compartment preceding the blocking site of the ionophore. PMID:6231921

  1. Dynamics of proteolytic activity of blood enzymes in combined burn and radiation injury and in its treatment with local cryoeffect and with wound closing preparation RZP-3

    International Nuclear Information System (INIS)

    Gertman, V.Z.

    1982-01-01

    The proteolytic activity of trypsine-like proteases of blood at acute injury period doesn't increase as in animals with mere burn and in animals with nure irradiation as well. It can be explained by the absence of adequate stress reaction of the organism due to the sharp decrease of organism reactivity in case of combined burn and radiation injury. Application of low temperatures and combination of cryogenic effect and RZP-3 preparation promotes proteolytic activity increase in animals with combined burn and radiation injury in the climax of radiation sickness. The normalization of the factor was observed at late periods of the investigation. It can be regarded as recovery of the organism reactivity

  2. Sedimentation, proteolytic activity and proteolysis of whole UHT milk during storage

    Directory of Open Access Journals (Sweden)

    Cláudia Lúcia de Oliveira Pinto

    2017-09-01

    Full Text Available The UHT milk destabilization during storage may be assigned, among other factors, to the activity of endogenous and, or bacterial proteases. This technological problem is a major obstacle in the dairy chain. The aim of this study was to evaluate the microbiological quality of raw milk and its effect on the formation of sediment, degree of proteolysis and proteolytic activity of whole UHT during storage. Mean counts of mesophilic bacteria, psychrotrophic, psychrotrophic proteolytic and Pseudomonas spp. from samples of raw milk were respectively 5.5 x 106 CFU/mL, 3.0 x 106 CFU/mL, 8.0 x 105 CFU/mL and 1.1 x 106 CFU/mL. The UHT treatment has reduced 93.2% of proteolytic activity detected comparing to the proteolytic activity of raw milk. The increase in sediment mass, degree of proteolysis and proteolytic activity of whole UHT milk has been detected during storage. All UHT milk samples have presented low count of mesophilic bacteria and remained thermally stable without evidence of gelation. Considering the residual activity of proteases in UHT milk and its relevance to sedimentation, proteolytic activity and proteolysis, the use of raw milk with low levels of proteolytic psychrotrophic bacteria hould be considered to prevent technological problems as sedimentation and gelation, which reduce significantly product acceptance by consumers besides causing industrial losses.

  3. Proteolytic receptor cleavage in the pathogenesis of blood rheology and co-morbidities in metabolic syndrome. Early forms of autodigestion.

    Science.gov (United States)

    Mazor, Rafi; Schmid-Schönbein, Geert W

    2015-01-01

    Abnormal blood rheological properties seldom occur in isolation and instead are accompanied by other complications, often designated as co-morbidities. In the metabolic syndrome with complications like hypertension, diabetes and lack of normal microvascular blood flow, the underlying molecular mechanisms that simultaneously lead to elevated blood pressure and diabetes as well as abnormal microvascular rheology and other cell dysfunctions have remained largely unknown. In this review, we propose a new hypothesis for the origin of abnormal cell functions as well as multiple co-morbidities. Utilizing experimental models for the metabolic disease with diverse co-morbidities we summarize evidence for the presence of an uncontrolled extracellular proteolytic activity that causes ectodomain receptor cleavage and loss of their associated cell function. We summarize evidence for unchecked degrading proteinase activity, e.g. due to matrix metalloproteases, in patients with hypertension, Type II diabetes and obesity, in addition to evidence for receptor cleavage in the form of receptor fragments and decreased extracellular membrane expression levels. The evidence suggest that a shift in blood rheological properties and other co-morbidities may in fact be derived from a common mechanism that is due to uncontrolled proteolytic activity, i.e. an early form of autodigestion. Identification of the particular proteases involved and the mechanisms of their activation may open the door to treatment that simultaneously targets multiple co-morbidities in the metabolic syndrome.

  4. Macrophage-mediated proteolytic remodeling of the extracellular matrix in atherosclerosis results in neoepitopes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Register, Thomas

    2010-01-01

    in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM...... of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage...

  5. How Does Reading Performance Modulate the Impact of Orthographic Knowledge on Speech Processing? A Comparison of Normal Readers and Dyslexic Adults

    Science.gov (United States)

    Pattamadilok, Chotiga; Nelis, Aubéline; Kolinsky, Régine

    2014-01-01

    Studies on proficient readers showed that speech processing is affected by knowledge of the orthographic code. Yet, the automaticity of the orthographic influence depends on task demand. Here, we addressed this automaticity issue in normal and dyslexic adult readers by comparing the orthographic effects obtained in two speech processing tasks that…

  6. Clinical implications of proteolytic activity imbalance in breast cancer diagnosis.

    Science.gov (United States)

    Swellam, Menha; Soliman, Hanan A; Abdelmaksoud, Mohamed D E; Nageeb, Amira M; El Arab, Lobna R Ezz; Boshnak, Hussein

    2014-01-01

    Matrix metalloproteinase-9 and its tissue inhibitor TIMP-1 have been documented as putative tumor markers because of their involvement in cancer invasion and metastasis. The aim of our study was to elucidate the diagnostic efficacy of proteolytic activity markers among traditional tumor markers (CEA and CA15.3) and clinicopathological variables. Serum samples were withdrawn from 160 individuals (80 patients with primary breast cancer, 40 patients with benign breast lesions and 40 individuals serve as healthy controls). MMP-9 and TIMP-1 were measured by ELISA and gelatin zymography. The best cutoff points for MMP-9 and TIMP-1 were depicted by receiver operating characteristic (ROC) curve. The positivity rates and the median levels for MMP-9 and TIMP-1 showed significant difference among the three investigated groups (Phormonal receptor status (ER, and PgR). MMP zymography results were comparable to those from ELISA. The sensitivity and the specificity of MMP-9, TIMP-1 and MMP-9/TIMP-1 were superior to traditional tumor markers (CEA and CA15.3) especially for early stages (T1) and low grade breast cancer patients. These findings indicate that investigated biomarkers are constructive for early diagnosis of breast cancer and MMP-9/TIMP-1 ratio might be a new significant marker in predicting breast cancer development.

  7. In vitro study of proteolytic degradation of rat histidine decarboxylase.

    Science.gov (United States)

    Olmo, M T; Urdiales, J L; Pegg, A E; Medina, M A; Sánchez-Jiménez, F

    2000-03-01

    Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.

  8. Proteolytic activities in yeast after UV irradiation. Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Schwencke, J.; Moustacchi, E.

    1982-04-01

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD/sup +/ yeast cells after a dose of 50 Jm/sup -2/ of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD/sup +/ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.

  9. New Data Pre-processing on Assessing of Obstructive Sleep Apnea Syndrome: Line Based Normalization Method (LBNM)

    Science.gov (United States)

    Akdemir, Bayram; Güneş, Salih; Yosunkaya, Şebnem

    Sleep disorders are a very common unawareness illness among public. Obstructive Sleep Apnea Syndrome (OSAS) is characterized with decreased oxygen saturation level and repetitive upper respiratory tract obstruction episodes during full night sleep. In the present study, we have proposed a novel data normalization method called Line Based Normalization Method (LBNM) to evaluate OSAS using real data set obtained from Polysomnography device as a diagnostic tool in patients and clinically suspected of suffering OSAS. Here, we have combined the LBNM and classification methods comprising C4.5 decision tree classifier and Artificial Neural Network (ANN) to diagnose the OSAS. Firstly, each clinical feature in OSAS dataset is scaled by LBNM method in the range of [0,1]. Secondly, normalized OSAS dataset is classified using different classifier algorithms including C4.5 decision tree classifier and ANN, respectively. The proposed normalization method was compared with min-max normalization, z-score normalization, and decimal scaling methods existing in literature on the diagnosis of OSAS. LBNM has produced very promising results on the assessing of OSAS. Also, this method could be applied to other biomedical datasets.

  10. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    Science.gov (United States)

    Laura, Richard P.; Dong, David; Reynolds, Wanda F.; Maki, Richard A.

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  11. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    Directory of Open Access Journals (Sweden)

    Richard P Laura

    Full Text Available Among the human heme-peroxidase family, myeloperoxidase (MPO has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO after it exits the endoplasmic reticulum (ER. Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

  12. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    Science.gov (United States)

    Laura, Richard P; Dong, David; Reynolds, Wanda F; Maki, Richard A

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

  13. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Directory of Open Access Journals (Sweden)

    Keyvan Pakshir

    2016-12-01

    Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species.

  14. The influence of radiation on bacterial cells and their proteolytic properties

    International Nuclear Information System (INIS)

    Szulc, M.; Stefaniakowa, A.; Stanczak, B.; Peconek, J.

    1980-01-01

    The suspension of bacterial cells and their spores were exposed to X rays in the environment with and without protein. The doses of radiation ranged from 1 to 100 Gy and in case of spores of B. subtilis from 50 to 1000 Gy. It was found that irradiation to Proteus vulgaris, Pseudomonas fluorescens and Ps. aeruginosa caused an inconsiderable decrease of proteolytic properties of the generation originated from irradiated bacteria. Irradiation of B. subtilis spores did not influence the proteolytic activity of bacterial cells derived from the exposed spores. The degree of wasting away of bacteria exposed to the same radiation was higher than the rate of proteolytic properties decrease. The presence of protein in the surroundings had no influence on proteolytic characteristics of new generations. (author)

  15. Data concerning the proteolytic resistance and oxidative stress in LAN5 cells after treatment with BSA hydrogels

    Directory of Open Access Journals (Sweden)

    Pasquale Picone

    2016-12-01

    Full Text Available Proteolytic resistance is a relevant aspect to be tested in the formulation of new nanoscale biomaterials. The action of proteolytic enzymes is a very fast process occurring in the range of few minutes. Here, we report data concerning the proteolytic resistance of a heat-set BSA hydrogel obtained after 20-hour incubation at 60 °C prepared at the pH value of 3.9, pH at which the hydrogel presents the highest elastic character with respect to gel formed at pH 5.9 and 7.4 “Heat-and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold” (G. Navarra, C. Peres, M. Contardi, P. Picone, P.L. San Biagio, M. Di Carlo, D. Giacomazza, V. Militello, 2016 [1]. We show that the BSA hydrogel produced by heating treatment is protected by the action of proteinase K enzyme. Moreover, we show that LAN5 cells cultured in presence of BSA hydrogels formed at pH 3.9, 5.9 and 7.4 did not exhibit any oxidative stress, one of the first and crucial events causing cell death “Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases?” (M. Di Carlo, D. Giacomazza, P. Picone, D. Nuzzo, P.L. San Biagio, 2012 [2] “Effect of zinc oxide nanomaterials induced oxidative stress on the p53 pathway” (M.I. Setyawati, C.Y. Tay, D.T. Leaong, 2013 [3].

  16. Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements

    OpenAIRE

    Koren, Erez; Apte, Anjali; Sawant, Rupa R.; Grunwald, Jacob; Torchilin, Vladimir P.

    2011-01-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studi...

  17. Proteolytic shedding of the macrophage scavenger receptor CD163 in multiple sclerosis

    DEFF Research Database (Denmark)

    Fabriek, Babs O; Møller, Holger J; Vloet, Rianka P M

    2007-01-01

    MMP-9 levels in controls, but not in MS patients. Moreover, evidence was obtained for CD163-cleaving MMP activity in plasma of MS patients. Finally, the increased proteolytic shedding of CD163 correlated to reduced plasma levels of circulating inflammatory cytokines. Collectively, our results provide...... evidence for proteolytic shedding of CD163 in MS and suggest a possible link to cytokine production. Udgivelsesdato: 2007-Jul...

  18. Process-oriented dose assessment model for 14C due to releases during normal operation of a nuclear power plant

    International Nuclear Information System (INIS)

    Aquilonius, Karin; Hallberg, Bengt

    2005-01-01

    Swedish nuclear utility companies are required to assess doses due to releases of radionuclides during normal operation. In 2001, calculation methods used earlier were updated due to new authority regulations. The isotope 14 C is of special interest in dose assessments due to the role of carbon in the metabolism of all life forms. Earlier, factors expressing the ratio between concentration of 14 C in air and in various plants were used. In order to extend the possibility to take local conditions into account, a process-oriented assessment model for uptake of carbon and doses from releases of 14 C to air was developed (POM 14 C). The model uses part of Daisy which has been developed to model the turnover of carbon in crops. [Hansen, S., Jensen, H.E., Nielsen, N.E., Svendsen, H., 1993. Description of the Soil Plant System Model DAISY, Basic Principles and Modelling Approach. Simulation Model for Transformation and Transport of Energy and Matter in the Soil Plant Atmosphere System. Jordbruksfoerlaget, The Royal Veterianary and Agricultural University, Copenhagen, Denmark]. The main objectives were to test model performance of the former method, and to investigate if taking site specific parameters into account to a greater degree would lead to major differences in the results. Several exposure pathways were considered: direct consumption of locally grown cereals, vegetables, and root vegetables, as well as consumption of milk and meat from cows having eaten fodder cereals and green fodder from the area around the nuclear plant. The total dose of the earlier model was compared with that of POM 14 C. The result of the former was shown to be slightly higher than the latter, but POM 14 C confirmed that the earlier results were of a reasonable magnitude. When full account of local conditions was taken, e.g. as regards solar radiation, temperature, and concentration of 14 C in air at various places in the surroundings of each nuclear plant, a difference in dose between

  19. Use of Proteolytic Enzymes in the Treatment of Proteinaceous Esophageal Food Impaction.

    Science.gov (United States)

    Morse, Christopher R; Wang, Howard; Donahue, Dean M; Garrity, Julie M; Allan, James S

    2016-01-01

    Proteinaceous esophageal food impaction typically requires endoscopic intervention. An alternative approach is the use of proteolytic enzymes. Concerns regarding the use of proteolytic enzymes include the risk of perforation and aspiration pneumonitis. We retrospectively reviewed our series of 69 patients treated with papain to determine the safety and efficacy of proteolytic enzymes. Patients were retrospectively reviewed if treated for an esophageal food impaction from 1999 through 2008. Median age was 56 years (range 19-91 years), with 46 male and 23 female patients. In 27 patients (39%) this was their first presentation, in 14 (20%) it was the second, and 28 (41%) had multiple previous episodes. Meat was the cause in 49 (71%), chicken in 6 (9%), fish in 3 (4%), and unspecified in 11 (16%). All patients presented with dysphagia for solids, 56 (81%) could not tolerate liquids. Papain solution, 1 tsp in 8 oz of water, was given to patients in an unlimited quantity. Papain was successful in relieving the obstruction in 60 patients (87%). The remaining 9 patients (13%) underwent endoscopy with successful retrieval. No patient suffered a perforation, either with papain ingestion or endoscopy. There were no episodes of pneumonitis or pneumonia. We have used proteolytic enzymes with a high success rate and with minimal complication. Further, if proteolytic enzymes fail, endoscopy can be performed safely and effectively. We recommend the use of proteolytic enzymes as the initial management in all patients with proteinaceous food impaction of the esophagus. Published by Elsevier Inc.

  20. The function of sentence accents and given/new information in speech processing: different strategies for normal-hearing and hearing-impaired listeners?

    Science.gov (United States)

    van Donselaar, W; Lentz, J

    1994-01-01

    Two experiments were carried out to investigate how the correspondence between sentence accentuation and distribution of information is used in human word processing. A forced-choice task with target words embedded in sentences was employed for this purpose. Target words provided either 'given' or 'new' information, and were either accented or unaccented. The subjects had to choose between two words that differed in the last consonant by one phonetic feature (e.g., mouth/mouse). The first experiment involved both normal-hearing and hearing-impaired listeners. A fixed amount of noise was used to reduce the quality of speech for normal-hearing listeners, in order to enable a comparison between the two listener groups. The results of the first experiment showed different processing patterns for normal-hearing and hearing-impaired listeners. The hearing-impaired listeners were more accurate with words that were properly accented for their information value, whereas the normal-hearing subjects were more accurate with accented than unaccented words regardless of their information value. A new group of normal-hearing subjects was tested in a second experiment with speech of more severely reduced quality. The results indicated that, under these circumstances, the normal-hearing listeners changed their strategy and also showed an interaction between information value and accent. It seems that, as speech becomes less intelligible, listeners depend increasingly on linguistic expectations stemming from the correlation between information value and accentuation.

  1. Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing.

    Science.gov (United States)

    Parker, M D; Barrett, P I; Shepherd, J; Price, L J; Bull, S D

    2015-01-01

    Under many of the conditions studied, a two-strain cocktail of non-toxigenic Clostridium spp. was found to be suitable as a surrogate for non-proteolytic Clostridium botulinum, and has the potential for use in chilled food challenge tests measuring growth. Non-toxigenic surrogates could also be used in thermal process screening studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  3. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD).

    Science.gov (United States)

    Yu, Wei-Hsuan; Huang, Po-Tsang; Lou, Kuo-Long; Yu, Shuan-Su C; Lin, Chen

    2012-05-29

    The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are

  4. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    Science.gov (United States)

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  5. Lateralized speech perception in normal-hearing and hearing-impaired listeners and its relationship to temporal processing

    DEFF Research Database (Denmark)

    Locsei, Gusztav; Pedersen, Julie Hefting; Laugesen, Søren

    2016-01-01

    This study investigated the role of temporal fine structure (TFS) coding in spatially complex, lateralized listening tasks. Speech reception thresholds (SRTs) were measured in young normal-hearing (NH) and two groups of elderly hearing-impaired (HI) listeners in the presence of speech-shaped noise...... and different interfering talker conditions. The HI subjects had either a mild or moderate hearing loss above 1.5 kHz and reduced audibility was compensated for individually in the speech tests. The target and masker streams were presented as coming from the same or from the opposite side of the head...... with normal hearing in the low-frequency range might have elevated SRTs, the binaural benefit they experience due to spatial separation of competing sources can remain similar to that of NH listeners....

  6. Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico

    Directory of Open Access Journals (Sweden)

    Miguel Borja

    2018-01-01

    Full Text Available Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s, such as Mojave toxin, and snake venom metalloproteinases (SVMPs. In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus are limited and little is known about the biological and proteolytic activities in this species. Tissue (34 and venom (29 samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR and protein (by ELISA levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28 and Hide Powder Azure proteolytic analysis (n = 27. Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II, with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I, without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.

  7. Fungal inhibitors of proteolytic enzymes: classification, properties, possible biological roles, and perspectives for practical use.

    Science.gov (United States)

    Dunaevsky, Y E; Popova, V V; Semenova, T A; Beliakova, G A; Belozersky, M A

    2014-06-01

    Peptidase inhibitors are ubiquitous regulatory proteins controlling catalytic activity of proteolytic enzymes. Interest in these proteins increased substantially after it became clear that they can be used for therapy of various important diseases including cancer, malaria, and autoimmune and neurodegenerative diseases. In this review we summarize available data on peptidase inhibitors from fungi, emphasizing their properties, biological role, and possible practical applications of these proteins in the future. A number of fungal peptidase inhibitors with unique structure and specificity of action have no sequence homology with other classes of peptidase inhibitors, thus representing new and specific candidates for therapeutic use. The main classifications of inhibitors in current use are considered. Available data on structure, mechanisms and conditions of action, and diversity of functions of peptidase inhibitors of fungi are analyzed. It is mentioned that on one side the unique properties of some inhibitors can be used for selective inhibition of peptidases responsible for initiation and development of pathogenic processes. On the other side, general inhibitory activity of other inhibitors towards peptidases of various catalytic classes might be able to provide efficient defense of transgenic plants against insect pests by overcoming compensatory synthesis of new peptidases by these pests in response to introduction of a fungal inhibitor. Together, the data analyzed in this review reveal that fungal inhibitors extend the spectrum of known peptidase inhibitors potentially suitable for use in medicine and agriculture. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry.

    Science.gov (United States)

    Gallagher, Elyssia S; Hudgens, Jeffrey W

    2016-01-01

    Biological processes are the result of noncovalent, protein-ligand interactions, where the ligands range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Amide groups within proteins constantly exchange protons with water. When immersed in heavy water (D2O), mass spectrometry (MS) can measure the change of mass associated with the hydrogen to deuterium exchange (HDX). Protein-ligand interactions modify the hydrogen exchange rates of amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This chapter describes a protocol for conducting bottom-up, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interface. This tutorial outlines the fundamental theory governing hydrogen exchange; provides practical information regarding the preparation of protein samples and solutions; and describes the exchange reaction, reaction quenching, enzymatic digestion, chromatographic separation, and peptide analysis by MS. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Additionally, two HDX-MS case studies are used to illustrate protein-ligand interactions involving: (1) a continuous sequence of interacting residues and (2) a set of discontinuously numbered residues, residing spatially near each other. © 2016 Elsevier Inc. All rights reserved.

  9. A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance

    Directory of Open Access Journals (Sweden)

    Mehdi Kabbage

    2016-04-01

    Full Text Available The Bcl-2-associated athanogene (BAG family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1, including four with a domain organization similar to animal BAGs (BAG1-4. The remaining three members (BAG5-7 contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2. The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants.

  10. Proteolytic activity in some freshwater animals and associated microflora in a wide pH range.

    Science.gov (United States)

    Kuz'mina, V V; Zolotareva, G V; Sheptitskiy, V A

    2017-04-01

    Proteolytic activity in some freshwater animals (crustacean plankton, sandhopper Amphipoda sp., larvae of chironomids Chironomus sp., oligochaetes Oligohaeta sp., dreissena Dreissena polymorpha, roach Rutilus rutilus heckelii, rudd Scardinius erythrophthalmus, ruff Acerina cernua, and monkey goby Neogobius fluviatilis) prevailing within the food of fishes of various ecological groups as well as in their associated microflora in a wide pH range was investigated. It has been shown that the optimum pH of proteases in the animals' whole organism varies: 6.0 for sandhopper; 8.0 for chironomid larvae, oligochaetes, monkey goby, and ruff; 8.0-9.0 for zooplankton; and 10.0 for roach and rudd. The optimum pH of associated microflora proteases is 6.0 for monkey goby; 7.0 for sandhopper and roach and ruff ; 8.0-9.0 for oligochaetes; 9.0 for zooplankton; and 10.0 for chironomid larvae and rudd. The compensatory role of food items and enteric microbiota proteases in digestive processes in fish of different ecological groups at low pH is discussed.

  11. Enzymatic surface hydrolysis of polyamide 6,6 with mixtures of proteolytic and lipolytic enzymes.

    Science.gov (United States)

    Parvinzadeh Gashti, Mazeyar; Assefipour, Reza; Kiumarsi, Amir; Parvinzadeh Gashti, Mahyar

    2013-01-01

    This study investigated the changes induced on nylon 6,6 fabric by a mixture of proteolytic and lipolytic enzymes. Technical measurements were studied including those of Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), weight loss (WL), bending lengths (BL), scanning electron microscopy (SEM), moisture absorbency (MA), and reflectance spectroscopy (RS). For this purpose, nylon 6,6 fabrics were treated separately with different concentrations of protease and lipase mixtures in solution. The dyeing process was then carried out on the treated fabrics with two reactive and acid dyes. The intensity of major peaks in the FTIR spectra of the protease-treated samples is in favor of chemical changes the polypeptide functional groups in the fabrics. Thermal studies also show a significant decrease in the thermal degradation temperature of the treated polymer at temperatures higher than 400°C. The protease and lipase mixtures decreased the sample weight, while lipase intensified the weight loss comparing with protease. It was observed that the concentration of lipase enzyme had a direct influence on the darkness of dyed samples.

