ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2008
A. Rajendran
Abstract Bacillus sphaericus,MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010,U/L was obtained during a fermentation period of 24,h under optimized conditions of 30,°C in a medium with an initial pH of 7 and at a shaking rate of 120,rpm. The maximum biomass obtained in the batch fermentation was 2.55,g/L after 16,h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking-Piret model for the production of protease and the Monod-incorporated modified Luedeking-Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R2) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively. [source]

Protease,proteoglycan complexes of mouse and human mast cells and importance of their ,-tryptase,heparin complexes in inflammation and innate immunity

IMMUNOLOGICAL REVIEWS, Issue 1 2007
Richard L. Stevens
Summary:, Approximately 50% of the weight of a mature mast cell (MC) consists of varied neutral proteases stored in the cell's secretory granules ionically bound to serglycin proteoglycans that contain heparin and/or chondroitin sulfate E/diB chains. Mouse MCs express the exopeptidase carboxypeptidase A3 and at least 15 serine proteases [designated as mouse MC protease (mMCP) 1,11, transmembrane tryptase/tryptase ,/protease serine member S (Prss) 31, cathepsin G, granzyme B, and neuropsin/Prss19]. mMCP-6, mMCP-7, mMCP-11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MCs. One of the challenges ahead is to understand why MCs express so many different protease,proteoglycan macromolecular complexes. MC-like cells that contain tryptase,heparin complexes in their secretory granules have been identified in the Ciona intestinalis and Styela plicata urochordates that appeared approximately 500 million years ago. Because sea squirts lack B cells and T cells, it is likely that MCs and their tryptase,proteoglycan granule mediators initially appeared in lower organisms as part of their innate immune system. The conservation of MCs throughout evolution suggests that some of these protease,proteoglycan complexes are essential to our survival. In support of this conclusion, no human has been identified that lacks MCs. Moreover, transgenic mice lacking the ,-tryptase mMCP-6 are unable to combat a Klebsiella pneumoniae infection effectively. Here we summarize the nature and function of some of the tryptase,serglycin proteoglycan complexes found in mouse and human MCs. [source]

Putative role of proteolysis and in,ammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures,

JOURNAL OF APPLIED TOXICOLOGY, Issue 3 2003
F. M. Cowan
Abstract Despite the contrasts in chemistry and toxicity, for blister and nerve chemical warfare agents there may be some analogous proteolytic and in,ammatory mediators and pathological pathways that can be pharmacological targets for a single-drug multi-threat medical countermeasure. The dermal,epidermal separation caused by proteases and bullous diseases compared with that observed following exposure to the blister agent sulfur mustard (2,2,-dichlorodiethyl sul,de) has fostered the hypothesis that sulfur mustard vesication involves proteolysis and in,ammation. In conjunction with the paramount toxicological event of cholinergic crisis that causes acute toxicity and precipitates neuronal degeneration, both anaphylactoid reactions and pathological proteolytic activity have been reported in nerve-agent-intoxicated animals. Two classes of drugs already have demonstrated multi-threat activity for both nerve and blister agents. Serine protease inhibitors can prolong the survival of animals intoxicated with the nerve agent soman and can also protect against vesication caused by the blister agent sulfur mustard. Poly (ADP-ribose) polymerase (PARP) inhibitors can reduce both soman-induced neuronal degeneration and sulfur-mustard-induced epidermal necrosis. Protease and PARP inhibitors, like many of the other countermeasures for blister and nerve agents, have potent primary or secondary anti-in,ammatory pharmacology. Accordingly, we hypothesize that drugs with anti-in,ammatory actions against either nerve or blister agent might also display multi-threat ef,cacy for the in,ammatory pathogenesis of both classes of chemical warfare agent. Published in 2003 by John Wiley & Sons, Ltd. [source]

Activation of ERK signaling upon alternative protease nexin-1 internalization mediated by syndecan-1

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2006
Xiaobiao Li
Abstract Protease nexin-1 (PN-1), an inhibitor of serine proteases, contributes to tissue homeostasis and influences the behavior of some tumor cells. The internalization of PN-1 protease complexes is considered to be mediated by the low-density lipoprotein receptor related protein 1 (LRP1). In this study, both wild-type and LRP1,/, mouse embryonic fibroblasts (MEF) were shown to internalize PN-1. Receptor associated protein (RAP) interfered with PN-1 uptake only in wild-type MEF cells, indicating that another receptor mediates PN-1 uptake in the absence of LRP1. In LRP1,/, MEF cells, inhibitor sensitivity and kinetic values (t1/2 at 45 min) of PN-1 uptake showed a similarity to syndecan-1-mediated endocytosis. In these cells, PN-1 uptake was increased by overexpression of full-length syndecan-1 and decreased by RNA interference targeting this proteoglycan. Most important, in contrast to PKA activation known to be triggered by LRP1-mediated internalization, our study shows that syndecan-1-mediated internalization of PN-1 stimulated the Ras-ERK signaling pathway. J. Cell. Biochem. 99: 936,951, 2006. © 2006 Wiley-Liss, Inc. [source]