  12. Biophysical and biochemical models of mechanisms of cellular development via the cellular cycle in normal tissue, cancerous tissue, and inflammatory processes.

    Science.gov (United States)

    Ponizovskiy, M R

    2013-01-01

    The significant separate biochemical discoveries of pro- and anti-apoptotic -autophagy, and -proliferative processes in normal and pathology cells were learned in detail from the point of view of biophysics, physical chemistry, and thermodynamics, which made possible the proposal that a common mechanism relates to all of these processes: intracellular balances in catabolic and anabolic processes interconnect with extracellular balances, promoting and maintaining the stability of internal medium and internal energy of cells as well as normal cell development. Nevertheless, violations to these interconnections of intracellular and extracellular balances promote pathologic processes. The study of cellular cycle mechanisms in normal cells explained the mechanisms of the maintenance of stability of the internal medium and internal energy of cells as a component of the overall stability of an organism. It explained the development of the cellular cycle as the oscillating changes in the flow of energy and substances. In addition, violations to mechanisms of the maintenance of stability of the cellular internal medium and internal energy in cancer tissue were elucidated and compared with violations of these mechanisms in inflammatory processes. All of this eliminated a lot of doubts and queries that were expressed by the authors of some experiments.

  13. The odontoid process invagination in normal subjects, Chiari malformation and Basilar invagination patients: Pathophysiologic correlations with angular craniometry.

    Science.gov (United States)

    Ferreira, Jânio A; Botelho, Ricardo V

    2015-01-01

    Craniometric studies have shown that both Chiari malformation (CM) and basilar invagination (BI) belong to a spectrum of malformations. A more precise method to differentiate between these types of CVJM is desirable. The Chamberlain's line violation (CLV) is the most common method to identify BI. The authors sought to clarify the real importance of CLV in the spectrum of craniovertebral junction malformations (CVJM) and to identify possible pathophysiological relationships. We evaluated the CLV in a sample of CVJM, BI, CM patients and a control group of normal subjects and correlated their data with craniocervical angular craniometry. A total of 97 subjects were studied: 32 normal subjects, 41 CM patients, 9 basilar invagination type 1 (BI1) patients, and 15 basilar invagination type 2 (BI2) patients. The mean CLV violation in the groups were: The control group, 0.16 ± 0.45 cm; the CM group, 0.32 ± 0.48 cm; the BI1 group, 1.35 ± 0.5 cm; and the BI2 group, 1.98 ± 0.18 cm. There was strong correlation between CLV and Boogard's angle (R = 0.82, P = 0.000) and the clivus canal angle (R = 0.7, P = 0.000). CM's CLV is discrete and similar to the normal subjects. BI1 and BI2 presented with at least of 0.95 cm CLV and these violations were strongly correlated with a primary cranial angulation (clivus horizontalization) and an acute clivus canal angle (a secondary craniocervical angle).

  14. [Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

    Science.gov (United States)

    Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S

    2014-01-01

    The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

  15. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    Science.gov (United States)

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Psychometric Evaluation of Children with Auditory Processing Disorder (APD): Comparison with Normal-Hearing and Clinical Non-APD Groups

    Science.gov (United States)

    Iliadou, Vasiliki; Bamiou, Doris Eva

    2012-01-01

    Purpose: To investigate the clinical utility of the Children's Auditory Processing Performance Scale (CHAPPS; Smoski, Brunt, & Tannahill, 1992) to evaluate listening ability in 12-year-old children referred for auditory processing assessment. Method: This was a prospective case control study of 97 children (age range = 11;4 [years;months] to…

  17. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    Science.gov (United States)

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  18. Rupture Process During the Mw 8.1 2017 Chiapas Mexico Earthquake: Shallow Intraplate Normal Faulting by Slab Bending

    Science.gov (United States)

    Okuwaki, R.; Yagi, Y.

    2017-12-01

    A seismic source model for the Mw 8.1 2017 Chiapas, Mexico, earthquake was constructed by kinematic waveform inversion using globally observed teleseismic waveforms, suggesting that the earthquake was a normal-faulting event on a steeply dipping plane, with the major slip concentrated around a relatively shallow depth of 28 km. The modeled rupture evolution showed unilateral, downdip propagation northwestward from the hypocenter, and the downdip width of the main rupture was restricted to less than 30 km below the slab interface, suggesting that the downdip extensional stresses due to the slab bending were the primary cause of the earthquake. The rupture front abruptly decelerated at the northwestern end of the main rupture where it intersected the subducting Tehuantepec Fracture Zone, suggesting that the fracture zone may have inhibited further rupture propagation.

  19. Supplemental selenium improves wheat grain yield and quality through alterations in biochemical processes under normal and water deficit conditions.

    Science.gov (United States)

    Nawaz, Fahim; Ashraf, M Y; Ahmad, R; Waraich, E A; Shabbir, R N; Bukhari, M A

    2015-05-15

    The paper mainly reported the effects of exogenous selenium (Se) supply (Se seed priming, Se fertigation and Se foliar spray) on physiological and antioxidant system of wheat aiming to clarify its effect on yield and nutritional quality of wheat under both normal and water deficit conditions. Water stress markedly decreased the grain Se, iron (Fe), phosphorous (P), zinc (Zn) and magnesium (Mg) contents. Supplemental Se (Na2SeO4) improved the yield and quality of water stressed plants due to enhancement in the production of osmoprotectants and increased activity of antioxidant enzymes. The foliar spray of Se was more effective than Se fertigation and Se seed treatment. To the best of our knowledge, this is the first elaborate study that involved various Se application methods to evaluate the efficiency of Se supply to plants that would be crucial to develop better understanding of Se translocation and accumulation within crop plants under drought stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein

    Science.gov (United States)

    Hoashi, Toshihiko; Sato, Shinichi; Yamaguchi, Yuji; Passeron, Thierry; Tamaki, Kunihiko; Hearing, Vincent J.

    2010-01-01

    Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation-related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane-bound glycoprotein that shows high homology with a well-known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART-1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART-1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi-modified form of GPNMB and that the PKC and Ca2+ intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.—Hoashi, T., Sato, S., Yamaguchi, Y., Passeron, T., Tamaki, K., Hearing, V. J. Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein. PMID:20056711

  1. Use of vaccinia virus vectors to study protein processing in human disease. Normal nerve growth factor processing and secretion in cultured fibroblasts from patients with familial dysautonomia.

    OpenAIRE

    Edwards, R H; Rutter, W J

    1988-01-01

    Familial dysautonomia is a hereditary disorder that affects autonomic and sensory neurons. Nerve growth factor (NGF) is required for the normal development of sympathetic and sensory neurons and it has been postulated that an abnormality involving NGF may be responsible for familial dysautonomia. Previous studies have shown that the beta-NGF gene is not linked to the disease. However, NGF appears to be abnormal by immunochemical assays; the putative altered form of NGF could result from a dis...

  2. Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

    Science.gov (United States)

    Settachaimongkon, Sarn; Nout, M J Robert; Antunes Fernandes, Elsa C; Hettinga, Kasper A; Vervoort, Jacques M; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J; van Valenberg, Hein J F

    2014-05-02

    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Gene expression of proteolytic systems and growth regulators of skeletal muscle in horses with myopathy associated with pituitary pars intermedia dysfunction.

    Science.gov (United States)

    Aleman, Monica; Nieto, Jorge E

    2010-06-01

    To investigate gene expression of the major proteolytic systems and growth regulators in skeletal muscle of horses with myopathy associated with pituitary pars intermedia dysfunction (PPID). 14 horses with PPID-associated myopathy and 7 healthy control horses. Horses with PPID and controls were age matched (15 to 28 years old). Muscle biopsy specimens were collected from both groups and processed for RNA and cDNA extraction. Validation of the most stable housekeeping genes for skeletal muscle was performed and used to compare gene expression of the following proteolytic systems: cysteine aspartate protease-dependent systems (caspases), lysosomal-dependent systems (cathepsins), non-lysosomal calcium protease-dependent systems (calpains), and ubiquitin-proteasome-dependent systems (ubiquitins). Gene expression of negative regulators of muscle growth (myostatin and inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha) was also determined. No significant difference between groups was detected in expression of the major proteolytic systems except for m-calpain, which was greater in horses with PPID. No differences in gene expression of myostatin and interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha were detected between groups. Greater expression of m-calpain may suggest that calpains play an important role in development of muscle atrophy in horses with PPID. However, because posttranslational events may alter protein activation, inactivation, and functions not studied here, other mechanisms of muscle atrophy cannot be excluded.

  4. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    Directory of Open Access Journals (Sweden)

    Gastón Ezequiel Ortiz

    2016-01-01

    Full Text Available A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea, quotient energy (Q10, Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

  5. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    Science.gov (United States)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  6. Proteolytic activity in Fasciola hepatica is reduced by the administration of cathepsin L mimotopes.

    Science.gov (United States)

    Villa-Mancera, A; Quiroz-Romero, H; Correa, D; Alonso, R A

    2011-03-01

    The objective of this study was to assess the proteolytic activity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolytic activity in hepatic tissue was reduced after the immunization with phage clones.

  7. Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

    A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum...... for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase...

  8. Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity.

    Science.gov (United States)

    Lü, Fan; Bize, Ariane; Guillot, Alain; Monnet, Véronique; Madigou, Céline; Chapleur, Olivier; Mazéas, Laurent; He, Pinjing; Bouchez, Théodore

    2014-01-01

    Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.

  9. Brunovsky Normal Form of Monod Kinetics Models and Growth Rate Control of a Fed-batch Cultivation Process

    Directory of Open Access Journals (Sweden)

    Pavlov Y.

    2007-12-01

    Full Text Available A mathematical methodology that gives assistance to design of fed-batch stabilization and control is presented. The methodology is based both on Utility theory and optimal Control theory. The Utility theory deals with the expressed subjective preferences and allows for the expert preferences to be taken in consideration in complex biotechnological systems as criteria for control and optimization. The Control theory is used for parameters stabilization of a fed-batch cultivation process. The control is written based on information of the growth rate. The simulations show good efficiency of the control laws.

  10. Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate

    International Nuclear Information System (INIS)

    Maliopoulou, T.B.; Dionyssiou-Asteriou, A.; Loucopoulos, D.

    1980-01-01

    A simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5- 3 H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products. (Auth.)

  11. Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

    Science.gov (United States)

    El Najjar, Farah; Lampe, Levi; Baker, Michelle L.; Wang, Lin-Fa; Dutch, Rebecca Ellis

    2015-01-01

    Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing. PMID:25706132

  12. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  13. The difference between the crystallization processes induced by mechanical milling and annealing under normal and high pressure in amorphous Fe-N alloy

    CERN Document Server

    LiuLi; Guo Xing Yuan; Zhao Xu Dong; Yao Bin; Su Wen Hui

    2002-01-01

    An amorphous Fe-N alloy was prepared by ball milling a mixture of Fe and h-BN. Its crystallization processes induced by mechanical milling (MM) and annealing under normal and high pressure were studied. The crystallization product of the amorphous Fe-N alloy induced by MM and annealing at temperatures between 690 and 800 K under pressures of 3-4 GPa is epsilon-Fe sub x N, while the thermal crystallization product under normal pressure is gamma'-Fe sub 4 N. The difference between the crystallization products produced by mechanical and thermal crystallization is attributed to the effects of local pressure and local temperature produced by ball collisions.

  14. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella.

    Science.gov (United States)

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C; Austin, John; Overall, Christopher M; Doxey, Andrew C

    2017-09-12

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per  ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.So far no enzymatic activity has been attributed to flagellin, the major component of bacterial flagella. Here the authors use bioinformatic analysis and identify a metallopeptidase insertion in flagellins from 74 bacterial species and show that recombinant flagellin and flagellar filaments have proteolytic activity.

  15. Proteolytic stability in colloidal systems : interaction of proteins with the solid-water interface

    NARCIS (Netherlands)

    Maste, M.C.L.

    1996-01-01


    Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of

  16. Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

    Directory of Open Access Journals (Sweden)

    Dewi Fitriani

    2017-09-01

    Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when β-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

  17. Effect of Four Commonly Used Dissolution Media Surfactants on Pancreatin Proteolytic Activity.

    Science.gov (United States)

    Guncheva, Maya; Stippler, Erika

    2017-05-01

    Proteolytic enzymes are often used in dissolution testing of cross-linked gelatin capsules that do not conform to the dissolution specification. Their catalytic activity, however, can be affected when they are added to a dissolution media containing solubility enhancers, such as surfactants. The aim of this study was to assess the activity of pancreatic proteases in presence of four commonly used surfactants. We found that pancreatin exhibits remarkable proteolytic activity in the presence of Tween 80, even at the concentrations as high as 250 times its critical micelle concentration (cmc) in water, whereas, Triton X-100 enhanced the proteolytic activity of pancreatin when added at concentrations above its cmc in water. Both surfactants are non-ionic surfactants. On the other hand, sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), which are ionic surfactants, have a detrimental effect on the proteolytic activity of pancreatin. For example, a 50% reduction of the pancreatin activity was found in samples which contain a minor amount of SDS (0.05% w/v) in comparison to a surfactant-free reaction. Additionally, no activity was observed for the pancreatin-SDS samples which were incubated for 30 min at 40°C prior to testing. CTAB had an impact on pancreatin activity at concentrations higher than its cmc. Data from this manuscript can be used as a benchmark for optimization of the dissolution procedures that require use of both surfactants and enzymes.

  18. Genetics of Proteolytic Enzymes of Lactococci and Their Role in Cheese Flavor Development

    NARCIS (Netherlands)

    Kok, Jan

    In recent years, knowledge of the genetics and biochemistry of the enzymes that constitute the proteolytic system of starter lactococci has increased tremendously. This paper summarizes the data obtained largely in the last 5 yr of intensive research by various research groups operative in this

  19. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    Science.gov (United States)

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  20. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability

    Science.gov (United States)

    Tur-Arlandis, Gema; Rodriguez-Larrea, David; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2010-01-01

    We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level. PMID:20303845

  2. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  3. Matrix metalloproteinase-14 triggers an anti-inflammatory proteolytic cascade in endotoxemia.

    Science.gov (United States)

    Aguirre, Alina; Blázquez-Prieto, Jorge; Amado-Rodriguez, Laura; López-Alonso, Inés; Batalla-Solís, Estefanía; González-López, Adrián; Sánchez-Pérez, Moisés; Mayoral-Garcia, Carlos; Gutiérrez-Fernández, Ana; Albaiceta, Guillermo M

    2017-05-01

    ᅟ: Matrix metalloproteinases can modulate the inflammatory response through processing of cyto- and chemokines. Among them, MMP-14 is a non-dispensable collagenase responsible for the activation of other enzymes, triggering a proteolytic cascade. To identify the role of MMP-14 during the pro-inflammatory response, wildtype and Mmp14 -/- mice were challenged with lipopolysaccharide. MMP-14 levels decreased after endotoxemia. Mutant animals showed 100% mortality, compared to 50% in wildtype mice. The increased mortality was related to a more severe lung injury, an impaired lung MMP-2 activation, and increased levels of the alarmin S100A9. There were no differences in the expression of other mediators including Il6, Cxcl2, Tgfb, Il10, or S100a8. A similar result was observed in lung explants of both genotypes cultured in presence of lipopolysaccharide. In this ex vivo model, exogenous activated MMP-2 ameliorated the observed increase in alarmins. Samples from septic patients showed a decrease in serum MMP-14 and activated MMP-2 compared to non-septic critically ill patients. These results demonstrate that the MMP-14-MMP-2 axis is downregulated during sepsis, leading to a proinflammatory response involving S100A9 and a more severe lung injury. This anti-inflammatory role of MMP-14 could have a therapeutic value in sepsis. • MMP-14 levels decrease in lungs from endotoxemic mice and serum from septic patients. • Mmp14 -/- mice show increased lung injury and mortality following endotoxemia. • Absence of Mmp14 decreases activated MMP-2 and increases S100A9 levels in lung tissue. • MMP-14 ameliorates inflammation by promoting S100A9 cleavage by activated MMP-2.

  4. Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

    Directory of Open Access Journals (Sweden)

    Sojka Daniel

    2010-12-01

    Full Text Available Abstract Background Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes. Results This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding. Conclusions Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.

  5. An EMG-to-Force Processing approach for estimating in vivo hip muscle forces in normal human walking.

    Science.gov (United States)

    Bogey, Ross A; Barnes, Lee A

    2016-10-12

    The force of a single muscle is not directly measurable without invasive methods. Yet invasive techniques are not appropriate for clinical use, thus a non-invasive technique that combined the electromyographic (EMG) signal and a neuromuscular model was developed to determine in vivo active muscle forces at the hip. The EMG-to-force processing (EFP) model included active and passive moment components, and the net EFP moment was compared with the hip moment obtained with standard inverse dynamics techniques ("gold standard"). The two methods were compared at percent gait cycle intervals, and the correlation coefficient between methods was excellent (r2=91). The closeness of fit confirms that the approach is a reasonable approximation of net moment and, possibly, individual muscle forces. The greatest estimated hip force was produced by a hip abductor. A novel finding was that the hip adductors did not behave a single synergistic group. The Adductor Magnus synergistically assisted other hip extensors, and produced forces that were out-of-phase with the other hip adductor forces. Rectus Femoris was only active during hip flexion (not knee extension).

  6. Proteolytic compartmentalization and activity in the midgut of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae

    Directory of Open Access Journals (Sweden)

    S. Sorkhabi-Abdolmaleki

    2013-04-01

    Full Text Available Digestive proteolytic activity in the alimentary canal of Andrallus spinidens, a potential biocontrol agent of lepidopteran larvae, was studied by considering enzyme compartmentalization and diversity. The alimentary canal of adults consists of a foregut, a four- sectioned midgut, namely V1 to V4 (ventriculus, and a hindgut. The optimal pH for general proteolytic activity was found to be at pH 8 with a small peak at pH 6. Results revealed that there are several specific proteases in the midgut of A. spinidens, including trypsin-like, chymotrypsin-like, and elastase as serine proteases, and cathepsins B, L and D as cysteine proteases, in addition to two exopeptidases of carboxy- and aminopetidases. Compartmentalization of digestive proteolytic activity showed that V3 is the main area of proteolytic secretion for both general and specific proteases and that V4 has the lowest enzymatic role, so that four out of the eight specific proteases found showed no activity in this section. The lowest and the highest proteolytic activity was found to be in the 1st and 4th nymphal instars, respectively. Using the specific inhibitors phenylmethylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, Ntosyl- L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinyl-leucylamido-( 4-guanidino-butane, cystatin, phenanthroline and ethylendiamidetetraacetic acid, we verified the presence of all specific proteases noted using both biochemical assays and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Our findings demonstrated that A. spinidens could utilize several caterpillars because of the presence of various of proteases in its midgut.

  7. The putative role of proteolytic pathways in the pathogenesis of Type 1 diabetes mellitus: the 'autophagy' hypothesis.

    Science.gov (United States)

    Fierabracci, Alessandra

    2014-05-01

    Autoimmune diseases are a heterogeneous group of disorders affecting different organs and tissues. New tools, such as genome-wide association studies, have provided evidence for new susceptibility loci and candidate genes in the disease process including common susceptibility genes involved in the immunological synapse and T cell activation. Close linkages have been found in a number of diseases, including ankylosing spondylitis, multiple sclerosis, Crohn's disease and insulin-dependent diabetes mellitus (Type 1 diabetes mellitus). Evidence for some association with Type 1 diabetes was previously found in the region containing 5q15/ERAP1 (endoplasmic reticulum aminopeptidase 1) (rs30187, ARTS1). Recent data suggest that in eukaryotic cells in addition to the ubiquitin/proteasome system another proteolytic pathway may have a significant role in the autoimmunity process, i.e. the autophagic pathway which constitutes the principal regulated catabolic process mediated by lysosomes. Autophagy could play a role in MHC class I and class II self-antigen presentation at the basis of the autoimmunity process. Furthermore cross-talk among different proteolytic pathways was recently highlighted i.e. components processed in the ubiquitin/proteasome system possibly engaged in autophagic pathways. T1D is an autoimmune disease characterised by the destruction of pancreatic beta cells by autoreactive T cells. Immunological abnormalities can precede months to years the initial symptoms and clinical diagnosis. Our hypothesis suggests that in the autoimmune process autophagy can intervene at different levels, during the thymic selection process of T lymphocytes causing escape of autoreactive T cells, at the initiation stage of the disease, in the preclinical period or subsequently to the disease onset having a role at the level of perpetuation of the autoimmunity process. Supporting evidence derives from the already reported discovery of polymorphisms in autophagy-related genes in

  8. What work has to be done to implement collaborative care for depression? Process evaluation of a trial utilizing the Normalization Process Model.

    Science.gov (United States)

    Gask, Linda; Bower, Peter; Lovell, Karina; Escott, Diane; Archer, Janine; Gilbody, Simon; Lankshear, Annette J; Simpson, Angela E; Richards, David A

    2010-02-10

    There is a considerable evidence base for 'collaborative care' as a method to improve quality of care for depression, but an acknowledged gap between efficacy and implementation. This study utilises the Normalisation Process Model (NPM) to inform the process of implementation of collaborative care in both a future full-scale trial, and the wider health economy. Application of the NPM to qualitative data collected in both focus groups and one-to-one interviews before and after an exploratory randomised controlled trial of a collaborative model of care for depression. Findings are presented as they relate to the four factors of the NPM (interactional workability, relational integration, skill-set workability, and contextual integration) and a number of necessary tasks are identified. Using the model, it was possible to observe that predictions about necessary work to implement collaborative care that could be made from analysis of the pre-trial data relating to the four different factors of the NPM were indeed borne out in the post-trial data. However, additional insights were gained from the post-trial interview participants who, unlike those interviewed before the trial, had direct experience of a novel intervention. The professional freedom enjoyed by more senior mental health workers may work both for and against normalisation of collaborative care as those who wish to adopt new ways of working have the freedom to change their practice but are not obliged to do so. The NPM provides a useful structure for both guiding and analysing the process by which an intervention is optimized for testing in a larger scale trial or for subsequent full-scale implementation.

  9. Understanding the challenges to implementing case management for people with dementia in primary care in England: a qualitative study using Normalization Process Theory.

    Science.gov (United States)

    Bamford, Claire; Poole, Marie; Brittain, Katie; Chew-Graham, Carolyn; Fox, Chris; Iliffe, Steve; Manthorpe, Jill; Robinson, Louise

    2014-11-08

    Case management has been suggested as a way of improving the quality and cost-effectiveness of support for people with dementia. In this study we adapted and implemented a successful United States' model of case management in primary care in England. The results are reported elsewhere, but a key finding was that little case management took place. This paper reports the findings of the process evaluation which used Normalization Process Theory to understand the barriers to implementation. Ethnographic methods were used to explore the views and experiences of case management. Interviews with 49 stakeholders (patients, carers, case managers, health and social care professionals) were supplemented with observation of case managers during meetings and initial assessments with patients. Transcripts and field notes were analysed initially using the constant comparative approach and emerging themes were then mapped onto the framework of Normalization Process Theory. The primary focus during implementation was on the case managers as isolated individuals, with little attention being paid to the social or organizational context within which they worked. Barriers relating to each of the four main constructs of Normalization Process Theory were identified, with a lack of clarity over the scope and boundaries of the intervention (coherence); variable investment in the intervention (cognitive participation); a lack of resources, skills and training to deliver case management (collective action); and limited reflection and feedback on the case manager role (reflexive monitoring). Despite the intuitive appeal of case management to all stakeholders, there were multiple barriers to implementation in primary care in England including: difficulties in embedding case managers within existing well-established community networks; the challenges of protecting time for case management; and case managers' inability to identify, and act on, emerging patient and carer needs (an essential, but

  10. Proteolytic and lipolytic microbiota of refrigerated raw milk from northeast and southern regions of Brazil

    Directory of Open Access Journals (Sweden)

    Jose Carlos Ribeiro Junior

    2015-12-01

    Full Text Available The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20 or small (26 according to the average daily production. All producers of Maranhão were considered small (<500L/day. The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05 lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently

  11. Motor Nerve Arborization Requires Proteolytic Domain of Damage-Induced Neuronal Endopeptidase (DINE) during Development.

    Science.gov (United States)

    Matsumoto, Sakiko; Kiryu-Seo, Sumiko; Kiyama, Hiroshi

    2016-04-27

    Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease, which we originally identified as a nerve regeneration-associated molecule. Abundant expression of DINE is observed in regenerating neurons, as well as in developing spinal motor neurons. In line with this, DINE-deficient (DINE KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form proper neuromuscular junctions (NMJ), which lead to death shortly after birth. However, it is unclear whether protease activity of DINE is involved in motor nerve terminal arborization and how DINE participates in the process. To address these issues, we performed an in vivo rescue experiment in which three types of motor-neuron specific DINE transgenic mice were crossed with DINE KO mice. The DINE KO mice, which overexpressed wild-type DINE in motor neurons, succeeded in rescuing the aberrant nerve terminal arborization and lethality after birth, while those overexpressing two types of protease domain-mutated DINE failed. Further histochemical analysis showed abnormal behavior of immature Schwann cells along the DINE-deficient axons. Coculture experiments of motor neurons and Schwann cells ensured that the protease domain of neuronal DINE was required for proper alignment of immature Schwann cells along the axon. These findings suggest that protease activity of DINE is crucial for intramuscular innervation of motor nerves and subsequent NMJ formation, as well as proper control of interactions between axons and immature Schwann cells. Damage-induced neuronal endopeptidase (DINE) is a membrane-bound metalloprotease; expression is abundant in developing spinal motor neurons, as well as in nerve-injured neurons. DINE-deficient (KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form a neuromuscular junction, leading to death immediately after birth. To address whether proteolytic activity of DINE is involved in this

  12. Fasciola hepatica proteolytic activity in liver revealed by in situ zymography.

    Science.gov (United States)

    Alcalá-Canto, Yazmín; Ibarra-Velarde, Froylán; Gracia-Mora, Jesús; Sumano-López, Héctor

    2005-07-01

    Fasciola hepatica secretes cysteine proteases that play a role in facilitating parasite migration. The aim of this study was to detect the inhibition of the proteolytic activity of F. hepatica cysteine proteases in the liver of C57BL/6 cathepsin B knockout mice (cat B-/-) and wild-type controls (cat B+/+) by intraperitoneal administration of N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine, (E-64) using the film in situ zymography (FIZ) technique and image analysis. The FIZ technique revealed that intraperitoneal administration of E-64 dramatically reduced (85%) F. hepatica proteolytic activity in the liver of experimentally infected mice with no discernable side effects. These results suggest the usefulness of the FIZ for determining in vivo activity of F. hepatica proteases, as well as their inhibition by intraperitoneal administration of E-64 in hepatic tissue of infected mice.

  13. PROTEOLYTIC AND FIBRINOLYTIC ACTIVITIES OF HALOPHILIC LACTIC ACID BACTERIA FROM TWO INDONESIAN FERMENTED FOODS

    Directory of Open Access Journals (Sweden)

    Asep A. Prihanto

    2013-04-01

    Full Text Available Exploration of fermented foods as sources of fibrinolytic enzymes is increased in the last decades. Terasi and Jambal roti is Indonesian traditional fermented fish products, which were famous in Java Island. Both are important products in Indonesian dishes, especially in Java. Investigation on halophilic lactic acid bacteria using MRS and M-17 agar obtained seventy four isolated strains. Their proteolytic and fibrinolytic activities were determined using skim milk agar and plasminogen-free fibrin plate. Twenty five isolates showed protease activities, while only four of them secreted fibrinolitic enzyme. The highest proteolytic and fibrinolytic activity was shown by TB1 strain, which is identified as Bacillus coagulans. The 16s rDNA is still in investigating to confirm the TB1 strain identity.

  14. Comparative analysis of antimicrobial and proteolytic activity of lactic acid bacteria isolated from Zlatar cheese

    Directory of Open Access Journals (Sweden)

    Topisirović Ljubiša

    2007-01-01

    Full Text Available Traditional artisan Zlatar cheese belongs to the group of white, semi hard home-made cheeses, which are produced from no pasteurized cow's milk, without addition of any known bacterial starter culture. In total, 253 Gram-positive and catalase negative lactic acid bacteria (LAB were isolated. Results showed that 70 out of 253 analyzed isolates produced antimicrobial compounds known as bacteriocins. Most isolates from genera Lactococcus and Enterococcus, and isolates belonging to species Lactobacillus plantarum and Lb. brevis, do not synthesize extracellular proteinase. In contrast, isolates from subspecies Lb. paracasei subsp. paracasei showed very good proteolytic activity. It was observed that good proteolytic activity of isolates was not in correlation with their good antimicrobial activity in the most of isolates.

  15. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

    OpenAIRE

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J.; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C.; Austin, John; Overall, Christopher M.; Doxey, Andrew C.

    2017-01-01

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species...

  16. Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

    OpenAIRE

    Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

    2012-01-01

    Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that the...

  17. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Twine Susan M

    2009-03-01

    Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism

  18. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Science.gov (United States)

    Pakshir, K; Mohamadi, T; Khodadadi, H; Motamedifar, M; Zomorodian, K; Alipour, S; Motamedi, M

    2016-01-01

    Background and Purpose: Globally, dermatophytes are the most common filamentous group of fungi causing cutaneous mycoses. Dermatophytes were shown to secrete a multitude of enzymes that play a role in their pathogenesis. There is limited data on co-hemolytic (CAMP-like) effect of different bacterial species on dermatophyte species. In this study, we sought to the evaluate exoenzyme activity and co-hemolytic effect of four bacteria on clinical dermatophytes isolated from patients in Shiraz, Iran. Materials and Methods: A total of 84 clinical dermatophyte species were isolated from patients suffering dermatophytosis and identified by conventional methods. Hemolytic activity was evaluated with Columbia 5% sheep blood agar. Proteolytic activity was determined by plate clearance assay method, using gelatin 8% agar. CAMP-like factor was evaluated with four bacteria, namely, S. areus, S. saprophyticus, S. pyogenes, and S. agalactiae. Fisher's exact test was run for statistical analysis. Results: T. mentagrophytes was the most predominant agent (27 [32.1%]) followed by T. verrucosum(20 [23.8%]), T. tonsurans (10 [11.9%]), Microsporum canis (7 [8.3%]), T. rubrum (6 [7.1%]), E. floccosum (6 [7.1%]), M. gypseum (5 [6%]), and T. violaceum (3[3.6%]). The most common clinical area of dermatophytosis was the skin. All the isolates expressed the zone of incomplete alpha hemolysis. All the isolates had CAMP- positive reaction with S. aureus and the other bacteria were CAMP-negative. All the isolates expressed proteolytic activity and no significant differences were noted among diverse genera of dermatophytes and severities of proteolytic activity. Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species. PMID:28959790

  19. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo

    DEFF Research Database (Denmark)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai

    2017-01-01

    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major...... of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection....

  20. Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis.

    Science.gov (United States)

    Pilon, F M; Visôtto, L E; Guedes, R N C; Oliveira, M G A

    2013-08-01

    The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar-Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-L-Arg-p-nitroanilide (L-BApNA) and N-α-p-tosyl-L-Arg methyl ester (L-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-L-lysine chloromethyl ketone (TLCK)] as well as CaCl2, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.

  1. The role of lysosomal proteolytic enzymes in invasion and dissemination of malignant melanoma

    International Nuclear Information System (INIS)

    Bassalyk, L.S.; Tsanev, P.E.; Parshikova, S.M.; Demidov, L.V.

    1992-01-01

    Preoperative chemo- and radiation therapy was followed by a decrease in lysosomal cathepsins activity in metastatic lymph nodes which, however, did not reach the level established for intact lymph nodes. The pathogenetic role of proteolytic endopeptidases in invasion and sissemination of malignant melanoma is discussed as well as the value of their level measurement for assessing metastatic potential of tumor and prognosis of disease of disease on the basis of tumor site, degree of invasion regional lymph node status

  2. 'It Opened My Eyes'-examining the impact of a multifaceted chlamydia testing intervention on general practitioners using Normalization Process Theory.

    Science.gov (United States)

    Yeung, Anna; Hocking, Jane; Guy, Rebecca; Fairley, Christopher K; Smith, Kirsty; Vaisey, Alaina; Donovan, Basil; Imrie, John; Gunn, Jane; Temple-Smith, Meredith

    2018-03-28

    Chlamydia is the most common notifiable sexually transmissible infection in Australia. Left untreated, it can develop into pelvic inflammatory disease and infertility. The majority of notifications come from general practice and it is ideally situated to test young Australians. The Australian Chlamydia Control Effectiveness Pilot (ACCEPt) was a multifaceted intervention that aimed to reduce chlamydia prevalence by increasing testing in 16- to 29-year-olds attending general practice. GPs were interviewed to describe the effectiveness of the ACCEPt intervention in integrating chlamydia testing into routine practice using Normalization Process Theory (NPT). GPs were purposively selected based on age, gender, geographic location and size of practice at baseline and midpoint. Interview data were analysed regarding the intervention components and results were interpreted using NPT. A total of 44 GPs at baseline and 24 at midpoint were interviewed. Most GPs reported offering a test based on age at midpoint versus offering a test based on symptoms or patient request at baseline. Quarterly feedback was the most significant ACCEPt component for facilitating a chlamydia test. The ACCEPt intervention has been able to moderately normalize chlamydia testing among GPs, although the components had varying levels of effectiveness. NPT can demonstrate the effective implementation of an intervention in general practice and has been valuable in understanding which components are essential and which components can be improved upon.

  3. PROTEOLYTIC ATTACK OF THE CHROMOPROTEINS OF PORPHYRA BY MARINE BACTERIAL ENZYMES1

    Science.gov (United States)

    Merkel, Joseph R.; Braithwaite, Gordon D.; Kritzler, Henry

    1964-01-01

    Merkel, Joseph R. (College of Charleston, Charleston, S.C.), Gordon D. Braithwaite, and Henry Kritzler. Proteolytic attack of the chromoproteins of Porphyra by marine bacterial enzymes. J. Bacteriol. 88:974–980. 1964.—Three chromoproteins were isolated from the marine red alga, Porphyra leucosticta (Thuret), and were purified by adsorption chromatography on calcium phosphate gel and by ammonium sulfate fractionation. The algal chromoproteins were subjected to the proteolytic action of cultures of actively growing marine bacteria, marine bacterial culture filtrates, trypsin, and various marine bacterial enzyme preparations. Digestion of the chromoproteins was followed through various incubation intervals by measuring the loss of chromophore color and the increase in either ninhydrin-positive material (570 mμ) or 280-mμ light-absorbing material. There was good correlation between the loss of color and enzymatic hydrolysis of the proteins. this was particularly evident when a marine pseudomonad proteinase preparation was used as the proteolytic agent. In every digestion mixture, phycoerythrin was more resistant to degradation than was phycocyanin or allophycocyanin. Phycoerythrin was also more resistant to heat than was phycocyanin or allophycocyanin. PMID:14219062

  4. Growth, viability, and proteolytic activity of bifidobacteria in whole camel milk.

    Science.gov (United States)

    Abu-Taraboush, H M; al-Dagal, M M; al-Royli, M A

    1998-02-01

    Four species of bifidobacteria were evaluated for growth, viability, and proteolytic activity in whole camel milk, and comparison was made with whole cow milk. Growth of all species in either whole milk was characterized by the appearance of two logarithmic phases following anaerobic incubation at 37 degrees C for 36 h. The growth rate of Bifidobacterium longum 15,707 was higher in camel milk than in bovine milk and was higher for Bifidobacterium angulatum 27,535 in bovine milk than in camel milk. Bifidobacterium bifidum 2715 and Bifidobacterium breve 2258 showed the same trend as B. angulatum 27,535 after 16 h of postinoculation. Viable counts of all species except B. bifidum 2715 increased in the fermented whole milks during the first 3 d of storage at 4 degrees C. However, such counts did not change in unfermented milk, except for B. longum 15,707, which showed an increase in viable counts after 12 d of storage. Viability of all species in both fermented and unfermented milks was unaffected during refrigerated storage for 15 d. All species except B. longum 15,707 showed higher proteolytic activity in fermented camel milk than in bovine milk. However, proteolytic activity of all species except B. breve 2258 started at d 9 in unfermented camel milk only and increased sharply until the end of the storage period.

  5. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

    Science.gov (United States)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur

    2017-10-13

    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

  6. Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging

    DEFF Research Database (Denmark)

    Karring, Henrik; Runager, Kasper; Valnickova, Zuzana

    2010-01-01

    Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging....... TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 mug TGFBIp per mg wet tissue. Two-dimentional (2-D......) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (corneas (>6 years). Matrix...

  7. Proteolytic activities in fillets of selected underutilized Australian fish species.

    Science.gov (United States)

    Ahmed, Z; Donkor, O; Street, W A; Vasiljevic, T

    2013-09-01

    The hydrolytic activity of major endogenous proteases, responsible for proteolysis of myofibrillar proteins during post-mortem storage, may be an indicator of the textural quality of fish which influences consumer purchasing behaviour and thus market value of the final product. Furthermore, it may also influence the type and bioactive properties of the peptides released during post-mortem proteolysis of myofibrillar proteins. This study compared the activities of cathepsins B, B+L, D, H and calpain-like enzymes in crude muscle extracted from 16 Australian underutilized fish species. Fish species had a significant effect on the activity of these enzymes with barracouta showing the highest cathepsins B, B+L, D and H activities. Activities of cathepsins B and B+L were higher than cathepsin H for all studied species. The more commercially important rock ling and tiger flathead demonstrated higher cathepsin B+L activity, whereas gemfish and eastern school whiting showed higher activity towards cathepsin B. Underutilized fish species showing higher endogenous protease activities may be suitable for fish sauce production, whereas those with lower protease activities for surimi processing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Proteolytic activities in yeast after UV irradiation. Pt. 2

    Energy Technology Data Exchange (ETDEWEB)

    Schwencke, J.; Moustacchi, E.

    1982-04-01

    When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains.

  9. Proteolytic Degradation of Bovine Submaxillary Mucin (BSM) and Its Impact on Adsorption and Lubrication at a Hydrophobic Surface

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Svensson, Birte; Abou Hachem, Maher

    2015-01-01

    The effects of proteolytic digestion on bovinesubmaxillary mucin (BSM) were investigated in terms ofchanges in size, secondary structure, surface adsorption, and lubricating properties. Two proteases with distinctly different cleavage specificities, namely trypsin and pepsin, were employed. SDS...

  10. Procesos de negociación de significado en una escuela normal mexicana Processes of negotiation of meaning in a mexican program of teacher education

    Directory of Open Access Journals (Sweden)

    Pedro Antonio Estrada Rodríguez

    2008-12-01

    Full Text Available En este trabajo, analizamos algunos procesos de negociación de significado y desarrollo de micropolíticas en las experiencias de docentes y directivos en una escuela normal mexicana con la reforma a la educación normal de 1997. En dicho plan de estudios se estipula que los estudiantes realicen prácticas escolares durante el último año de la carrera. El estudio se enfocó en las actividades que realizaron los docentes de manera previa a las prácticas del preservicio con la primera generación de estudiantes del plan de 1997 de la Licenciatura en Educación Primaria. En el artículo mostramos cómo los docentes y el personal directivo de la normal del estudio, transitaron por un proceso de negociación de significado y generación de micropolíticas en su relación con la propuesta curricular nacional y la forma en que se organizaron bajo un esquema que concebimos como comunidad de práctica cultivada.Neste trabalho analisamos alguns processos de negociação de significado e desenvolvimento de micro-políticas nas experiências dos docentes e autoridades em uma escola normal mexicana após a reforma à educação normal de 1997. Neste programa de estudos estipula-se que os estudantes realizem os estágios escolares durante o último ano da faculdade. O estudo foi focado nas atividades que realizaram os docentes de forma prévia aos estágios do pré-serviço com a primeira geração de estudantes do plano de 1997 do Bacharelado em Educação Primária (Ensino Básico. No artigo mostramos como os docentes e autoridades da normal do estudo, transitaram por um processo de negociação de significado e geração de micro-política na sua realização com a proposta curricular nacional e a maneira com a qual foram organizados, sob um esquema que concebemos como comunidade de prática cultivada.In this paper, we analyze the experiences of teachers and principals in a Mexican teachers' college following the teacher education reform of 1997. In

  11. Pursuing Normality

    DEFF Research Database (Denmark)

    Madsen, Louise Sofia; Handberg, Charlotte

    2018-01-01

    BACKGROUND: The present study explored the reflections on cancer survivorship care of lymphoma survivors in active treatment. Lymphoma survivors have survivorship care needs, yet their participation in cancer survivorship care programs is still reported as low. OBJECTIVE: The aim of this study...... implying an influence on whether to participate in cancer survivorship care programs. Because of "pursuing normality," 8 of 9 participants opted out of cancer survivorship care programming due to prospects of "being cured" and perceptions of cancer survivorship care as "a continuation of the disease...

  12. Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species

    Czech Academy of Sciences Publication Activity Database

    Caccia, S.; Chakroun, M.; Vinokurov, Konstantin; Ferré, J.

    2014-01-01

    Roč. 67, AUG 2014 (2014), s. 76-84 ISSN 0022-1910 R&D Projects: GA ČR(CZ) GAP502/11/1471 Grant - others: Spanish Ministry of Science and Innovation(ES) AGL2009-13340-C02-01; Generalitat Valenciana and by European FEDER funds(ES) ACOMP/2009/313; Generalitat Valenciana and by European FEDER funds(ES) PROMETEO 2011/044; Marie Curie Grant(ES) PIEF-GA-2008-219993; European Union FP7(US) MOBITAG, GA 229518 Institutional support: RVO:60077344 Keywords : mode of action * serine peptidases * midgut peptidases Subject RIV: ED - Physiology Impact factor: 2.470, year: 2014 http://www.sciencedirect.com/science/article/pii/S0022191014001231

  13. Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression

    Science.gov (United States)

    2011-09-01

    Matriptase overexpression causes increased persistence of LNCap cell migration on Ln-332; 6) Cell adhesion of prostate cancer cells is not affected by hepsin... laryngeal , colon, tracheal, and cervical cancers [12,13]. Interest- ingly, Ln-332 expression is instead decreased or lost in prostate cancer [14–17]. As...migration is directionally persistent, as in the cases of highly invasive cancers like neuroepithelial tumors [73] or epithelial cell over- expressing

  14. Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression

    Science.gov (United States)

    2010-09-01

    38). In vitro studies have identified blood coagulation factors as substrates of hepsin (39). Prohepatocyte growth factor and prourokinase-type...ml), Ln-332 coincubated with hepsin and KD1, PBS, hepsin (1.3 M), or KD1 (5.6 M) overnight at 4 °C. Tran- swells were then blocked with 5% milk in

  15. Use of a proteolytic enzyme in cocoa (Theobroma cacao L. processing

    Directory of Open Access Journals (Sweden)

    Edy Sousa de Brito

    2004-08-01

    Full Text Available Protein hydrolysis using an exogenous protease on cocoa nibs was performed to verify the formation of precursors and the effect on cocoa flavour. An experimental design was used to check the influence of temperature (30 to 70 ºC and enzyme : substrate ratio [E/S] (97.5 to 1267.5 U g-1 of protein. The % Degree of Hydrolysis (% DH was affected mainly by [E/S] leading to a 4-fold increase (from 5 to 20 % after 6 hours of treatment. During cocoa nibs roasting, there was a greater consumption of hydrolysis compounds in the sample treated with protease as compared to the control, indicating their participation in the Maillard reaction. An increased perception of chocolate flavour and bitter taste was observed in a product formulated with protease treated cocoa.Foi feita uma hidrólise da proteína dos nibs de cacau usando-se uma protease para verificar a formação de precursores e o efeito sobre o sabor do cacau. Um desenho experimental foi usado para verificar a influência da temperatura (30 a 70 ºC e razão enzima : substrato [E/S] (97,5 a 1267,5 U g-1 de proteína. O grau de hidrólise % (%DH foi afetado principalmente pela [E/S], tendo sofrido um aumento de 4 vezes (de 5 para 20 % após 6 horas de tratamento. Durante a torração dos nibs houve um consumo maior dos compostos de hidrólise na amostra tratada com protease em comparação com o controle, indicando a participação desses compostos na reação de Maillard. Foi observado um aumento na percepção do sabor de chocolate e do gosto amargo em um produto formulado com o cacau tratado com a protease.

  16. Proteolytic processing of anti-Müllerian hormone differs between human fetal testes and adult ovaries

    DEFF Research Database (Denmark)

    Mamsen, Linn; Petersen, TS; Jeppesen, JV

    2015-01-01

    and specificity of a panel of five novel high-affinity AMH monoclonal antibodies. Two recognize the mature C-terminal form of AMH, whereas three recognize the active pro-mature form of AMH in human tissue. The antibodies were tested on fetal male testicular and mesonephric tissue aged 8-19 weeks post conception...... (pc), fetal male serum aged 16-26 weeks pc and human immature GCs by immunofluorescence, immunohistochemistry, ELISA and western blotting. The active pro-mature forms of AMH were expressed in both Sertoli cells from human fetal testis and human immature GCs. In contrast, the mature C-terminal form...... of AMH was hardly detected in Sertoli cells, but was readily detected in GCs. This particular form was also located to the nucleus in GCs, whereas the other investigated AMH forms remained in the cytoplasm. Interestingly, the distribution of the AMH forms in the fetal serum of boys showed...

  17. Normality in Analytical Psychology

    Science.gov (United States)

    Myers, Steve

    2013-01-01

    Although C.G. Jung’s interest in normality wavered throughout his career, it was one of the areas he identified in later life as worthy of further research. He began his career using a definition of normality which would have been the target of Foucault’s criticism, had Foucault chosen to review Jung’s work. However, Jung then evolved his thinking to a standpoint that was more aligned to Foucault’s own. Thereafter, the post Jungian concept of normality has remained relatively undeveloped by comparison with psychoanalysis and mainstream psychology. Jung’s disjecta membra on the subject suggest that, in contemporary analytical psychology, too much focus is placed on the process of individuation to the neglect of applications that consider collective processes. Also, there is potential for useful research and development into the nature of conflict between individuals and societies, and how normal people typically develop in relation to the spectrum between individuation and collectivity. PMID:25379262

  18. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation...... of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same...

  19. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  20. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

    Science.gov (United States)

    Xie, Nan; Khabbazi, Samira; Nassar, Zeyad D; Gregory, Kye; Vithanage, Tharindu; Anand-Apte, Bela; Cabot, Peter J; Sturgess, David; Shaw, Paul N; Parat, Marie-Odile

    2017-12-01

    Opioids modulate the tumor microenvironment with potential functional consequences for tumor growth and metastasis. We evaluated the effects of morphine administration on the circulating proteolytic profile of tumor-free mice. Serum from morphine-treated (1 or 10 mg/kg, i.p. every 12 h) or saline-treated mice was collected at different time points and tested ex vivo in endothelial, lymphatic endothelial, and breast cancer cell migration assays. Serum from mice that were treated with 10 mg/kg morphine for 3 d displayed reduced chemotactic potential for endothelial and breast cancer cells, and elicited reduced cancer cell invasion through reconstituted basement membrane compared with serum from saline controls. This was associated with decreased circulating matrix metalloproteinase 9 (MMP-9) and increased circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3/4 as assessed by zymography and reverse zymography. By using quantitative RT-PCR, we confirmed morphine-induced alterations in MMP-9 and TIMP expression and identified organs, including the liver and spleen, in which these changes originated. Pharmacologic inhibition of MMP-9 abrogated the difference in chemotactic attraction between serum from saline-treated and morphine-treated mice, which indicated that reduced proteolytic ability mediated the decreased migration toward serum from morphine-treated mice. This novel mechanism may enable morphine administration to promote an environment that is less conducive to tumor growth, invasion, and metastasis.-Xie, N., Khabbazi, S., Nassar, Z. D., Gregory, K., Vithanage, T., Anand-Apte, B., Cabot, P. J., Sturgess, D., Shaw, P. N., Parat, M.-O. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion. © FASEB.

  1. Regions with different evoked frequency band responses during early-stage visual processing distinguish mild Alzheimer dementia from mild cognitive impairment and normal aging.

    Science.gov (United States)

    Haupt, M; González-Hernández, J A; Scherbaum, W A

    2008-09-19

    Although diagnostic procedures have been developed for detection of mild cognitive impairment (MCI) and mild Alzheimer dementia (AD), more valid noninvasive tools are needed. In this work, we apply a procedure based on the evidences that different evoked frequency band responses may emerge from different sources during early-stage visual processing in a mental state-specific manner, while subjects were passively viewing a visual stimulus. In this case, spatial differences should arise across mental conditions such as mild Alzheimer dementia, mild cognitive impairment, and normal aging. With the use of EEG source image we found three different neural patterns in aged individuals: (1) left hippocampus and midbrain in mild AD, (2) left lateral orbitofrontal gyrus, left nucleus accumbens, caudate nucleus, thalamus, posterior cinguli, right precuneous, right superior parietal lobe in MCI, and (3) right lateral-medial orbitofrontal gyrus, caudate nucleus, thalamus, right lateral occipitotemporal gyrus in elderly controls. Although preliminary, these results show remarkably robust differences that distinguish between an age-matched control group, a group with MCI, and a group with mild AD. Because the method applied in this work differentiates among clinical entities with varying severity of cognitive decline, it may eventually serve as an electrophysiological marker in the early detection of neurodegeneration.

  2. Synergetic effects of solution-processable fluorinated graphene and PEDOT as a hole-transporting layer for highly efficient and stable normal-structure perovskite solar cells.

    Science.gov (United States)

    Yu, Jae-Hun; Lee, Cheol-Ho; Joh, Han-Ik; Yeo, Jun-Seok; Na, Seok-In

    2017-11-16

    We demonstrate that a bi-interlayer consisting of water-free poly(3,4-ethylenedioxythiophene) (PEDOT) and fluorinated reduced graphene oxide (FrGO) noticeably enhances the efficiency and the stability of the normal-structure perovskite solar cells (PeSCs). With simple and low temperature solution-processing, the PeSC employing the PEDOT + FrGO interlayer exhibits a significantly improved power conversion efficiency (PCE) of 14.9%. Comprehensive investigations indicate that the enhanced PCE is mostly attributed to the retarded recombination in the devices. The minimized recombination phenomena are related to the interfacial dipoles at the PEDOT/FrGO interface, which facilitates the electron-blocking and the higher built-in potential in the devices. Furthermore, the PEDOT + FrGO device shows a better stability by maintaining 70% of the initial PCE over the 30 days exposure to ambient conditions. This is because the more hydrophobic graphitic sheets of the FrGO on the PEDOT further protect the perovskite films from oxygen/water penetration. Consequently, the introduction of composite interfacial layers including graphene derivatives can be an effective and versatile strategy for high-performing, stable, and cost-effective PeSCs.

  3. Guidelines and training initiatives that support communication in cross-cultural primary-care settings: appraising their implementability using Normalization Process Theory.

    Science.gov (United States)

    de Brún, Tomas; de-Brún, Mary O'Reilly; van Weel-Baumgarten, Evelyn; van Weel, Chris; Dowrick, Christopher; Lionis, Christos; O'Donnell, Catherine A; Burns, Nicola; Mair, Frances S; Saridaki, Aristoula; Papadakaki, Maria; Princz, Christine; van den Muijsenbergh, Maria; MacFarlane, Anne

    2015-08-01

    Guidelines and training initiatives (G/TIs) available to support communication in cross-cultural primary health care consultations are not routinely used. We need to understand more about levers and barriers to their implementation and identify G/TIs likely to be successfully implemented in practice. To report a mapping process used to identify G/TIs and to prospectively appraise their implementability, using Normalization Process Theory (NPT). RESTORE is a 4-year EU FP-7 project. We used purposeful and network sampling to identify experts in statutory and non-statutory agencies across Austria, England, Greece, Ireland, Scotland and the Netherlands who recommended G/TI data from the grey literature. In addition, a peer review of literature was conducted in each country. Resulting data were collated using a standardized Protocol Mapping Document. G/TIs were identified for inclusion by (i) initial elimination of incomplete G/TI material; (ii) application of filtering criteria; and (iii) application of NPT. 20 G/TIs met selection criteria: 8 guidelines and 12 training initiatives. Most G/TIs were identified in the Netherlands (n = 7), followed by Ireland (n = 6) and England (n = 5). Fewer were identified in Scotland (n = 2), and none in Greece or Austria. The majority (n = 13) were generated without the inclusion of migrant service users. All 20 were prospectively appraised for potential implementability by applying NPT. NPT is useful as a means of prospectively testing G/TIs for implementability. Results indicate a need to initiate meaningful engagement of migrants in the development of G/TIs. A European-based professional standard for development and assessment of cross-cultural communication resources is advised. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Radionuclide method for determination of gastric content proteolytic activity, (Clinical investigation)

    International Nuclear Information System (INIS)

    Sergienko, V.B.; Popova, L.V.; Khodarev, N.N.; Mel'nikov, N.A.

    1988-01-01

    Clinical testing of a new tubeless radionuclide rapid method for measuring gastric content proteolytic activity (GCPA) using a protein (gelatin) capsule filled with a radiopharmaceutical (RP) labelled with 99m Tc is described. The solution time of a capsule with a radiotracer determined by its clearance, corresponded to GCPA measured after Mett's method. The testing has shown that the above method is physiological, causes no complications and unpleasant sensations in patients, is simple to perform, and can be recommended for a wide clinical use in gastroenterology

  5. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    International Nuclear Information System (INIS)

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-01-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis

  6. A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

    International Nuclear Information System (INIS)

    Rucklidge, G.J.; Milne, G.

    1990-01-01

    A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue

  7. Normal Coagulation

    Science.gov (United States)

    2014-09-04

    fibrinolytic process and replaced by cellular reconstruction of the perfo- rated vessel. Fibrinolysis Proteins Clot formation is integrated with clot...Res 50:1488–1494, 1990. 226. Evans DM, et al: Time and dose dependency of the suppression of pulmonary metastases of rat mammary cancer by amiloride...Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary

  8. Exploring drivers and challenges in implementation of health promotion in community mental health services: a qualitative multi-site case study using Normalization Process Theory.

    Science.gov (United States)

    Burau, Viola; Carstensen, Kathrine; Fredens, Mia; Kousgaard, Marius Brostrøm

    2018-01-24

    There is an increased interest in improving the physical health of people with mental illness. Little is known about implementing health promotion interventions in adult mental health organisations where many users also have physical health problems. The literature suggests that contextual factors are important for implementation in community settings. This study focused on the change process and analysed the implementation of a structural health promotion intervention in community mental health organisations in different contexts in Denmark. The study was based on a qualitative multiple-case design and included two municipal and two regional provider organisations. Data were various written sources and 13 semi-structured interviews with 22 key managers and frontline staff. The analysis was organised around the four main constructs of Normalization Process Theory: Coherence, Cognitive Participation, Collective Action, and Reflexive Monitoring. Coherence: Most respondents found the intervention to be meaningful in that the intervention fitted well into existing goals, practices and treatment approaches. Cognitive Participation: Management engagement varied across providers and low engagement impeded implementation. Engaging all staff was a general problem although some of the initial resistance was apparently overcome. Collective Action: Daily enactment depended on staff being attentive and flexible enough to manage the complex needs and varying capacities of users. Reflexive Monitoring: During implementation, staff evaluations of the progress and impact of the intervention were mostly informal and ad hoc and staff used these to make on-going adjustments to activities. Overall, characteristics of context common to all providers (work force and user groups) seemed to be more important for implementation than differences in the external political-administrative context. In terms of research, future studies should adopt a more bottom-up, grounded description of context

  9. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

    Science.gov (United States)

    Solari, Fiorella A; Mattheij, Nadine J A; Burkhart, Julia M; Swieringa, Frauke; Collins, Peter W; Cosemans, Judith M E M; Sickmann, Albert; Heemskerk, Johan W M; Zahedi, René P

    2016-10-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca 2+ -dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca 2+ -dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular

  10. Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

    Science.gov (United States)

    Tryggvesson, Anders; Ståhlberg, Frida M; Mogk, Axel; Zeth, Kornelius; Clarke, Adrian K

    2012-09-01

    The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

  11. Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis.

    Science.gov (United States)

    de Souza Carvalho, F R; Carrijo-Carvalho, L C; Chudzinski-Tavassi, A M; Foronda, A S; de Freitas, D

    2011-04-01

    Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea. © 2010 European Society of Clinical Microbiology and Infectious Diseases. No claim to original US government works.

  12. Capillary electrophoresis of the proteolytic activity of the stratum corneum obtained by tape stripping.

    Science.gov (United States)

    Papoiu, Alexandru D P; Yosipovitch, Gil; Shihabi, Zak

    2010-10-01

    Serine proteases and some cathepsins are present in the stratum corneum. They are known to play a significant role in the pathophysiological mechanism of several dermatological conditions (e.g. atopic dermatitis) and in the induction of itch. Tape stripping of skin is a simple technique used in the investigation of skin barrier function and in the penetration of topically applied drugs. Herein, we show that CE, under stacking conditions, is a well-suited technique to measure the proteolytic activity of enzymes in the stratum corneum. Disks of about 6 mm (id) were cut from adhered tapes and submerged directly in a buffer containing the appropriate peptide substrate. After incubation, the split peptides were separated and detected directly by CE at 214 nm in a borate buffer. The esterase activity on N-benzoyl-tyrosine ethyl ester and the amidase activity on succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and the splitting of hemoglobin were detected by CE. The esterase activity was the highest when compared to the proteolytic activities. Skin scratching increased the enzymatic activity adhered to the tapes. The CE offered over the traditional end-point colorimetric methods the ability to measure the low enzymatic activity and the ability to detect the released peptides directly. This technique is simple, non-invasive, easy to perform and uses non-expensive substrates. It can be useful in quantifying cathepsins and serine proteases in the skin.

  13. Cell-penetrating TAT peptide in drug delivery systems: proteolytic stability requirements.

    Science.gov (United States)

    Koren, Erez; Apte, Anjali; Sawant, Rupa R; Grunwald, Jacob; Torchilin, Vladimir P

    2011-07-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a 'short' PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function.

  14. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Directory of Open Access Journals (Sweden)

    Gizeli S. Gimenez

    2014-01-01

    Full Text Available Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

  15. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Science.gov (United States)

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  16. Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench.

    Science.gov (United States)

    Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

    2017-06-01

    Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. © 2017 Beckers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. The challenge of transferring an implementation strategy from academia to the field: a process evaluation of local quality improvement collaboratives in Dutch primary care using the normalization process theory.

    Science.gov (United States)

    Trietsch, Jasper; van Steenkiste, Ben; Hobma, Sjoerd; Frericks, Arnoud; Grol, Richard; Metsemakers, Job; van der Weijden, Trudy

    2014-12-01

    A quality improvement strategy consisting of comparative feedback and peer review embedded in available local quality improvement collaboratives proved to be effective in changing the test-ordering behaviour of general practitioners. However, implementing this strategy was problematic. We aimed for large-scale implementation of an adapted strategy covering both test ordering and prescribing performance. Because we failed to achieve large-scale implementation, the aim of this study was to describe and analyse the challenges of the transferring process. In a qualitative study 19 regional health officers, pharmacists, laboratory specialists and general practitioners were interviewed within 6 months after the transfer period. The interviews were audiotaped, transcribed and independently coded by two of the authors. The codes were matched to the dimensions of the normalization process theory. The general idea of the strategy was widely supported, but generating the feedback was more complex than expected and the need for external support after transfer of the strategy remained high because participants did not assume responsibility for the work and the distribution of resources that came with it. Evidence on effectiveness, a national infrastructure for these collaboratives and a general positive attitude were not sufficient for normalization. Thinking about managing large databases, responsibility for tasks and distribution of resources should start as early as possible when planning complex quality improvement strategies. Merely exploring the barriers and facilitators experienced in a preceding trial is not sufficient. Although multifaceted implementation strategies to change professional behaviour are attractive, their inherent complexity is also a pitfall for large-scale implementation. © 2014 John Wiley & Sons, Ltd.

  18. Non-linear pressure/temperature-dependence of high pressure thermal inactivation of proteolytic Clostridium botulinum type B in foods.

    Directory of Open Access Journals (Sweden)

    Maximilian B Maier

    Full Text Available The effect of high pressure thermal (HPT processing on the inactivation of spores of proteolytic type B Clostridium botulinum TMW 2.357 in four differently composed low-acid foods (green peas with ham, steamed sole, vegetable soup, braised veal was studied in an industrially feasible pressure range and temperatures between 100 and 120°C. Inactivation curves exhibited rapid inactivation during compression and decompression followed by strong tailing effects. The highest inactivation (approx. 6-log cycle reduction was obtained in braised veal at 600 MPa and 110°C after 300 s pressure-holding time. In general, inactivation curves exhibited similar negative exponential shapes, but maximum achievable inactivation levels were lower in foods with higher fat contents. At high treatment temperatures, spore inactivation was more effective at lower pressure levels (300 vs. 600 MPa, which indicates a non-linear pressure/temperature-dependence of the HPT spore inactivation efficiency. A comparison of spore inactivation levels achievable using HPT treatments versus a conventional heat sterilization treatment (121.1°C, 3 min illustrates the potential of combining high pressures and temperatures to replace conventional retorting with the possibility to reduce the process temperature or shorten the processing time. Finally, experiments using varying spore inoculation levels suggested the presence of a resistant fraction comprising approximately 0.01% of a spore population as reason for the pronounced tailing effects in survivor curves. The loss of the high resistance properties upon cultivation indicates that those differences develop during sporulation and are not linked to permanent modifications at the genetic level.

  19. Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein.

    Directory of Open Access Journals (Sweden)

    Magdalena Anna Krzyzaniak

    Full Text Available Respiratory Syncytial Virus (RSV is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549, we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.

  20. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.

    Science.gov (United States)

    Choi, Nack-Shick; Choi, Jong Hyun; Kim, Bo-Hye; Han, Yun-Jon; Kim, Joong Su; Lee, Seung-Goo; Song, Jae Jun

    2009-06-01

    A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.

  1. Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    Borges-Veloso Andre

    2012-06-01

    Full Text Available Abstract Background The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the

  2. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; 1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein

  3. Antioxidants, free radicals, storage proteins, and proteolytic activities in wheat (Triticum durum) seeds during accelerated aging.

    Science.gov (United States)

    Galleschi, Luciano; Capocchi, Antonella; Ghiringhelli, Silvia; Saviozzi, Franco; Calucci, Lucia; Pinzino, Calogero; Zandomeneghi, Maurizio

    2002-09-11

    Accelerated aging was performed by incubation of wheat seeds at 40 degrees C and 100% relative humidity for 3, 4, 6, 10, and 14 days. The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activity) and gluten (free radical content and flexibility) were evaluated. A decrease of germinability was found during aging, the germination being completely inhibited after 14 days. The lutein content decreased gradually, without going to zero, while that of free radicals increased. A reduction of soluble proteins and a degradation of glutenins and gliadins were observed, associated with a substantial increase of protease activity and a decrease in gluten flexibility. The results were discussed in reference to those previously obtained by natural aging of wheat seeds of the same species and cultivar.

  4. Proteolytic Activity in Reduced-Fat Cheddar Cheese Made with Lactic Acid Bacteria and Camel Chymosin

    DEFF Research Database (Denmark)

    Børsting, Mette Winther

    be the need of an extended ripening period to reach a similar cheese structure as in cheeses produced with BC. The aim of this project was to compensate for the lower proteolytic activity in cheese produced with CC compared to BC. Selection of dairy lactic acid bacteria (LAB) for cheese production with high...... for their ability to influence proteolysis and structure during cheese ripening. In an attempt to improve the screening methods and contribute to the development of a new classification system of Latcococcus lactic strains, the peptide profile formed by selected strains after growth in milk was analyzed...... mediated an increase in the total amount of amino acids as well as a shorter structure. A model system, used to study the retention of chymosin in a curd, showed that the retention of CC was less dependent on pH compared to BC, and the retention of CC was higher than BC in the pH interval 6...

  5. Proteolytic Activity in Reduced-Fat Cheddar Cheese Made with Lactic Acid Bacteria and Camel Chymosin

    DEFF Research Database (Denmark)

    Børsting, Mette Winther

    are characterized by a more firm structure, higher risk of bitterness and lower flavor intensity. The bitterness can be reduced by replacing bovine chymosin (BC) in cheese production with camel chymosin (CC), which has a lower general proteolysis. A disadvantage of the lower proteolytic activity of CC could...... for their ability to influence proteolysis and structure during cheese ripening. In an attempt to improve the screening methods and contribute to the development of a new classification system of Latcococcus lactic strains, the peptide profile formed by selected strains after growth in milk was analyzed...... to the reference cheeses. Lc. lactis strains which were previously defined as group d based on their cleavage specificity towards αS1-CN (f1-23), could be subdivided into three groups. This grouping was seen both in the variation of CEP amino acid sequences, and in the identified peptides after hydrolysis in milk...

  6. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

    Science.gov (United States)

    Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

    2016-04-15

    The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Microbiological and biochemical response of certain proteolytic bacterial isolates to varying levels of gamma irradiation

    International Nuclear Information System (INIS)

    El-Hifnawi, H.M.N.E.A.

    1997-01-01

    Amniotic membrane allo - and xeno grafts prepared from human foetal placenta, and their potential replacement of skin autotransplant, would significantly contribute to the success of clinical treatment of skin burns. Allo-and xenografts of human amniotic membrane should be ensured for their sterility, bio-mechanics and tissue antigenicity. The present study has been focused on sterilization and sterility assurance of the membrane grafts. Physico-chemical properties and antigenicity of the grafts await investigation. In the present study the isolation and identification of the bacteria contaminating the amniotic membrane allo-and xenografts prepared from human foetal placenta and the effect of gamma irradiation on it has been investigated. The proteolytic activity of these bacteria and the role of gamma irradiation in the control of bacterial activity were similarly investigated

  8. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  9. Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

    International Nuclear Information System (INIS)

    Doinel, C.; Ropars, C.; Salmon, C.

    1978-01-01

    Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

  10. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

    Science.gov (United States)

    Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

    2017-06-01

    Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds

  11. Evidence for proteolytic cleavage of brevican by the ADAMTSs in the dentate gyrus after excitotoxic lesion of the mouse entorhinal cortex

    Directory of Open Access Journals (Sweden)

    Gottschall Paul E

    2005-08-01

    Full Text Available Abstract Background Brevican is a member of the lectican family of aggregating extracellular matrix (ECM proteoglycans that bear chondroitin sulfate (CS chains. It is highly expressed in the central nervous system (CNS and is thought to stabilize synapses and inhibit neural plasticity and as such, neuritic or synaptic remodeling would be less likely to occur in regions with intact and abundant, lectican-containing, ECM complexes. Neural plasticity may occur more readily when these ECM complexes are broken down by endogenous proteases, the ADAMTSs (adisintegrin and metalloproteinase with thrombospondin motifs, that selectively cleave the lecticans. The purpose of these experiments was to determine whether the production of brevican or the ADAMTS-cleaved fragments of brevican were altered after deafferentation and reinnervation of the dentate gyrus via entorhinal cortex lesion (ECL. Results In the C57Bl6J mouse, synaptic density in the molecular layer of the dentate gyrus, as measured by synaptophysin levels in ELISA, was significantly attenuated 2 days (nearly 50% of contralateral and 7 days after lesion and returned to levels not different from the contralateral region at 30 days. Immunoreactive brevican in immunoblot was elevated 2 days after lesion, whereas there was a significant increase in the proteolytic product at 7, but not 30 days post-lesion. ADAMTS activity, estimated using the ratio of the specific ADAMTS-derived brevican fragment and intact brevican levels was increased at 7 days, but was not different from the contralateral side at 2 or 30 days after deafferentation. Conclusion These findings indicate that ADAMTS activity in the dentate outer molecular layer (OML is elevated during the initial synaptic reinnervation period (7 days after lesion. Therefore, proteolytic processing of brevican appears to be a significant extracellular event in the remodeling of the dentate after EC lesion, and may modulate the process of sprouting and

  12. Normality in Analytical Psychology

    Directory of Open Access Journals (Sweden)

    Steve Myers

    2013-11-01

    Full Text Available Although C.G. Jung’s interest in normality wavered throughout his career, it was one of the areas he identified in later life as worthy of further research. He began his career using a definition of normality which would have been the target of Foucault’s criticism, had Foucault chosen to review Jung’s work. However, Jung then evolved his thinking to a standpoint that was more aligned to Foucault’s own. Thereafter, the post Jungian concept of normality has remained relatively undeveloped by comparison with psychoanalysis and mainstream psychology. Jung’s disjecta membra on the subject suggest that, in contemporary analytical psychology, too much focus is placed on the process of individuation to the neglect of applications that consider collective processes. Also, there is potential for useful research and development into the nature of conflict between individuals and societies, and how normal people typically develop in relation to the spectrum between individuation and collectivity.

  13. Proteolytic extracts of three Bromeliaceae species as eco-compatible tools for leather industry.

    Science.gov (United States)

    Errasti, María Eugenia; Caffini, Néstor Oscar; López, Laura María Isabel

    2018-01-02

    Most tanneries use high proportions of Na 2 S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic H 2 S. Solid waste rich in protein is another environmental problem of tanneries. Enzymes are an interesting technological tool for industry due to their biodegradability, nontoxic nature, and nonpolluting effluent generation. In the leather industry, proteases have been chosen as a promising eco-friendly alternative to Na 2 S/CaO dehairing. Extracts with high proteolytic activity have been obtained from fruits of Bromeliaceae species: Bromelia balansae Mez (Bb), Bromelia hieronymi Mez (Bh), and Pseudananas macrodontes (Morr.) Harms (Pm). In this work, Bb, Bh, and Pm have been studied for application in the leather industry, focusing in their dehairing properties. Enzymatic activities were measured against collagen, keratin, elastin, and epidermis while a dehairing assay was performed by employing cowhide. All extracts showed similar activity on collagen and epidermis, while Bh and Pm were the most active against keratin at the same caseinolytic unit (CU) values; Bh was the only extract active against elastin. Bb (1 CU/ml), Bh (1.5 CU/ml), and Pm (0.5 CU/ml) were able to depilate cowhide. Desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface was clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry.

  14. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    Science.gov (United States)

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  15. The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes.

    Science.gov (United States)

    Nhan, Hoang S; Chiang, Karen; Koo, Edward H

    2015-01-01

    The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the -YENPTY- motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.

  16. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  17. Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

    Directory of Open Access Journals (Sweden)

    Mourah Samia

    2010-02-01

    Full Text Available Abstract SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22, a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

  18. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems.

    Science.gov (United States)

    Kemp, C M; Oliver, W T; Wheeler, T L; Chishti, A H; Koohmaraie, M

    2013-07-01

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

  19. Effect of auxin (iaa upon the proteolytic system in differentiating secondary xylem of pine (Pinus sylvestris L.

    Directory of Open Access Journals (Sweden)

    Krzysztof J. Rakowski

    2014-01-01

    Full Text Available Effects of decapitation and IAA on proteolytic activity were studied in main stem of 4-7 year-old Pinus sylvestris trees. Proteolytic activity in the extract from differentiating secondary xylem was found to be totally reduced in decapitated 2-3 year-old segments of the main stem after a few weeks. Simultaneous application of IAA in lanolin paste prevented this reduction. Proteolytic activity reduced totally after decapitation was restored within 2 days when auxin was applied. Analogous responses to decapitated and auxin application were observed in respect to cambial activity and protein level. The latter effects were not correlated in time with the effects upon the activity of proteases. The differences were especially visible when phloem continuity between the decapited stem segment and the rest of the tree crown was broken by ring-barking. The results suggest dependence of a proteolytic system on the shoot apical control. In this epigenetic system of control the role of auxin seems to be directly associated with the seasonal meristematic activity of the cambium, which was observed in earlier studies.

  20. Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity.

    Science.gov (United States)

    Tremonte, P; Reale, A; Di Renzo, T; Tipaldi, L; Di Luccia, A; Coppola, R; Sorrentino, E; Succi, M

    2010-11-01

    To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures. © 2010 The Authors. © 2010 The Society for Applied Microbiology.

  1. Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme

    NARCIS (Netherlands)

    Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.

    2005-01-01

    A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional

  2. Direct proteolytic cleavage of NLRP1B is necessary and sufficient for inflammasome activation by anthrax lethal factor.

    Directory of Open Access Journals (Sweden)

    Joseph Chavarría-Smith

    Full Text Available Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1 protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR or AIM2-like receptor (ALR protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.

  3. Asymptotic normalization coefficients from proton transfer reactions and astrophysical S factors for the (CNOC)-C-13(p, gamma)N-14 radiative capture process

    Czech Academy of Sciences Publication Activity Database

    Mukhamedzhanov, A. M.; Azhari, A.; Burjan, Václav; Gagliardi, C. A.; Kroha, Václav; Sattarov, A.; Tang, X.; Trache, L.; Tribble, R. E.

    2003-01-01

    Roč. 725, č. 22 (2003), s. 279-294 ISSN 0375-9474 R&D Projects: GA ČR GA202/01/0709; GA MŠk ME 385 Institutional research plan: CEZ:AV0Z1048901 Keywords : radiative capture reaction * asymptotic normalization coefficient Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 1.761, year: 2003

  4. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    International Nuclear Information System (INIS)

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B.

    2006-01-01

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

  5. Avaliação do processamento auditivo e da discriminação fonêmica em crianças com desenvolvimento fonológico normal e desviante Evaluation of auditory processing and phonemic discrimination in children with normal and disordered phonological development

    Directory of Open Access Journals (Sweden)

    Tiago Mendonça Attoni

    2010-12-01

    Full Text Available O processamento auditivo e a discriminação fonêmica são imprescindíveis para o processo comunicativo. Tipo de Estudo: retrospectivo. OBJETIVO: Analisar as respostas encontradas na avaliação do processamento auditivo e da discriminação fonêmica em crianças com desenvolvimento normal de fala e com desvio fonológico. MATERIAL E MÉTODO: Este estudo constitui-se da avaliação de 46 crianças, sendo 22 com desvio fonológico e 24 com desenvolvimento normal de fala. Foram aplicados os testes de escuta diótica, monótica e dicótica para avaliar o processamento auditivo e um teste que avalia a capacidade de discriminação fonêmica. DESENHO CIENTÍFICO: Transversal, contemporâneo. RESULTADOS: As crianças normais obtiveram valores considerados normais em todos os testes do processamento auditivo e índices máximos no teste de discriminação fonêmica. As crianças com desvio fonológico foram piores neste último, além de apresentarem alterações no processamento auditivo. CONCLUSÃO: Crianças com desvio fonológico apresentam alterações de processamento auditivo e discriminação fonêmica.Auditory processing and phonemic discrimination are essential for communication. Type of study: Retrospective. AIM: To evaluate auditory processing and phonemic discrimination in children with normal and disordered phonological development. MATERIAL AND METHODS: An evaluation of 46 children was carried out: 22 had phonological disorders and 24 had normally developing speech. Diotic , monotic and dichotic tests were applied to assess auditory processing and a test to evaluate phonemic discrimination abilities. DESIGN: Cross-sectional, contemporary. RESULTS: The values of normally-developing children were within the normal range in all auditory processing tests; these children attained maximum phonemic discrimination test scores. Children with phonological disorders performed worse in the latter, and presented disordered auditory processing

  6. Applying normalization process theory to understand implementation of a family violence screening and care model in maternal and child health nursing practice: a mixed method process evaluation of a randomised controlled trial.

    Science.gov (United States)

    Hooker, Leesa; Small, Rhonda; Humphreys, Cathy; Hegarty, Kelsey; Taft, Angela

    2015-03-28

    In Victoria, Australia, Maternal and Child Health (MCH) services deliver primary health care to families with children 0-6 years, focusing on health promotion, parenting support and early intervention. Family violence (FV) has been identified as a major public health concern, with increased prevalence in the child-bearing years. Victorian Government policy recommends routine FV screening of all women attending MCH services. Using Normalization Process Theory (NPT), we aimed to understand the barriers and facilitators of implementing an enhanced screening model into MCH nurse clinical practice. NPT informed the process evaluation of a pragmatic, cluster randomised controlled trial in eight MCH nurse teams in metropolitan Melbourne, Victoria, Australia. Using mixed methods (surveys and interviews), we explored the views of MCH nurses, MCH nurse team leaders, FV liaison workers and FV managers on implementation of the model. Quantitative data were analysed by comparing proportionate group differences and change within trial arm over time between interim and impact nurse surveys. Qualitative data were inductively coded, thematically analysed and mapped to NPT constructs (coherence, cognitive participation, collective action and reflexive monitoring) to enhance our understanding of the outcome evaluation. MCH nurse participation rates for interim and impact surveys were 79% (127/160) and 71% (114/160), respectively. Twenty-three key stakeholder interviews were completed. FV screening work was meaningful and valued by participants; however, the implementation coincided with a significant (government directed) change in clinical practice which impacted on full engagement with the model (coherence and cognitive participation). The use of MCH nurse-designed FV screening/management tools in focussed women's health consultations and links with FV services enhanced the participants' work (collective action). Monitoring of FV work (reflexive monitoring) was limited. The use of

  7. Normal Pressure Hydrocephalus (NPH)

    Science.gov (United States)

    ... local chapter Join our online community Normal Pressure Hydrocephalus (NPH) Normal pressure hydrocephalus is a brain disorder ... Symptoms Diagnosis Causes & risks Treatments About Normal Pressure Hydrocephalus Normal pressure hydrocephalus occurs when excess cerebrospinal fluid ...

  8. A Comparison of the Effects of Interference and Distortion in Visual Information Processing by Normal and Retarded Children and by Adults. Final Report.

    Science.gov (United States)

    Meyer, Philip A.

    Reported were four experiments which investigated the developmental and mental retardation aspects of an initial stage of visual information processing termed iconic memory. The stage was explained to involve processing visual stimuli prior to sensation and through to recognition. In three of the four experiments, the paradigm of visual masking…

  9. Mechanical stabilization of proteolytically degradable polyethylene glycol dimethacrylate hydrogels through peptide interaction.

    Science.gov (United States)

    Lim, Hyun Ju; Khan, Zara; Lu, Xi; Perera, T Hiran; Wilems, Thomas S; Ravivarapu, Krishna T; Smith Callahan, Laura A

    2018-03-09

    Balancing enhancement of neurite extension against loss of matrix support in synthetic hydrogels containing proteolytically degradable and bioactive signaling peptides to optimize tissue formation is difficult. Using a systematic approach, polyethylene glycol hydrogels containing concurrent continuous concentration gradients of the laminin derived bioactive signaling peptide, Ile-Lys-Val-Ala-Val (IKVAV), and collagen derived matrix metalloprotease degradable peptide, GPQGIWGQ, were fabricated and characterized. During proteolytic degradation of the concentration gradient hydrogels, the IKVAV and IWGQ cleavage fragment from GPQGIWGQ were found to interact and stabilize the bulk Young's Modulus of the hydrogel. Further testing of discrete samples containing GPQGIWGQ or its cleavage fragments, GPQG and IWGQ, indicates hydrophobic interactions between the peptides are not necessary for mechanical stabilization of the hydrogel, but changes in the concentration ratio between the peptides tethered in the hydrogel and salts and ions in the swelling solution can affect the stabilization. Encapsulation of human induced pluripotent stem cell derived neural stem cells did not reduce the mechanical properties of the hydrogel over a 14 day neural differentiation culture period, and IKVAV was found to maintain concentration dependent effects on neurite extension and mRNA gene expression of neural cytoskeletal markers, similar to previous studies. As a result, this work has significant implications for the analysis of biological studies in matrices, as the material and mechanical properties of the hydrogel may be unexpectedly temporally changing during culture due to interactions between peptide signaling elements, underscoring the need for greater matrix characterization during the degradation and cell culture. Greater emulation of the native extracellular matrix is necessary for tissue formation. To achieve this, matrices are becoming more complex, often including multiple

  10. Pregnancy-associated plasma protein-A (PAPP-A) modulates early developmental rate in zebrafish independent of its proteolytic activity

    DEFF Research Database (Denmark)

    Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu

    2013-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving IGF binding proteins, causing increased IGF bioavailability and hence local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene...... phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A....

  11. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Fernanda Cortez Lopes

    2011-01-01

    Full Text Available A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

  12. Computing H/D-Exchange rates of single residues from data of proteolytic fragments

    Directory of Open Access Journals (Sweden)

    Althaus Ernst

    2010-08-01

    Full Text Available Abstract Background Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant. The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct. Results In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution. Conclusions With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.

  13. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Science.gov (United States)

    Lopes, Fernanda Cortez; Silva, Lucas André Dedavid e; Tichota, Deise Michele; Daroit, Daniel Joner; Velho, Renata Voltolini; Pereira, Jamile Queiroz; Corrêa, Ana Paula Folmer; Brandelli, Adriano

    2011-01-01

    A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism. PMID:22007293

  14. Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota.

    Science.gov (United States)

    Cella, R; Carbonera, D; Orsi, R; Ferri, G; Iadarola, P

    1991-06-01

    The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

  15. Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers.

    Science.gov (United States)

    Moor, Andrea N; Vachon, David J; Gould, Lisa J

    2009-01-01

    Venous leg ulcers affect approximately 1% of the general population and 3.6% of those over the age of 65. The goal of the research described herein is to shorten the time to healing by developing wound care alternatives that are based on a comprehensive understanding of the venous ulcer wound environment. The proteolytic and inflammatory components in wound fluids and tissue biopsy samples were characterized in subjects with documented long-standing venous ulcers that had showed resistance to standard therapy. All wounds showed polymicrobial colonization with greater than 10(6) CFU/g. Myeloperoxidase, a measure of leukocyte infiltration, was also markedly elevated in these wounds. Zymography revealed the presence of both pro-matrix metalloproteinase (MMP)-2 and pro-MMP-9 in wound fluids and to a lesser extent in tissue biopsies. Using an immunocapture activity assay we reveal a sevenfold excess of MMP-9 in wound fluid as compared to tissue, with 73% in the activated form. In contrast, MMP-8 total protein levels were nearly equal in wound fluids and biopsies. Fibronectin, a critical component of the extracellular matrix, was shown to be degraded in both wound fluids and biopsy samples. Finally, the potential of a novel wound dressing to neutralize several constituents of this hostile wound environment is shown.

  16. Atorvastatin treatment decreases inflammatory and proteolytic activity in patients with hypercholesterolemia.

    Science.gov (United States)

    Ercan, Ertugrul; Tengiz, Istemihan; Altuglu, Imre; Sekuri, Cevat; Aliyev, Emil; Ercan, H Ekin; Akin, Mustafa

    2004-05-01

    Statins have anti-inflammatory and anti-platelet effects, which are known as non-lipid effects. Statin treatment can decrease endogenous inflammatory response. To study the effects of atorvastatin on matrix metalloproteinase-9 (MMP-9) and high sensitive C-reactive protein (hs-CRP) - markers of the proteinolytic and inflammatory activity. In this prospective study 44 patients with hypercholesterolemia were randomly assigned into 2 groups; Group 1 (n=22) treated with atorvastatin and diet for 2 months, and Group 2 (n=22) - diet alone. MMP-9 and hs-CRP were measured at baseline and two months later. Groups were matched for age, sex and baseline characteristics. Lipid levels decreased by 32% (LDL from 153.9+/-26.6 to 94.5+/-20.8 mg/dl, pdiet alone group. Atorvastatin lowered plasma CRP from 5.16+/-1.9 to 2.88+/-1.06 mg/L (pdiet alone group. MMP-9 levels did not show significant changes in Group 2 after two months of diet. Atorvastatin treatment decreases inflammatory and proteolytic activity in patients with hypercholesterolemia.

  17. Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

    Science.gov (United States)

    Shtenberg, Giorgi; Massad-Ivanir, Naama; Moscovitz, Oren; Engin, Sinem; Sharon, Michal; Fruk, Ljiljana; Segal, Ester

    2013-02-05

    A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

  18. Studies on the membrane-associated proteolytic activities of Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.

    1986-01-01

    The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl ≅ 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH ≅ 8.0. Incubation of whole membrane preparations with [ 3 H] DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind [ 125 I] ampicillin

  19. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    Science.gov (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  20. Occupational asthma after inhalation of dust of the proteolytic enzyme, papain.

    Science.gov (United States)

    Milne, J; Brand, S

    1975-11-01

    Papain is a proteolytic enzyme widely used by biochemists. In experiments on animals papain has been shown to cause emphysema either when they inhaled a single small dose or after intratracheal inhalation. Four food technologists were occupationally exposed to heavy concentrations of papain dust in air. Subjects 1 and 2 developed an immediate acute asthmatic reaction, and symptoms of obstructive airways disease persisted for some months while each remained in the same working area, presumably exposed to small gradually diminishing amounts of residual papain dust. Tests of respiratory function were carried out on all four subjects 1 1/2 years later and showed in subjects 1 and 3 minimal abnormality of bronchial reactivity and of ventilation distribution. Review of the literature reveals only two reports of asthma resulting from papain inhalation, although its antigenic and skin sensitizing qualities have been known and described for many years. It seems remarkable that a substance such as papain, shown to be a potent cause of lung damage in experimental animals, should have produced so little evidence of abnormality in our subjects after considerable exposure. Follow-up ventilatory function tests may cast further light on this but we postulate that the asthmatic response may be biologically protective and those lacking this reaction could later develop emphysema as a long-term outcome.

  1. Mutation in Transforming Growth Factor Beta Induced protein associated with Granular Corneal Dystrophy Type 1 Reduces the Proteolytic Susceptibility through Local Structural Stabilization#

    Science.gov (United States)

    Underhaug, Jarl; Koldsø, Heidi; Runager, Kasper; Nielsen, Jakob Toudahl; Sørensen, Charlotte S.; Kristensen, Torsten; Otzen, Daniel E.; Karring, Henrik; Malmendal, Anders; Schiøtt, Birgit; Enghild, Jan J.; Nielsen, Niels Chr.

    2014-01-01

    Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. PMID:24129074

  2. Antioxidants, free radicals, storage proteins, puroindolines, and proteolytic activities in bread wheat (Triticum aestivum) seeds during accelerated aging.

    Science.gov (United States)

    Calucci, Lucia; Capocchi, Antonella; Galleschi, Luciano; Ghiringhelli, Silvia; Pinzino, Calogero; Saviozzi, Franco; Zandomeneghi, Maurizio

    2004-06-30

    Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

  3. Parser Adaptation for Social Media by Integrating Normalization

    NARCIS (Netherlands)

    van der Goot, Rob; van Noord, Gerardus

    This work explores normalization for parser adaptation. Traditionally, normalization is used as separate pre-processing step. We show that integrating the normalization model into the parsing algorithm is beneficial. This way, multiple normalization candidates can be leveraged, which improves

  4. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Roč. 20, č. 12 (2011), s. 2004-2012 ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  5. Effects of reducing fat content on the proteolytic and rheological properties of Cheddar-like caprine milk cheese

    Science.gov (United States)

    High-moisture Cheddar-like cheeses made from caprine milk containing 3.6, 2.0, 1.0, and 0.1-0.5% fat were manufactured and their proteolytic and rheological properties compared after 1, 3, and 6 mo of aging at 4 deg C. The full-fat (FF), reduced fat (RF), low-fat (LF), and non-fat (NF) cheeses conta...

  6. Co-Segregation of Social Cognition, Executive Function and Local Processing Style in Children with ASD, Their Siblings and Normal Controls

    Science.gov (United States)

    Oerlemans, Anoek M.; Droste, Katharina; van Steijn, Daphne J.; de Sonneville, Leo M. J.; Buitelaar, Jan K.; Rommelse, Nanda N.

    2013-01-01

    Cognitive research proposes that social cognition (SC), executive functions (EF) and local processing style (weak CC) may be fruitful areas for research into the familial-genetic underpinnings of Autism Spectrum Disorders (ASD). The performance of 140 children with ASD, 172 siblings and 127 controls on tasks measuring SC (face recognition,…

  7. Changes in connectivity of the posterior default network node during visual processing in mild cognitive impairment: staged decline between normal aging and Alzheimer's disease.

    Science.gov (United States)

    Krajcovicova, Lenka; Barton, Marek; Elfmarkova-Nemcova, Nela; Mikl, Michal; Marecek, Radek; Rektorova, Irena

    2017-12-01

    Visual processing difficulties are often present in Alzheimer's disease (AD), even in its pre-dementia phase (i.e. in mild cognitive impairment, MCI). The default mode network (DMN) modulates the brain connectivity depending on the specific cognitive demand, including visual processes. The aim of the present study was to analyze specific changes in connectivity of the posterior DMN node (i.e. the posterior cingulate cortex and precuneus, PCC/P) associated with visual processing in 17 MCI patients and 15 AD patients as compared to 18 healthy controls (HC) using functional magnetic resonance imaging. We used psychophysiological interaction (PPI) analysis to detect specific alterations in PCC connectivity associated with visual processing while controlling for brain atrophy. In the HC group, we observed physiological changes in PCC connectivity in ventral visual stream areas and with PCC/P during the visual task, reflecting the successful involvement of these regions in visual processing. In the MCI group, the PCC connectivity changes were disturbed and remained significant only with the anterior precuneus. In between-group comparison, we observed significant PPI effects in the right superior temporal gyrus in both MCI and AD as compared to HC. This change in connectivity may reflect ineffective "compensatory" mechanism present in the early pre-dementia stages of AD or abnormal modulation of brain connectivity due to the disease pathology. With the disease progression, these changes become more evident but less efficient in terms of compensation. This approach can separate the MCI from HC with 77% sensitivity and 89% specificity.

  8. Short communication: Effects of Lactobacillus helveticus-fermented milk on the differentiation of cultured normal human epidermal keratinocytes.

    Science.gov (United States)

    Baba, H; Masuyama, A; Takano, T

    2006-06-01

    Effects of Lactobacillus helveticus-fermented milk whey on the differentiation of normal human epidermal keratinocytes were studied. Analysis using real-time reverse transcription-polymerase chain reaction revealed that addition of Lactobacillus helveticus-fermented milk whey to the culture medium enhanced mRNA expression of keratin 10, an early differentiation marker, as well as involucrin, a late differentiation marker. Whey of artificially acidified milk, prepared by the addition of dl-lactic acid to milk instead of fermentation, also promoted expression of both markers, but Lactobacillus helveticus-fermented milk whey was more effective in increasing expression of those markers. These results indicate that milk whey has the potential to induce multiple stages of keratinocyte differentiation and that fermentation with Lactobacillus helveticus increases that activity. Furthermore, we examined the expression of profilaggrin, which increases with epidermal terminal differentiation, and found that Lactobacillus helveticus-fermented milk whey enhanced expression of profilaggrin mRNA in a dose-dependent manner. Expression also occurred to a greater extent than with artificially acidified milk whey or other whey samples prepared with several lactic acid bacterial species. Because the proteolytically processed form of profilaggrin, filaggrin, is very important for normal epidermal hydration and flexibility, our results indicate that Lactobacillus helveticus-fermented milk whey has the potential to enhance the production of filaggrin-related natural moisturizing factor, because of its effect on the induction of epidermal differentiation, and is expected to be a useful skin moisturizing agent.

  9. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  10. Proteolytic activity of prostate-specific antigen (PSA) towards protein substrates and effect of peptides stimulating PSA activity.

    Science.gov (United States)

    Mattsson, Johanna M; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  11. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  12. Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Kottapalli, Bala; Lineback, D Scott; Talley, Ryan J; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

  13. An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

    Science.gov (United States)

    Papagianni, Maria

    2014-01-01

    A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

  14. Testing for normality

    CERN Document Server

    Thode, Henry C

    2002-01-01

    Describes the selection, design, theory, and application of tests for normality. Covers robust estimation, test power, and univariate and multivariate normality. Contains tests ofr multivariate normality and coordinate-dependent and invariant approaches.

  15. Efficient debridement of necrotic wounds using proteolytic enzymes derived from Antarctic krill: a double-blind, placebo-controlled study in a standardized animal wound model

    NARCIS (Netherlands)

    Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Bos, J. D.; Westerhof, W.

    1998-01-01

    Wound healing can be accelerated by removing necrotic tissue. Various methods of wound debridement have been developed, including enzymatic debridement. Recently potent proteolytic enzymes were isolated from the intestine of Euphausia superba (Antarctic krill) that might be useful for degrading

  16. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

    DEFF Research Database (Denmark)

    Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz

    2011-01-01

    Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens...

  17. Matrix Metalloproteinases and Neurotrauma: Evolving Roles in Injury and Reparative Processes

    Science.gov (United States)

    Zhang, Haoqian; Adwanikar, Hita; Werb, Zena; Noble-Haeusslein, Linda J.

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes. PMID:20400713

  18. Matrix metalloproteinases and neurotrauma: evolving roles in injury and reparative processes.

    Science.gov (United States)

    Zhang, Haoqian; Adwanikar, Hita; Werb, Zena; Noble-Haeusslein, Linda J

    2010-04-01

    Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes.

  19. The Normal Distribution From Binomial to Normal

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 2; Issue 6. The Normal Distribution From Binomial to Normal. S Ramasubramanian. Series Article Volume 2 Issue 6 June 1997 pp 15-24. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/002/06/0015-0024 ...

  20. Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count.

    Science.gov (United States)

    Albenzio, Marzia; Santillo, Antonella; Caroprese, Mariangela; Schena, Laura; Russo, Donatella Esterina; Sevi, Agostino

    2011-11-01

    This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.

  1. Screening for proteolytically active lactic acid bacteria and bioactivity of peptide hydrolysates obtained with selected strains.

    Science.gov (United States)

    Kliche, T; Li, B; Bockelmann, W; Habermann, D; Klempt, M; de Vrese, M; Wutkowski, A; Clawin-Raedecker, I; Heller, K J

    2017-10-01

    In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and β-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and β-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of β-casein. Peptide length was between 9 (β-CN(f 59-67)) and 22 amino acids (β-CN(f 117-138)).

  2. A novel immune evasion strategy of candida albicans: proteolytic cleavage of a salivary antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Timothy F Meiller

    Full Text Available Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps, involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the

  3. The E3 ligase AtCHIP positively regulates Clp proteolytic subunit homeostasis.

    Science.gov (United States)

    Wei, Jia; Qiu, Xiaoyun; Chen, Lin; Hu, Wenjun; Hu, Rongbin; Chen, Jian; Sun, Li; Li, Li; Zhang, Hong; Lv, Zhiqiang; Shen, Guoxin

    2015-09-01

    The caseinolytic peptidase (Clp) core proteins are essential for plant growth and development, especially for chloroplast function. Antisense or overexpression of ClpP4, which is one of the Clp core subunits, causes chlorotic phenotypes in Arabidopsis. An E3 ligase gene, AtCHIP, has previously been found to ubiquitylate ClpP4 in vitro. ClpP4 antisense and overexpressing plants that also overexpressed AtCHIP were constructed to explore the effect of AtCHIP on ClpP4. Overexpression of AtCHIP was found to rescue the chlorotic phenotypes of both ClpP4 antisense and overexpressing plants. The unbalanced levels of Clp core proteins in ClpP4 antisense and overexpressing plants with overexpression of AtCHIP were similar to wild-type levels, suggesting that AtCHIP regulates Clp core proteins. The results also show that AtCHIP can interact with ClpP3 and ClpP5 in yeast and ubiquitylate ClpP3 and ClpP5 in vitro. This suggests that AtCHIP is directly related to ClpP3 and ClpP5. Given these results, the inference is that through selective degradation of Clp subunits, AtCHIP could positively regulate homeostasis of Clp proteolytic subunits and maximize the production of functional chloroplasts. Similar results were obtained from transgenic tobacco plants, suggesting that regulation of the Clp protease by AtCHIP is conserved. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Selection of proteolytic bacteria with ability to inhibit Vibrio harveyi during white shrimp (Litopenaeus vannamei cultivation

    Directory of Open Access Journals (Sweden)

    Suntinanalert, P.

    2007-03-01

    Full Text Available Five isolates of bacteria with high proteolytic activity, isolated from water samples of intensive shrimp ponds in southern Thailand, were selected to test for the ability to control the shrimp pathogen Vibrioharveyi. 70 μl of each culture broth were investigated for their ability to inhibit V. harveyi using an agar well diffusion test but only one isolate W3 gave a reasonable sized inhibition zone of 21.62 mm. This zone wassimilar to that of oxolinic acid (2 μg and sulfamethoxazole (25 μg. The W3 isolate was identified as Pseudomonas sp. Shrimp cultivation in aquaria was conducted to investigate the inhibition of V. harveyi bythe isolate W3. The experiment consisted of a treatment of the shrimp culture with an inoculum of the isolate W3 and V. harveyi (biocontrol set, a positive control set (only inoculation of V. harveyi and a negativecontrol set as without inoculation. No mortality was found in the negative control. Shrimp mortality in the biocontrol set (33% was lower than that in the positive control set (40%; however, it showed no significantdifference (p>0.05. The average numbers of V. harveyi over 12 days of the biocontrol set were lower than those in the positive control set by about 1 log cycle although the numbers were not significantly different(p>0.05. The shrimp growth rate at day 32 of cultivation was in order of the biocontrol treatment (10.17% > the negative control treatment (9.44% > the positive control set (9.28%, but no significant difference (p>0.05 was observed among treatments.

  5. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    International Nuclear Information System (INIS)

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P.

    1989-01-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125 I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

  6. Preferred Orientation Contribution to the Anisotropic Normal State Resistivity in Superconducting Melt-Cast Processed Bi2Sr2CaCu2O8+δ

    Directory of Open Access Journals (Sweden)

    Aline Dellicour

    2017-05-01

    Full Text Available We describe how the contribution of crystallographic texture to the anisotropy of the resistivity of polycrystalline samples can be estimated by averaging over crystallographic orientations through a geometric mean approach. The calculation takes into account the orientation distribution refined from neutron diffraction data and literature values for the single crystal resistivity tensor. The example discussed here is a melt-cast processed Bi2Sr2CaCu2O8+δ (Bi-2212 polycrystalline tube in which the main texture component is a <010> fiber texture with relatively low texture strength. Experimentally-measured resistivities along the longitudinal, radial, and tangential directions of the Bi-2212 tube were compared to calculated values and found to be of the same order of magnitude. Calculations for this example and additional simulations for various texture strengths and single crystal resistivity anisotropies confirm that in the case of highly anisotropic phases such as Bi-2212, even low texture strengths have a significant effect on the anisotropy of the resistivity in polycrystalline samples.

  7. Preferred Orientation Contribution to the Anisotropic Normal State Resistivity in Superconducting Melt-Cast Processed Bi₂Sr₂CaCu₂O8+δ.

    Science.gov (United States)

    Dellicour, Aline; Vertruyen, Benedicte; Rikel, Mark O; Lutterotti, Luca; Pautrat, Alain; Ouladdiaf, Bachir; Chateigner, Daniel

    2017-05-15

    We describe how the contribution of crystallographic texture to the anisotropy of the resistivity of polycrystalline samples can be estimated by averaging over crystallographic orientations through a geometric mean approach. The calculation takes into account the orientation distribution refined from neutron diffraction data and literature values for the single crystal resistivity tensor. The example discussed here is a melt-cast processed Bi₂Sr₂CaCu₂O 8+δ (Bi-2212) polycrystalline tube in which the main texture component is a fiber texture with relatively low texture strength. Experimentally-measured resistivities along the longitudinal, radial, and tangential directions of the Bi-2212 tube were compared to calculated values and found to be of the same order of magnitude. Calculations for this example and additional simulations for various texture strengths and single crystal resistivity anisotropies confirm that in the case of highly anisotropic phases such as Bi-2212, even low texture strengths have a significant effect on the anisotropy of the resistivity in polycrystalline samples.

  8. Normalized modes at selected points without normalization

    Science.gov (United States)

    Kausel, Eduardo

    2018-04-01

    As every textbook on linear algebra demonstrates, the eigenvectors for the general eigenvalue problem | K - λM | = 0 involving two real, symmetric, positive definite matrices K , M satisfy some well-defined orthogonality conditions. Equally well-known is the fact that those eigenvectors can be normalized so that their modal mass μ =ϕT Mϕ is unity: it suffices to divide each unscaled mode by the square root of the modal mass. Thus, the normalization is the result of an explicit calculation applied to the modes after they were obtained by some means. However, we show herein that the normalized modes are not merely convenient forms of scaling, but that they are actually intrinsic properties of the pair of matrices K , M, that is, the matrices already "know" about normalization even before the modes have been obtained. This means that we can obtain individual components of the normalized modes directly from the eigenvalue problem, and without needing to obtain either all of the modes or for that matter, any one complete mode. These results are achieved by means of the residue theorem of operational calculus, a finding that is rather remarkable inasmuch as the residues themselves do not make use of any orthogonality conditions or normalization in the first place. It appears that this obscure property connecting the general eigenvalue problem of modal analysis with the residue theorem of operational calculus may have been overlooked up until now, but which has in turn interesting theoretical implications.Á

  9. Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.

    Science.gov (United States)

    Park, Seonhwa; Shin, Yu Mi; Song, Ji-Joon; Yang, Haesik

    2015-10-15

    Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. [Breast cancer invasion: the key role of normal cells of host tissues].

    Science.gov (United States)

    Foidart, J M; Polette, M; Birembaut, P; Noel, A

    1994-03-01

    Tumor progression is influenced by extracellular matrices and by soluble factors or cytokines locally produced by host tissue cells (fibroblasts, immune cells ...). Such factors may also accumulate in close association with some extracellular matrix molecules in the tumor. They may also be unmasked during breaking down of extracellular matrices. The most insidious aspect of tumors is their propensity to locally invade normal tissues of the host and to form secondary foci in organs at distant sites from the primary tumor called metastases. During this process, invasive cells come into contact with host tissue cells such as fibroblasts, endothelial cells, macrophages, lymphocytes. These cells are not the passive witnesses of the metastatic cascade but actively participate to the malignant invasion. Through soluble messages (cytokines) and through insoluble molecules of the extracellular matrix, neoplastic and normal cells mutually modulate their activities. Cancer cells regulate the biosynthetic activities of fibroblasts and alter in this way the scaffold of the tumor. Reciprocally, host cells secrete extracellular matrix proteins and cytokines which influence the growth and activities of tumor cells. They also produce at the periphery of tumor cells proteolytic enzymes which promote host tissue destruction and cancerous cells migration. Among these enzymes, matrix metalloproteinases appear to play a key role during invasion and metastasis. Tumors represent thus a complex ecosystem. Tumor cells interact with several components of the extracellular matrix and with host cells (immune cells, fibroblasts, endothelial cells). Such multiple cell-cell and cell-matrix interactions condition angiogenesis, tumor growth, destruction of host tissues, local migration of cancer cells and their metastatic dissemination. It is probable that a precise knowledge of the genes which are selectively activated in tumors under the influence of the host cells or of the tumor cells will

  11. Planar integrated optical waveguide used as a transducer to yield chemical information: detection of the activity of proteolytic enzymes e.g. serine-proteases

    Science.gov (United States)

    Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.

    2005-03-01

    The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

  12. The Vietnam-U.S. Normalization Process

    Science.gov (United States)

    2005-06-17

    agreement to resume U.S. adoptions of Vietnamese children , which Hanoi halted in 2002. There are a number of possible reasons Vietnam seeks to upgrade its...abandoned or orphaned children ) for Vietnam; agreed to restore direct telecommunications with Vietnam; agreed to allow U.S. commercial sales to meet...Commissioner for Refugees; (3) resolve the issue of the estimated several thousand Amerasians (whose fathers are Americans and whose mothers are

  13. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    OpenAIRE

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackieviru...

  14. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    NARCIS (Netherlands)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were

  15. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation...... of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing....

  16. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-01-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (PLactobacillus bulgaricus at the transcription level. PMID:25845365

  17. PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED, ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES

    Science.gov (United States)

    Winnick, Theodore; Davis, Alva R.; Greenberg, David M.

    1940-01-01

    1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion. PMID:19873154

  18. Loss of pulmonary elastic recoil in workers formerly exposed to proteolytic enzyme (alcalase) in the detergent industry.

    Science.gov (United States)

    Musk, A W; Gandevia, B

    1976-01-01

    Sixty-seven workers in the detergent industry whose exposure to proteolytic enzyme ceased in 1969 have been examined clinically and functionally. By comparison with 42 lightly and moderately exposed subjects, 13 heavily exposed subjects showed significant loss of pulmonary elastic recoil as evidenced by increased lung volumes and increased pulmonary compliance, but there were no differences in airways resistance or other parameters of lung function. No difference was found between the two groups in relation to symptoms on exposure, current exercise tolerance, skin reactivity to the proteolytic enzyme alcalase, trypsin inhibitor capacity, and other features. An increased clinical grade of breathlessness was associated with evidence of airways obstruction, but not of altered elastic recoil. Comparison of the data on lung mechanics with results obtained in 1970 suggests that partial recovery of pulmonary elastic recoil may have occurred in some cases. It is also suggested that diminished elastic recoil, in the absence of impairment of transfer factor at rest, may reflect altered physical properties of the lung fibre network without loss of effective surface area available for gas exchange. PMID:963000

  19. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

  20. NBT-PABA test to assess efficiency and kinetics of substituted proteolytic enzyme action in pancreatic duct ligated minipigs.

    Science.gov (United States)

    Mösseler, A; Bergemann, J; Becker, C; Stemme, K; Gregory, P C; Kamphues, J

    2008-06-01

    The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals.

  1. Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases

    Science.gov (United States)

    Matsui, Mihoko; Kawamata, Akinori; Kosugi, Makiko; Imura, Satoshi; Kurosawa, Norio

    2017-09-01

    Despite being an extreme environment, the water temperature of freshwater lakes in Antarctica reaches 10 °C in summer, accelerating biological activity. In these environments, proteolytic microbial decomposers may play a large role in protein hydrolysis. We isolated 71 microbial strains showing proteolytic activity at 4 °C from three Antarctic freshwater lakes. They were classified as bacteria (63 isolates) and eukaryotes (8 isolates). The bacterial isolates were classified into the genera Flavobacterium (28 isolates), Pseudomonas (14 isolates), Arthrobacter (10 isolates), Psychrobacter (7 isolates), Cryobacterium (2 isolates), Hymenobacter (1 isolate), and Polaromonas (1 isolate). Five isolates of Flavobacterium and one of Hymenobacter seemed to belong to novel species. All eukaryotic isolates belonged to Glaciozyma antarctica, a psychrophilic yeast species originally isolated from the Weddell Sea near the Joinville Island, Antarctica. A half of representative strains were psychrophilic and did not grow at temperatures above 25 °C. The protease secreted by Pseudomonas prosekii strain ANS4-1 showed the highest activity among all proteases from representative isolates. The results of inhibitor tests indicated that nearly all the isolates secreted metalloproteases. Proteases from four representative isolates retained more than 30% maximal activity at 0 °C. These results expand our knowledge about microbial protein degradation in Antarctic freshwater lakes.

  2. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus.

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-04-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (Pproteolytic system genes in Lactobacillus bulgaricus at the transcription level.

  3. Cathepsin G-mediated proteolytic degradation of MHC class I molecules to facilitate immune detection of human glioblastoma cells.

    Science.gov (United States)

    Palesch, David; Wagner, Johanna; Meid, Annika; Molenda, Nicole; Sienczyk, Marcin; Burkhardt, Jutta; Münch, Jan; Prokop, Lea; Stevanovic, Stefan; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Wirtz, Christian Rainer; Zimecki, Michal; Burster, Timo

    2016-03-01

    To mount an adaptive immune response, MHC I molecules present antigenic peptides to CTLs. Transcriptional reduction of MHC I molecules is a strategy of immune evasion, which impairs the detection of infected or tumorous cells by CTLs. Natural killer (NK) cells, on the other hand, eliminate target cells specifically in the absence of MHC I. Consequently, infected or tumorous cells partly retain their MHC I at the cell surface to avoid NK recognition. However, it remains unclear which protease degrades MHC I molecules and how these cells maintain a limited set of MHC I at the cell surface. Here, we demonstrate that cathepsin G (CatG), a serine protease, found in the endocytic compartment of APCs and, to a lesser extent, CatD and CatS proteolytically degrade MHC I molecules. Inhibition of CatG boosted MHC I expression at the cell surface of primary human immune cells. In contrast, human glioblastoma cells do not harbor active CatG and might have lost the ability to proteolytically degrade MHC I during tumorigenesis to avoid NK-mediated killing. Overexpression of CatG in glioblastoma cells resulted in a rapid and efficient MHC I degradation. In conclusion, CatG is an essential protease for regulating MHC I molecules and thus modulation of CatG activity might present a new avenue for therapeutic intervention.

  4. Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

    Science.gov (United States)

    Tulini, Fabricio L; Hymery, Nolwenn; Haertlé, Thomas; Le Blay, Gwenaelle; De Martinis, Elaine C P

    2016-02-01

    Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

  5. Normal pediatric postmortem CT appearances

    Energy Technology Data Exchange (ETDEWEB)

    Klein, Willemijn M.; Bosboom, Dennis G.H.; Koopmanschap, Desiree H.J.L.M. [Radboud University Medical Center, Department of Radiology and Nuclear Medicine, Nijmegen (Netherlands); Nievelstein, Rutger A.J. [University Medical Center Utrecht, Department of Radiology, Utrecht (Netherlands); Nikkels, Peter G.J. [University Medical Center Utrecht, Department of Pathology, Utrecht (Netherlands); Rijn, Rick R. van [Academic Medical Center, Department of Radiology, Amsterdam (Netherlands)

    2015-04-01

    Postmortem radiology is a rapidly developing specialty that is increasingly used as an adjunct to or substitute for conventional autopsy. The goal is to find patterns of disease and possibly the cause of death. Postmortem CT images bring to light processes of decomposition most radiologists are unfamiliar with. These postmortem changes, such as the formation of gas and edema, should not be mistaken for pathological processes that occur in living persons. In this review we discuss the normal postmortem thoraco-abdominal changes and how these appear on CT images, as well as how to differentiate these findings from those of pathological processes. (orig.)

  6. The proteolytic activity of pregnancy-associated plasma protein-A is potentially regulated by stanniocalcin-1 and -2 during human ovarian follicle development

    DEFF Research Database (Denmark)

    Jepsen, Rikke Malene Hg; Kløverpris, Søren; Bøtkjær, Jane Alrø

    2016-01-01

    STUDY QUESTION: Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER: The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation...... of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY: The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen...

  7. Corners of normal matrices

    Indian Academy of Sciences (India)

    The structure of general normal matrices is far more complicated than that of two special kinds — hermitian and unitary. There are many interesting theorems for hermitian and unitary matrices whose extensions to arbitrary normal matrices have proved to be extremely recalcitrant (see e.g., [1]). The problem whose study we ...

  8. Normalized medical information visualization.

    Science.gov (United States)

    Sánchez-de-Madariaga, Ricardo; Muñoz, Adolfo; Somolinos, Roberto; Castro, Antonio; Velázquez, Iker; Moreno, Oscar; García-Pacheco, José L; Pascual, Mario; Salvador, Carlos H

    2015-01-01

    A new mark-up programming language is introduced in order to facilitate and improve the visualization of ISO/EN 13606 dual model-based normalized medical information. This is the first time that visualization of normalized medical information is addressed and the programming language is intended to be used by medical non-IT professionals.

  9. Baby Poop: What's Normal?

    Science.gov (United States)

    ... I'm breast-feeding my newborn and her bowel movements are yellow and mushy. Is this normal for baby poop? Answers from Jay L. Hoecker, M.D. Yellow, mushy bowel movements are perfectly normal for breast-fed babies. Still, ...

  10. Making nuclear 'normal'

    International Nuclear Information System (INIS)

    Haehlen, Peter; Elmiger, Bruno

    2000-01-01

    The mechanics of the Swiss NPPs' 'come and see' programme 1995-1999 were illustrated in our contributions to all PIME workshops since 1996. Now, after four annual 'waves', all the country has been covered by the NPPs' invitation to dialogue. This makes PIME 2000 the right time to shed some light on one particular objective of this initiative: making nuclear 'normal'. The principal aim of the 'come and see' programme, namely to give the Swiss NPPs 'a voice of their own' by the end of the nuclear moratorium 1990-2000, has clearly been attained and was commented on during earlier PIMEs. It is, however, equally important that Swiss nuclear energy not only made progress in terms of public 'presence', but also in terms of being perceived as a normal part of industry, as a normal branch of the economy. The message that Swiss nuclear energy is nothing but a normal business involving normal people, was stressed by several components of the multi-prong campaign: - The speakers in the TV ads were real - 'normal' - visitors' guides and not actors; - The testimonials in the print ads were all real NPP visitors - 'normal' people - and not models; - The mailings inviting a very large number of associations to 'come and see' activated a typical channel of 'normal' Swiss social life; - Spending money on ads (a new activity for Swiss NPPs) appears to have resulted in being perceived by the media as a normal branch of the economy. Today we feel that the 'normality' message has well been received by the media. In the controversy dealing with antinuclear arguments brought forward by environmental organisations journalists nowadays as a rule give nuclear energy a voice - a normal right to be heard. As in a 'normal' controversy, the media again actively ask themselves questions about specific antinuclear claims, much more than before 1990 when the moratorium started. The result is that in many cases such arguments are discarded by journalists, because they are, e.g., found to be

  11. Stanniocalcin-1 Potently Inhibits the Proteolytic Activity of the Metalloproteinase Pregnancy-associated Plasma Protein-A

    DEFF Research Database (Denmark)

    Kløverpris, Søren; Mikkelsen, Jakob Hauge; Pedersen, Josefine Hvidkjær

    2015-01-01

    regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth...... that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue....... It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity...

  12. Reversed hydroxamate-bearing thermolysin inhibitors mimic a high-energy intermediate along the enzyme-catalyzed proteolytic reaction pathway.

    Science.gov (United States)

    Park, Jung Dae; Kim, Dong H

    2003-10-06

    A series of inhibitors that bear a reversed hydroxamate moiety have been evaluated as transition state analogue inhibitors for thermolysin. A linear correlation is observed between the K(i) values of these inhibitors and the kinetic parameters (K(M)/k(cat)) of the parallel series of related substrates, satisfying the criterion stipulated for transition state analogue inhibitors by Bartlett and Marlowe. Furthermore, examination of the binding mode of a related reversed hydroxamate bearing thermolysin inhibitor, in comparison with a transition state postulated for the enzyme-catalyzed proteolytic reaction revealed that the inhibitors under study mimic the electronic as well as the geometric characteristics of the transition state. On the basis of these results it may be concluded that the hydroxamate-bearing zinc protease inhibitors are a new type of transition state analogue inhibitors.

  13. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity

    Science.gov (United States)

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-02-01

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  14. [Effect of Azospirillum lectins on the Activity of Proteolytic Enzymes and Their Inhibitors in Wheat Seedling Roots].

    Science.gov (United States)

    Alen'kina, S A; Nikitina, V E

    2015-01-01

    The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

  15. [Proteolytic, procoagulant activity and electrophoretic characterization of the proteins of 2 toxic extracts of Loxosceles laeta venom].

    Science.gov (United States)

    Heredia, V; Arbaiza, E; Venegas, J; Yarlequé, A; Zavaleta, A

    1989-01-01

    Some biochemical properties and proteic components of the brown spider (Loxos celes laeta) venom were studied. The electrophoretic profiles of glandular venom and venom obtained through electrical stimulation were compared using two electrophoretic systems. The first, using a polyacrylamide gel with SDS in tubes, and the second, using an acrylamide gradient on slides. The glandular venom presented 20 and 35 bands respectively, while the venom obtained through electrical stimulation presented 19 and 24 bands. The molecular weight of the proteins detected ranged from 13.5 Kd to 220 Kd. A thermolabil proteolitic activity of casein was detected, and was optimum at pH 9. The effects of the divalent ions, calcium and magnesium, as well as that of chelating agents upon the proteolytic activity of the venom were analyzed. The venom had a procoagulant effect upon citrated human plasma, and was not able to activate the Factor X of the coagulation system in vitro.

  16. Induction of the staphylococcal proteolytic cascade by antimicrobial fatty acids in community acquired methicillin resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Benjamin Arsic

    Full Text Available Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA, and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16∶1 and linoleic acid (C18∶1 were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10-12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus.

  17. Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

    International Nuclear Information System (INIS)

    Smith, A.C.; Harmon, J.M.

    1987-01-01

    The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [ 3 H]dexamethasone 21-mesylate ([ 3 H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [ 3 H]DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [ 3 H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [ 3 H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments

  18. Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas Microbial quality of raw refrigerated milk and isolation of psychrotrophic proteolytic bacteria

    Directory of Open Access Journals (Sweden)

    Cláudia Lúcia de Oliveira Pinto

    2006-09-01

    Full Text Available A estocagem do leite cru refrigerado na fonte de produção reduz perdas econômicas por atividade acidificante de bactérias mesofílicas, mas permite a seleção de bactérias psicrotróficas relacionadas a problemas tecnológicos e econômicos na indústria de laticínios. Com o objetivo de avaliar a qualidade microbiológica de leite cru, foram analisadas amostras provenientes de tanques de refrigeração individual, coletivos e do silo de uma indústria processadora de leite. Além disso, bactérias psicrotróficas proteolíticas foram isoladas do leite cru refrigerado e caracterizadas quanto à reação ao Gram e fermentação de glicose. O leite cru refrigerado mantido no silo industrial não atendeu ao padrão microbiológico legal e apresentou contagens microbianas significativamente superiores às do leite mantido em tanques individuais ou coletivos. Diferença significativa na contaminação por mesófilos e psicrotrófilos proteolíticos e não proteolíticos e por Pseudomonas foi observada entre as amostras coletadas nos tanques de refrigeração e no silo industrial. A microbiota Gram-negativa foi isolada com maior freqüência, especialmente bactérias Gram-negativas não fermentadoras de glicose.Storing refrigerated raw milk in the source of production reduces economic losses which occur due to the acidifying activity of mesophilic bacteria. Nevertheless, this process selects psychrotrophic bacteria that cause technological and economic problems to the dairy industry. Samples of individual and collective refrigerated tanks and samples from an industrial silo in a dairy industry were collected and analyzed to evaluate the microbiological quality of raw milk. Furthermore, proteolytic psychrotrophic bacteria were isolated from refrigerated raw milk and characterized according to the Gram reaction and glucose fermentation. The refrigerated raw milk from the industrial silo was not compatible with legal microbiological standards, and

  19. Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides.

    Science.gov (United States)

    de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles

    2017-11-10

    The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

    Directory of Open Access Journals (Sweden)

    Mareš Michael

    2008-03-01

    Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

  1. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin.

    Science.gov (United States)

    Boehm, Manja; Hoy, Benjamin; Rohde, Manfred; Tegtmeyer, Nicole; Bæk, Kristoffer T; Oyarzabal, Omar A; Brøndsted, Lone; Wessler, Silja; Backert, Steffen

    2012-04-25

    Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni's HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori's HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  2. Proteolytic detergent enzymes enhance the allergic antibody responses of guinea pigs to nonproteolytic detergent enzymes in a mixture: implications for occupational exposure.

    Science.gov (United States)

    Sarlo, K; Ritz, H L; Fletcher, E R; Schrotel, K R; Clark, E D

    1997-10-01

    A guinea pig intratracheal test was developed to assess the respiratory allergenicity of enzymes used in the detergent industry. Information gained from this test was used in a process for setting operational exposure guidelines to protect worker health. Mixtures of enzyme proteins were given to guinea pigs once per week for 10 weeks to determine whether there were interactions among enzymes that affected the induction of antibody responses to each enzyme in the mixture. Passive cutaneous anaphylaxis antibody titers against each enzyme were measured in sera. Mixtures of two or three enzymes always consisted of a protease (Alcalase, Savinase; Novo Industri A/S) with an alpha-amylase (Termamyl; Novo Industri A/S), a lipase (Lipolase; Novo Industri A/S), or both. Control animals were exposed to single enzymes. The antibody titers to Termamyl and Lipolase were significantly greater in animals dosed with the protease-containing mixtures as compared with control animals dosed with a single enzyme. Antibody titers to the protease were unchanged in the presence of additional enzymes in the mixture. Complete inactivation of protease activity abrogated the enhanced antibody response to Lipolase. Inhalation exposure of guinea pigs to a mixture of Alcalase and Lipolase also resulted in higher antibody titers to Lipolase as compared with animals exposed by inhalation to Lipolase alone, showing that the enhanced response was not due to intratracheal delivery of antigen to the respiratory tract. These results show that proteolytic enzymes in a mixture enhance antibody responses to other enzymes. This should be considered when defining exposure guidelines for protease-containing enzyme mixtures.

  3. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    LENUS (Irish Health Repository)

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  4. Normal Female Reproductive Anatomy

    Science.gov (United States)

    ... an inner lining called the endometrium. Normal female reproductive system anatomy. Topics/Categories: Anatomy -- Gynecologic Type: Color, Medical Illustration Source: National Cancer Institute Creator: Terese Winslow (Illustrator) AV Number: CDR609921 Date Created: November 17, 2014 Date Added: ...

  5. Normal growth and development

    Science.gov (United States)

    A child's growth and development can be divided into four periods: Infancy Preschool years Middle childhood years Adolescence Soon after birth, an infant normally loses about 5% to 10% of their birth weight. By about age ...

  6. Normal pressure hydrocephalus

    Science.gov (United States)

    Hydrocephalus - occult; Hydrocephalus - idiopathic; Hydrocephalus - adult; Hydrocephalus - communicating; Dementia - hydrocephalus; NPH ... Ferri FF. Normal pressure hydrocephalus. In: Ferri FF, ed. ... Elsevier; 2016:chap 648. Rosenberg GA. Brain edema and disorders ...

  7. Normal Functioning Family

    Science.gov (United States)

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Español Text Size Email Print Share Normal Functioning Family Page Content Article Body Is there any way ...

  8. Normal Pressure Hydrocephalus

    Science.gov (United States)

    ... improves the chance of a good recovery. Without treatment, symptoms may worsen and cause death. What research is being done? The NINDS conducts and supports research on neurological disorders, including normal pressure hydrocephalus. Research on disorders such ...

  9. Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

    Science.gov (United States)

    Sjögren, Lars L E; Tanabe, Noriaki; Lymperopoulos, Panagiotis; Khan, Nadir Z; Rodermel, Steven R; Aronsson, Henrik; Clarke, Adrian K

    2014-04-18

    The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

  10. Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization, Interaction with the Clp Proteolytic Core, and Functional Importance*

    Science.gov (United States)

    Sjögren, Lars L. E.; Tanabe, Noriaki; Lymperopoulos, Panagiotis; Khan, Nadir Z.; Rodermel, Steven R.; Aronsson, Henrik; Clarke, Adrian K.

    2014-01-01

    The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import. PMID:24599948

  11. Proteolytic activation of the epithelial sodium channel ENaC in preeclampsia examined with urinary exosomes

    DEFF Research Database (Denmark)

    Nielsen, Maria Ravn; Rytz, Mie; Frederiksen-Møller, Britta

    2015-01-01

    OBJECTIVES: Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity...... as a positive control for the presence of collecting duct membrane. RESULTS: Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group (p... pregnancy and preeclampsia CONCLUSIONS: It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ-ENaC, 2) Urine exosome fraction displays a variable pattern of γ-ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women...

  12. PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX

    Energy Technology Data Exchange (ETDEWEB)

    Burke, R.L.; Alberts, B.M.; Hosoda, J.

    1980-07-01

    The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. (1) Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32*I protein for this synthesis. (2) Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. (3) Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. (4) The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3{prime}-OH end of a

  13. Monitoring the normal body

    DEFF Research Database (Denmark)

    Nissen, Nina Konstantin; Holm, Lotte; Baarts, Charlotte

    2015-01-01

    provides us with knowledge about how to prevent future overweight or obesity. This paper investigates body size ideals and monitoring practices among normal-weight and moderately overweight people. Methods : The study is based on in-depth interviews combined with observations. 24 participants were...... recruited by strategic sampling based on self-reported BMI 18.5-29.9 kg/m2 and socio-demographic factors. Inductive analysis was conducted. Results : Normal-weight and moderately overweight people have clear ideals for their body size. Despite being normal weight or close to this, they construct a variety...... of practices for monitoring their bodies based on different kinds of calculations of weight and body size, observations of body shape, and measurements of bodily firmness. Biometric measurements are familiar to them as are health authorities' recommendations. Despite not belonging to an extreme BMI category...

  14. Efecto Zeeman Normal

    OpenAIRE

    Calderón Chamochumbi, Carlos

    2015-01-01

    Se describe el Efecto Zeeman Normal y se presenta una derivación general del torque experimentado por un dipolo magnético debido a su interacción con un campo magnético externo. Los cálculos correspondientes al elemento diferencial de energía potencial magnética y de la energía potencial magnética convencional son estándares. ABSTRACT: The Normal Zeeman Effect is described and a general derivation of the torque undergone by a magnetic dipole due to its interactio...

  15. The normal holonomy group

    International Nuclear Information System (INIS)

    Olmos, C.

    1990-05-01

    The restricted holonomy group of a Riemannian manifold is a compact Lie group and its representation on the tangent space is a product of irreducible representations and a trivial one. Each one of the non-trivial factors is either an orthogonal representation of a connected compact Lie group which acts transitively on the unit sphere or it is the isotropy representation of a single Riemannian symmetric space of rank ≥ 2. We prove that, all these properties are also true for the representation on the normal space of the restricted normal holonomy group of any submanifold of a space of constant curvature. 4 refs

  16. Medically-enhanced normality

    DEFF Research Database (Denmark)

    Møldrup, Claus; Traulsen, Janine Morgall; Almarsdóttir, Anna Birna

    2003-01-01

    Objective: To consider public perspectives on the use of medicines for non-medical purposes, a usage called medically-enhanced normality (MEN). Method: Examples from the literature were combined with empirical data derived from two Danish research projects: a Delphi internet study and a Telebus......, to optimise economic, working and family conditions. The term "doping" does not cover or explain the use of medicines as enhancement among healthy non-athletes. Conclusion: We recommend wider use of the term medically-enhanced normality as a conceptual framework for understanding and analysing perceptions...... of what is considered rational medicine use in contemporary society....

  17. Striving for the unknown normal

    DEFF Research Database (Denmark)

    Nielsen, Mikka

    During the last decade, more and more people have received prescriptions for ADHD drug treatment, and simultaneously the legitimacy of the ADHD diagnosis has been heavily debated among both professionals and laymen. Based on an anthropological fieldwork among adults with ADHD, I illustrate how...... the ADHD diagnosis both answers and produces existential questions on what counts as normal behaviour and emotions. The diagnosis helps the diagnosed to identify, accept and handle problems by offering concrete explanations and solutions to diffuse experienced problems. But the diagnostic process......-analysing situation trying to answer: What is ADHD and what is me and my personal features? When are the drugs having the right effect? And since I have never been normal then how would I know what behavioural ideal I am striving for? Moreover, the public questioning of the diagnosis calls the individual...

  18. Corners of normal matrices

    Indian Academy of Sciences (India)

    ∗Department of Mathematics, University of Toronto, Toronto M5S 2E4, Canada. E-mail: rbh@isid.ac.in; choi@math.toronto.edu. To Kalyan Sinha on his sixtieth birthday. Abstract. We study various conditions on matrices B and C under which they can be the off-diagonal blocks of a partitioned normal matrix. Keywords.

  19. Normalized information distance

    NARCIS (Netherlands)

    Vitányi, P.M.B.; Balbach, F.J.; Cilibrasi, R.L.; Li, M.; Emmert-Streib, F.; Dehmer, M.

    2009-01-01

    The normalized information distance is a universal distance measure for objects of all kinds. It is based on Kolmogorov complexity and thus uncomputable, but there are ways to utilize it. First, compression algorithms can be used to approximate the Kolmogorov complexity if the objects have a string

  20. Plasminogen-dependent and -independent proteolytic activity of murine endothelioma cells with targeted inactivation of fibrinolytic genes.

    Science.gov (United States)

    Lijnen, H R; Wagner, E F; Collen, D

    1997-02-01

    Plasminogen-dependent and -independent proteolytic activity of marine endothelioma (End) cells that were derived from mice with targeted inactivation of the tissue-type plasminogen activator (t-PA-/-), urokinase-type plasminogen activator (u-PA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) genes was studied with the use of fibrin and extracellular matrix degradation assays. In a buffer milieu, the activation rate of plasminogen (final concentration 0.25 microM) with wild-type and t-PA-/- End cells (3 x 10(4) to 4 x 10(6) cells/ml) was comparable, but it was about 4-fold reduced with u-PA-/- End cells and 3-fold enhanced with PAI-1-/- End cells. Plasminogen activation was markedly reduced by addition of amiloride or of anti-murine u-PA antibodies but not by addition of anti-murine t-PA antibodies, and it was not stimulated by addition of fibrin. Lysis of 125I-fibrin labeled matrix in the presence of plasminogen was comparable with wild-type, t-PA-/- and PAI-1-/- End cells (50% lysis in 3 h with 0.7 to 1.5 x 10(6) cells/ml), but was significantly reduced with u-PA-/- End cells (50% lysis in 20 h with 0.87 x 10(6) cells/ml). Lysis of 3H-proline labeled extracellular matrix in the presence of plasminogen with wild-type, t-PA-/- and PAI-1-/- End cells (20% lysis in 48 h with 3 to 5 x 10(6) cells/ml) was comparable, but it was virtually abolished with u-PA-/- End cells. In the absence of plasminogen, lysis of both the fibrin and the extracellular matrix by all four cell types was drastically reduced and was virtually abolished by addition of phenylmethylsulfonylfluoride or 1,10 phenanthroline. These data indicate that the proteolytic activity of the transformed murine endothelioma cells, measured in plasminogen activation or matrix degradation assays, is essentially u-PA-related and largely plasminogen-dependent.

  1. Meta-analysis of D-values of proteolytic Clostridium botulinum and its surrogate strain Clostridium sporogenes PA 3679.

    Science.gov (United States)

    Diao, Mamadou Moctar; André, Stéphane; Membré, Jeanne-Marie

    2014-03-17

    Foodborne botulism is a serious disease resulting from ingestion of preformed Clostridium botulinum neurotoxin in foodstuff. Since the 19th century, the heat resistance of this spore forming bacteria has been extensively studied in order to guarantee the public health associated with low acidic, ambient stable products. The most largely used heat resistance parameters in thermal settings of such products are the D121.1°C values (time required to have a 10-fold decrease of the spore count, at 121.1°C) and the z-values (temperature increase to have a 10-fold decrease of D-values). To determine D121.1°C and z-values of proteolytic C. botulinum and its nontoxigenic surrogate strain C. sporogenes PA3679, a dataset of 911 D-values was collected from 38 scientific studies. Within a meta-analysis framework, a mixed-effect linear model was developed with the log D-value (min) as response and the heat treatment temperature as explicative variable. The studies (38), the C. botulinum strains (11), and the heat treatment media (liquid media and various food matrices, split into nine categories in total) were considered as co-variables having a random effect. The species (C. botulinum and C. sporogenes) and the pH (five categories) were considered as co-variables having a fixed effect. Overall, the model gave satisfactory results with a residual standard deviation of 0.22. The heat resistance of proteolytic C. botulinum was found significantly lower than the C. sporogenes PA 3679 one: the mean D-values at the reference temperature of 121.1°C, in liquid media and pH neutral, were estimated to 0.19 and 1.28min for C. botulinum and C. sporogenes, respectively. On the other hand, the mean z-values of the two species were similar: 11.3 and 11.1°C for C. botulinum and C. sporogenes, respectively. These results will be applied to thermal settings of low-acid ambient stable products. Copyright © 2014. Published by Elsevier B.V.

  2. Timely Activation of Budding Yeast APCCdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

    Science.gov (United States)

    Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.

    2014-01-01

    Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles

  3. Proteolytic activity of selected moulds in the first fermentation of black-seeded soysauce

    Science.gov (United States)

    Yulifianti, R.; Ginting, E.

    2018-01-01

    Black-seeded soybean is preferred as normally it has higher protein content and would give black colour to the soyfiltrate for making soysauce. Mould is usually used in the first fermentation of soysauce making to prepare koji with high soluble protein as a media for the subsequent bacteria fermentation in brine solution. Black-seeded soybean of Detam 1 variety was used as the soysauce ingredient. The trial was a randomized complete design with four replicates. The treatments were (1) Rhizopus oligosporus starter (in flour form) with two day-fermentation, (2) similar R. oligosporus starter with three-day fermentation, (3) Aspergillus sojae (pure culture) with three-day fermentation and (4) A. sojae in flour form with three-day fermentation. The black-seeded soybean had 100-grain weight of 11.7 g, high protein content of 42.5% dw and fat content of 14.9% dw. Koji prepared using R. oligosporus starter with two-day fermentation had the lowest protein content (48.9% dw). Both starters of A. sojae culture and flour gave the highest soluble protein content of koji (41.0-41.5% dw), followed by R. oligosporus starter with 3 day-fermentation (35.2% dw). Whilst the lowest value was noted in koji prepared using R. oligosporus starter with two day-fermentation (30.8% dw). This suggests that both A. sojae starters had similar proteolitic activity and higher than that of R. oligosporus starter. In terms of practical application and maintenance of the mould viability by soysauce processors, the use of A. sojae flour starter with three-day fermentation is suggested.

  4. Comparação do processo de reparo ósseo em tíbias de ratas normais e osteopênicas Bone repair process in normal and osteopenic female rats' tibiae: a comparative study

    Directory of Open Access Journals (Sweden)

    Angélica Rossi Sartori

    2008-01-01

    Full Text Available O objetivo foi comparar a consolidação óssea em tíbias de ratas normais e osteopênicas. 49 ratas albinas fêmeas, linhagem Wistar, peso médio de 160 (± 20g e 100 dias foram distribuídas em 2 grupos: Ooforectomizado (OOF e Pseudo-ooforectomizado (Grupo controle - SHAM. 30 dias após a ooforectomia e/ou cirurgia simulada, todas foram submetidas à produção de lesão óssea cortical. Foram sacrificadas na 2ª, 4ª, 6ª e 8ª semanas. Os osteoblastos foram contados. O peso aumentou progressivamente, porém as OOF apresentaram maior peso (pThe purpose was to compare tibial bone union in normal and osteopenic female rats. Forty-nine Wistar albino female rats weighing 160 g (±20g and 100 days were distributed into 2 groups: Oophorectomized (OOF and Pseudo-oophorectomized (SHAM. Thirty days later, a cortical injury was produced in all the animals. They were sacrificed in the 2nd, 4th, 6th and 8th weeks. Osteoblasts count was performed. Progressive weight increase was observed, but the OOF group was shown to have gained more weight (p£0.05 than the SHAM group, at the time of the second surgery. After 15 days post-injury, the animals in the OOF group presented a higher number of osteoblasts (p£0.05 compared to the SHAM group. Thirty days after injury, the number of osteoblasts was reduced, but both groups showed similar amounts. Forty-five days after injury, despite a constant reduction, the number of osteoblasts in the OOF group remained high when compared to SHAM (p£0.05 group. After 60 days, we found less osteoblasts in the SHAM group, suggesting an advanced bone repair process. The osteopenic animals showed an early accelerated response, which became equivalent between both groups 30 days after injury. However, after that period, they showed a delayed osteoid mineralization, suggesting delayed late bone repair process.

  5. The Early Diagnosis of Hypothyroidism as a Factor in the Progression of Osteoarthritis and Comorbid Processes, the Features of Phytocorrection

    Directory of Open Access Journals (Sweden)

    L.O. Voloshynа

    2016-10-01

    Full Text Available Objective: to study the prevalence and forms of hypothyroidism in patients with osteoarthritis (OA and to investigate the effect of phytomedication zobofit on the functional and morphological status of the thyroid gland, the manifestations of comorbid diseases, oxidative status, fibrinolytic and proteolytic activity of the blood. Materials and methods. In 312 patients with OA, we have studied the manifestations of comorbid processes, and among them — hypothyroidism phenomena. We have used clinical, biochemical, radioimmunological, ultrasound methods. Results. All patients with OA after 50 years had the increasing number and severity of comorbid processes. Among them, 4.48 % of patients has symptomatic and 13.78 % — subclinical hypothyroidism. Symptoms of hypothyroidism were marked in people over 55, especially — over 60 years. Subclinical hypothyroidism slightly worsened clinical symptoms of OA and comorbid processes, but enhanced the effects of oxidative stress, reduction of fibrinolytic and increase of proteolytic activity of the blood, impaired glomerular filtration rate of the kidneys. The above is considered as non-specific pathophysiological basis for the progression of identified ailments. Three-month application of the herbal medication zobofit in most patients has normalized thyroid homeostasis, improved overall outcomes of the treatment for underlying and comorbid illnesses, reduced the severity of metabolic disorders. Conclusions. In OA patients aged over 50 years with a high level of comorbidity, it is necessary to carry out screening for hypothyroidism. If you noticed the signs of subclinical hypothyroidism in this population of patients, it is appropriate to use multicomponental phytomedications with diverse thyroid-stimulating action.

  6. Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

    Science.gov (United States)

    Senger, Moritz; Stripp, Sven T; Soboh, Basem

    2017-07-14

    Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Normal Weight Dyslipidemia

    DEFF Research Database (Denmark)

    Ipsen, David Hojland; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-01-01

    Objective: The liver coordinates lipid metabolism and may play a vital role in the development of dyslipidemia, even in the absence of obesity. Normal weight dyslipidemia (NWD) and patients with nonalcoholic fatty liver disease (NAFLD) who do not have obesity constitute a unique subset...... of individuals characterized by dyslipidemia and metabolic deterioration. This review examined the available literature on the role of the liver in dyslipidemia and the metabolic characteristics of patients with NAFLD who do not have obesity. Methods: PubMed was searched using the following keywords: nonobese......, dyslipidemia, NAFLD, NWD, liver, and metabolically obese/unhealthy normal weight. Additionally, article bibliographies were screened, and relevant citations were retrieved. Studies were excluded if they had not measured relevant biomarkers of dyslipidemia. Results: NWD and NAFLD without obesity share a similar...

  8. Idiopathic Normal Pressure Hydrocephalus

    Directory of Open Access Journals (Sweden)

    Basant R. Nassar BS

    2016-04-01

    Full Text Available Idiopathic normal pressure hydrocephalus (iNPH is a potentially reversible neurodegenerative disease commonly characterized by a triad of dementia, gait, and urinary disturbance. Advancements in diagnosis and treatment have aided in properly identifying and improving symptoms in patients. However, a large proportion of iNPH patients remain either undiagnosed or misdiagnosed. Using PubMed search engine of keywords “normal pressure hydrocephalus,” “diagnosis,” “shunt treatment,” “biomarkers,” “gait disturbances,” “cognitive function,” “neuropsychology,” “imaging,” and “pathogenesis,” articles were obtained for this review. The majority of the articles were retrieved from the past 10 years. The purpose of this review article is to aid general practitioners in further understanding current findings on the pathogenesis, diagnosis, and treatment of iNPH.

  9. Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

    DEFF Research Database (Denmark)

    Kooij, Pepijn Wilhelmus; Rogowska-Wrzesinska, Adelina; Hoffmann, Daniel

    2014-01-01

    The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen...... limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal...

  10. Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models

    DEFF Research Database (Denmark)

    Almholt, Kasper; Hebsgaard, Josephine B; Nansen, Anneline

    2018-01-01

    Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test...... the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes...... mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target...

  11. Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

    Science.gov (United States)

    Capocchi, Antonella; Muccilli, Vera; Casani, Simone; Foti, Salvatore; Galleschi, Luciano; Fontanini, Debora

    2011-04-01

    In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

  12. A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway.

    Science.gov (United States)

    Lehmann, Lorenz H; Jebessa, Zegeye H; Kreusser, Michael M; Horsch, Axel; He, Tao; Kronlage, Mariya; Dewenter, Matthias; Sramek, Viviana; Oehl, Ulrike; Krebs-Haupenthal, Jutta; von der Lieth, Albert H; Schmidt, Andrea; Sun, Qiang; Ritterhoff, Julia; Finke, Daniel; Völkers, Mirko; Jungmann, Andreas; Sauer, Sven W; Thiel, Christian; Nickel, Alexander; Kohlhaas, Michael; Schäfer, Michaela; Sticht, Carsten; Maack, Christoph; Gretz, Norbert; Wagner, Michael; El-Armouche, Ali; Maier, Lars S; Londoño, Juan E Camacho; Meder, Benjamin; Freichel, Marc; Gröne, Hermann-Josef; Most, Patrick; Müller, Oliver J; Herzig, Stephan; Furlong, Eileen E M; Katus, Hugo A; Backs, Johannes

    2018-01-01

    The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

  13. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    Science.gov (United States)

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Neuroethics beyond Normal.

    Science.gov (United States)

    Shook, John R; Giordano, James

    2016-01-01

    An integrated and principled neuroethics offers ethical guidelines able to transcend conventional and medical reliance on normality standards. Elsewhere we have proposed four principles for wise guidance on human transformations. Principles like these are already urgently needed, as bio- and cyberenhancements are rapidly emerging. Context matters. Neither "treatments" nor "enhancements" are objectively identifiable apart from performance expectations, social contexts, and civic orders. Lessons learned from disability studies about enablement and inclusion suggest a fresh way to categorize modifications to the body and its performance. The term "enhancement" should be broken apart to permit recognition of enablements and augmentations, and kinds of radical augmentation for specialized performance. Augmentations affecting the self, self-worth, and self-identity of persons require heightened ethical scrutiny. Reversibility becomes the core problem, not the easy answer, as augmented persons may not cooperate with either decommissioning or displacement into unaccommodating societies. We conclude by indicating how our four principles of self-creativity, nonobsolescence, empowerment, and citizenship establish a neuroethics beyond normal that is better prepared for a future in which humans and their societies are going so far beyond normal.

  15. Ethics and "normal birth".

    Science.gov (United States)

    Lyerly, Anne Drapkin

    2012-12-01

    The concept of "normal birth" has been promoted as ideal by several international organizations, although debate about its meaning is ongoing. In this article, I examine the concept of normalcy to explore its ethical implications and raise a trio of concerns. First, in its emphasis on nonuse of technology as a goal, the concept of normalcy may marginalize women for whom medical intervention is necessary or beneficial. Second, in its emphasis on birth as a socially meaningful event, the mantra of normalcy may unintentionally avert attention to meaning in medically complicated births. Third, the emphasis on birth as a normal and healthy event may be a contributor to the long-standing tolerance for the dearth of evidence guiding the treatment of illness during pregnancy and the failure to responsibly and productively engage pregnant women in health research. Given these concerns, it is worth debating not just what "normal birth" means, but whether the term as an ideal earns its keep. © 2012, Copyright the Authors Journal compilation © 2012, Wiley Periodicals, Inc.

  16. In vitro determination of the antibiotic susceptibility of biofilm-forming Pseudomonas aeruginosa and Staphylococcus aureus: possible role of proteolytic activity and membrane lipopolysaccharide.

    Science.gov (United States)

    Masadeh, Majed M; Mhaidat, Nizar M; Alzoubi, Karem H; Hussein, Emad I; Al-Trad, Esra'a I

    2013-01-01

    We carried out a comprehensive overview of inhibitory effects of selected antibiotics on planktonic and biofilm cells of Staphylococcus aureus (ATCC 29213) and Pseudomonas aeruginosa (ATCC 27853) strains. The possible involvement of protease activity and the lipopolysaccharide (LPS) profile of P. aeruginosa were also analyzed. Biofilm cells of both strains were more resistant to antibiotics than their planktonic counterparts. Protease activity was increased in both strains in the biofilm forms. Challenge with sublethal doses of antibiotics also increased proteolytic activity of biofilm cells. Additionally, the LPS profile of P. aeruginosa showed pattern alterations of the biofilm that can contribute to biofilm resistance and survival. These observations provide evidence for the involvement of bacterial proteolytic activity and LPS profile in the resistance of biofilm bacteria to antibiotics compared to their planktonic counterparts.

  17. Acoustic Reflexes in Normal-Hearing Adults, Typically Developing Children, and Children with Suspected Auditory Processing Disorder: Thresholds, Real-Ear Corrections, and the Role of Static Compliance on Estimates.

    Science.gov (United States)

    Saxena, Udit; Allan, Chris; Allen, Prudence

    2017-06-01

    Previous studies have suggested elevated reflex thresholds in children with auditory processing disorders (APDs). However, some aspects of the child's ear such as ear canal volume and static compliance of the middle ear could possibly affect the measurements of reflex thresholds and thus impact its interpretation. Sound levels used to elicit reflexes in a child's ear may be higher than predicted by calibration in a standard 2-cc coupler, and lower static compliance could make visualization of very small changes in impedance at threshold difficult. For this purpose, it is important to evaluate threshold data with consideration of differences between children and adults. A set of studies were conducted. The first compared reflex thresholds obtained using standard clinical procedures in children with suspected APD to that of typically developing children and adults to test the replicability of previous studies. The second study examined the impact of ear canal volume on estimates of reflex thresholds by applying real-ear corrections. Lastly, the relationship between static compliance and reflex threshold estimates was explored. The research is a set of case-control studies with a repeated measures design. The first study included data from 20 normal-hearing adults, 28 typically developing children, and 66 children suspected of having an APD. The second study included 28 normal-hearing adults and 30 typically developing children. In the first study, crossed and uncrossed reflex thresholds were measured in 5-dB step size. Reflex thresholds were analyzed using repeated measures analysis of variance (RM-ANOVA). In the second study, uncrossed reflex thresholds, real-ear correction, ear canal volume, and static compliance were measured. Reflex thresholds were measured using a 1-dB step size. The effect of real-ear correction and static compliance on reflex threshold was examined using RM-ANOVA and Pearson correlation coefficient, respectively. Study 1 replicated previous

  18. [Role of the ribosomes in controlling cellular differentiation and secondary metabolism in sporulating bacteria. I. Sporogenesis, antibiotic formation and the proteolytic activity of streptomycin-resistant mutants].

    Science.gov (United States)

    Lukin, A A; Korolev, V I

    1981-01-01

    A ribosomal mutant Bacillus subtilis IG1 resistant to 100 mkg/ml of streptomycin was isolated. The strA mutation is cotransduced with the cysA gene and, consequently, maps in the ribosomal cluster. The mutation does not influence cell division but does reduce a level of sporulation as well as its antibiotic and proteolytic activity. Involvement of ribosomes in the control of sporulation and secondary metabolism of spore forming bacteria is discussed.

  19. Investigation of proteolytic enzymes expression in brain tissue and cultivated retinal pigment epithelial cells at transgenic animal model of Hintington´s disease

    Czech Academy of Sciences Publication Activity Database

    Ardan, Taras; Kocurová, Gabriela; Motlík, Jan

    2015-01-01

    Roč. 78, Suppl 2 (2015), s. 12-12 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : Huntington´s disease * transgenic porcine model * proteolytic enzymes Subject RIV: EB - Genetics ; Molecular Biology

  20. In vitro determination of the antibiotic susceptibility of biofilm-forming Pseudomonas aeruginosa and Staphylococcus aureus: possible role of proteolytic activity and membrane lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Masadeh MM

    2013-03-01

    Full Text Available Majed M Masadeh,1 Nizar M Mhaidat,2 Karem H Alzoubi,2 Emad I Hussein,3 Esra’a I Al-Trad41Department of Pharmaceutical Technology, 2Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan; 3Department of Biological Sciences, Yarmouk University, Irbid, Jordan; 4Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, JordanAbstract: We carried out a comprehensive overview of inhibitory effects of selected antibiotics on planktonic and biofilm cells of Staphylococcus aureus (ATCC 29213 and Pseudomonas aeruginosa (ATCC 27853 strains. The possible involvement of protease activity and the lipopolysaccharide (LPS profile of P. aeruginosa were also analyzed. Biofilm cells of both strains were more resistant to antibiotics than their planktonic counterparts. Protease activity was increased in both strains in the biofilm forms. Challenge with sublethal doses of antibiotics also increased proteolytic activity of biofilm cells. Additionally, the LPS profile of P. aeruginosa showed pattern alterations of the biofilm that can contribute to biofilm resistance and survival. These observations provide evidence for the involvement of bacterial proteolytic activity and LPS profile in the resistance of biofilm bacteria to antibiotics compared to their planktonic counterparts.Keywords: biofilm, Pseudomonas aeruginosa, Staphylococcus aureus, proteolytic activity, lipopolysaccharide

  1. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    Science.gov (United States)

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  2. PROTEOLYTIC DEGRADATION OF POLY (ADP-RIBOSE POLYMERASE IN RATS WITH CARRAGEENAN-INDUCED GASTROENTEROCOLITIS

    Directory of Open Access Journals (Sweden)

    Tkachenko A. S.

    2017-12-01

    Full Text Available The aim of the research was to study the activity of poly (ADP-ribose polymerase in small intestinal homogenate of rats with chronic carrageenan-induced gastroenterocolitis, as well as mechanisms of regulation of the enzyme in this pathology. Twenty Wistar Albino Glaxo rats were divided into two groups. Animals of group 1 (n = 10 consumed 1 % carrageenan solution for 28 days, which resulted in the development of gastroenterocolitis confirmed morphologically. The control group consisted of intact animals (n = 10. The activity of poly (ADP-ribose polymerase (PARP in the homogenate of small intestine, as well as caspase-3, matrix metalloproteinase-2 (MMP-2 and matrix metalloproteinase-9 (MMP-9 serum levels were determined. Obtained data were statistically processed using the Mann-Whitney U test and calculating median and interquartile range (Me, 25th–75th percentile with the help of the GraphPad Prism 5 application. The development of carrageenan-induced gastroenterocolitis was accompanied by an increase in caspase-3, MMP-2, MMP-9 concentrations in blood serum and a decrease in the activity of PARP in small intestinal homogenates. The reduced activity of PARP in chronic carrageenan-induced gastroenterocolitis may be due to the proteolysis of this enzyme under the action of caspase-3, MMP-2, and MMP-9.

  3. Tag team: Roles of miRNAs and Proteolytic Regulators in Ensuring Robust Gene Expression Dynamics.

    Science.gov (United States)

    Weaver, Benjamin P; Han, Min

    2018-01-01

    Lack of prominent developmental defects arising from loss of many individual miRNAs is consistent with the observations of collaborative networks between miRNAs and roles for miRNAs in regulating stress responses. However, these characteristics may only partially explain the seemingly nonessential nature of many miRNAs. Non-miRNA gene expression regulatory mechanisms also collaborate with miRNA-induced silencing complex (miRISC) to support robust gene expression dynamics. Genetic enhancer screens have revealed roles of miRNAs and other gene repressive mechanisms in development or other cellular processes that were masked by genetic redundancy. Besides discussing the breadth of the non-miRNA genes, we use LIN-28 as an example to illustrate how distinct regulatory systems, including miRNAs and multiple protein stability mechanisms, work at different levels to target expression of a given gene and provide tissue-specific and stage-specific regulation of gene expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

    Directory of Open Access Journals (Sweden)

    Karina Mastropasqua Rebello

    2012-09-01

    Full Text Available Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1 were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3 were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

  5. Isolation of proteolytic bacteria from mealworm (Tenebrio molitor) exoskeletons to produce chitinous material.

    Science.gov (United States)

    da Silva, Fernanda Kerche Paes; Brück, Dieter W; Brück, Wolfram M

    2017-09-15

    The use of insects as a source of protein is becoming an important factor for feeding an increasing population. After protein extraction for food use, the insect exoskeleton may offer the possibility for the production of added value products. Here, the aim was to isolate bacteria from the surface of farmed mealworms (Tenebrio molitor Linnaeus, 1758) for the production of chitinous material from insect exoskeletons using microbial fermentation. Isolates were screened for proteases and acid production that may aid deproteination and demineralisation of insects through fermentation to produce chitin. Selected isolates were used single-step (isolated bacteria only) or two-step fermentations with Lactobacillus plantarum (DSM 20174). Two-step fermentations with isolates from mealworm exoskeletons resulted in a demineralisation of 97.9 and 98.5% from deproteinated mealworm fractions. Attenuated total reflectance-Fourier- transform infrared spectroscopy analysis showed that crude chitin was produced. However, further optimisation is needed before the process can be upscaled. This is, to our knowledge, the first report using microbial fermentation for the extraction of chitin from insects. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Doxycycline indirectly inhibits proteolytic activation of tryptic kallikrein-related peptidases and activation of cathelicidin.

    Science.gov (United States)

    Kanada, Kimberly N; Nakatsuji, Teruaki; Gallo, Richard L

    2012-05-01

    The increased abundance and activity of cathelicidin and kallikrein 5 (KLK5), a predominant trypsin-like serine protease (TLSP) in the stratum corneum, have been implicated in the pathogenesis of rosacea, a disorder treated by the use of low-dose doxycycline. Here we hypothesized that doxycycline can inhibit activation of tryptic KLKs through an indirect mechanism by inhibition of matrix metalloproteinases (MMPs) in keratinocytes. The capacity of doxycycline to directly inhibit enzyme activity was measured in surface collections of human facial skin and extracts of cultured keratinocytes by fluorescence polarization assay against fluorogenic substrates specific for MMPs or TLSPs. Doxycycline did inhibit MMP activity but did not directly inhibit serine protease activity against a fluorogenic substrate specific for TLSPs. However, when doxycycline or other MMP inhibitors were added to live keratinocytes during the production of tryptic KLKs, this treatment indirectly resulted in decreased TLSP activity. Furthermore, doxycycline under these conditions inhibited the generation of the cathelicidin peptide LL-37 from its precursor protein hCAP18, a process dependent on KLK activity. These results demonstrate that doxycycline can prevent cathelicidin activation, and suggest a previously unknown mechanism of action for doxycycline through inhibiting generation of active cathelicidin peptides.

  7. Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

    Directory of Open Access Journals (Sweden)

    Ohad Iosefson

    2015-08-01

    Full Text Available In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.

  8. Normal radiological findings

    International Nuclear Information System (INIS)

    Moeller, T.B.

    1987-01-01

    This book is intended for learners in radiology, presenting a wealth of normal radiological findings together with a systematic guide for appraisal and interpretation, and for formulation of reports. The text examples and criteria given will help beginners in learning to 'read' a radiograph, and to verify their conclusions by means of checklists and standard reports. The case material covers numerous illustrations from the following sectors: Skeletal radiography, mammography, tomography, contrast radiography, organ examination by intravenous techniques, arthrography and angiography, and specialized radiography, (ECB) With 184 figs [de

  9. Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

    Directory of Open Access Journals (Sweden)

    Yumiko Obayashi

    2017-10-01

    conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement.

  10. Normal and Abnormal Behavior in Early Childhood

    OpenAIRE

    Spinner, Miriam R.

    1981-01-01

    Evaluation of normal and abnormal behavior in the period to three years of age involves many variables. Parental attitudes, determined by many factors such as previous childrearing experience, the bonding process, parental psychological status and parental temperament, often influence the labeling of behavior as normal or abnormal. This article describes the forms of crying, sleep and wakefulness, and affective responses from infancy to three years of age.

  11. Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Margaret V Powers-Fletcher

    Full Text Available Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.

  12. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Holger B Kramer

    2010-05-01

    Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

  13. Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics.

    Science.gov (United States)

    Shangguan, Lulu; Zhang, Lingyi; Xiong, Zhichao; Ren, Jun; Zhang, Runsheng; Gao, Fangyuan; Zhang, Weibing

    2015-04-03

    The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Normal Untreated Jurkat Cells

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

  15. Normal shoulder: MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kieft, G.J.; Bloem, J.L.; Obermann, W.R.; Verbout, A.J.; Rozing, P.M.; Doornbos, J.

    1986-06-01

    Relatively poor spatial resolution has been obtained in magnetic resonance (MR) imaging of the shoulder because the shoulder can only be placed in the periphery of the magnetic field. The authors have devised an anatomically shaped surface coil that enables MR to demonstrate normal shoulder anatomy in different planes with high spatial resolution. In the axial plane anatomy analogous to that seen on computed tomographic (CT) scans can be demonstrated. Variations in scapular position (produced by patient positioning) may make reproducibility of sagittal and coronal plane images difficult by changing the relationship of the plane to the shoulder anatomy. Oblique planes, for which the angle is chosen from the axial image, have the advantage of easy reproducibility. Obliquely oriented structures and relationships are best seen in oblique plane images and can be evaluated in detail.

  16. Chapter 1: Direct Normal Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Myer, Daryl R.

    2016-04-15

    This chapter addresses the quantitative and qualitative aspects of the solar resource, the direct solar radiation. It discusses the total or integrated broadband direct beam extraterrestrial radiation (ETR). This total integrated irradiance is comprised of photons of electromagnetic radiation. The chapter also discusses the impact of the atmosphere and its effect upon the direct normal irradiance (DNI) beam radiation. The gases and particulates present in the atmosphere traversed by the direct beam reflect, absorb, and scatter differing spectral regions and proportions of the direct beam, and act as a variable filter. Knowledge of the available broadband DNI beam radiation resource data is essential in designing a concentrating photovoltaic (CPV) system. Spectral variations in the DNI beam radiation affect the performance of a CPV system depending on the solar cell technology used. The chapter describes propagation and scattering processes of circumsolar radiation (CSR), which includes the Mie scattering from large particles.

  17. Institutionalizing Normal: Rethinking Composition's Precedence in Normal Schools

    Science.gov (United States)

    Skinnell, Ryan

    2013-01-01

    Composition historians have recently worked to recover histories of composition in normal schools. This essay argues, however, that historians have inadvertently misconstrued the role of normal schools in American education by inaccurately comparing rhetorical education in normal schools to rhetorical education in colleges and universities.…

  18. Regulatory processes in Aspergillus niger

    DEFF Research Database (Denmark)

    Poulsen, Lars

    some disadvantages as well, those are byproduct formation, secretion of proteolytic enzymes and formation of mycotoxins. The aim of this project was to reduce these disadvantages, though investigating the regulatory processes. The first objective was to study the regulatory events leading to A. niger...... characteristics, as a lower oxalic ii acid formation and wild type growth performance; it was therefore argued that this strain could be an attractive alternative to ΔprtT. Finally, in order to characterize the formation of the carcinogenic mycotoxin fumonisin, a reporter strain of A. niger was constructed, where...

  19. A zoo of computable binary normal sequences.

    Science.gov (United States)

    Pincus, Steve; Singer, Burton H

    2012-11-20

    Historically there has been a virtual absence of constructive methods to produce broad classes of "certifiably random" infinite sequences, despite considerable interest in this endeavor. Previously, we proved a theorem that yielded explicit algorithms to produce diverse sets of normal numbers, reasonable candidates for random sequences, given their limiting equidistribution of subblocks of all lengths. Herein, we develop this algorithmic approach much further, systematizing the normal number generation process in several ways. We construct delineated, distinct sets of normal numbers (classified by the extent to which initial segments deviate from maximal irregularity), with virtually any allowable specified rate of convergence to 0 of this deviation, encompassing arbitrarily fast and slow rates, and accommodating asymmetric behavior above or below a centered median. As a corollary, we provide an explicit construction of a normal number that satisfies the Law of the Iterated Logarithm. We also produce distinct families of "biased" normal numbers, with virtually any specified rate of convergence of the bias (to 0). This latter theory is in part motivated by the remarkable observation that the binary version of Champernowne's number, which is also normal, is biased-any initial segment has more 1s than 0s. Finally, we construct an interesting normal sequence with arbitrarily fast convergence to equidistribution of singleton blocks, yet arbitrarily slow convergence of pairs, which has profound implications both for probability theory, and for metrics to evaluate the "near-randomness" of sequences.

  20. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells

    International Nuclear Information System (INIS)

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-01-01

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O 6 -methylguanine–DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross-linking repair in a p