Stabilization and Partial Purification of a Protease from Ginger Rhizome (Zingiber offinale Roscoe)

JOURNAL OF FOOD SCIENCE, Issue 3 2005
Pitaya Adulyatham
ABSTRACT: Ginger protease (GP) or zingibain is of interest as a meat tenderizing agent. The objective of this research was to investigate food-compatible methods for stabilizing GP during storage or enzyme fractionation. Crude GP extracted from fresh ginger had a half-life (t1/2) of 2.1 (±0.16) d at 5°C decreasing to 20 min at 30°C. Addition of ascorbate (0.2% w/v) increased the t1/2 for GP from 2 to 20 d at 5°C. Dithiothreitol or Ethylenediaminetetraacetic acid (EDTA) had no effect on GP stability. Acetone powder preparations from ginger yielded GP with t1/2 of 18 mo at 5°C. Crude GP extracted from acetone powder was sufficiently stabilized to allow fractionation by ion exchange chromatography without the addition of toxic or expensive additives. GP was partially purified 252-fold with a recovery of 61%. The nomimal molecular weight of GP was 34.8 kDa compared with 25.1 kDa for papain. This work shows that the stability of GP can be greatly improved, increasing its attractiveness as a commercial product. Some possible routes of GP deactivation and stabilization are discussed. [source]

Location of Caspase 3-like Protease in the Development of Sieve Element and Tracheary Element of Stem in Cucurbita moschata

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 12 2008
Xia Hao
Abstract The casepase is considered to regulate the process of programmed cell death in the development of organisms. In this study, caspase 3-like protease was detected by immunohistochemistry and immunoelectron microscopy during the development of sieve element and tracheary element of stem in Cucurbita moschata Duch. Antibody with brown color (under light microscopy) and gold particles (under transmission electron microscopy) for detecting caspase 3-like protease was mainly displayed in inner phloem, external phloem and xylem in the region close to procambium. From the results it was considered that caspase 3-like protease did exist in vascular elements and played different roles during the development of sieve and tracheary elements, and different types of programmed cell death might be carried out. The caspase 3-like protease mainly participated in making cytoplasmic streaming cease and in degrading P-protein bodies; however, it rarely participated in the function for signal transferring in the developmental sieve element. However, it might induce calcium accumulation for rupturing the tonoplast in the signal of PCD in the developmental tracheary element. [source]

Detection of drug resistance mutations as a predictor of subsequent virological failure in patients with HIV-1 viral rebounds of less than 1,000 RNA copies/ml

JOURNAL OF MEDICAL VIROLOGY, Issue 9 2007
Chris Verhofstede
Abstract In order to evaluate the usefulness of resistance testing after a viral rebound with plasma HIV RNA levels of less than 1,000 copies (c)/ml, genotyping was performed on 39 samples from patients on highly active antiretroviral therapy (HAART) showing a viremia of over 50 c/ml up to a maximum of 1,000 c/ml after at least one undetectable viral load result. Protease and reverse transcriptase (RT) sequences were obtained for all 39 samples. In 10 (25.6%) of the samples, mutations not seen before the initiation of the regimen were observed. The M184V/I mutation was the most prevalent but in several patients a combination of multiple mutations was detected. Follow-up samples were available for 34 patients. In six (85.71%) out of seven patients with new mutations, the viral load on the follow-up visit remained detectable, indicating true failure, compared to 6 (31.6%) true failures out of 19 patients in whom only wild type virus was detected (P,=,0.02) and three (37.5%) out of eight patients in whom only the mutations already present at the initiation of HAART were seen (P,=,0.08). The results indicate that reliable resistance testing can be performed on samples with a viral burden of less than 1,000 c/ml and demonstrate that multiple drug resistance mutations can be selected at low viral load rebounds. Most importantly, detection of resistance mutations in viral rebound samples was predictive of subsequent virological failure. J. Med. Virol. 79:1254,1260, 2007. © Wiley-Liss, Inc. [source]

Antifungal Activity of a Bowman,Birk-type Trypsin Inhibitor from Wheat Kernel

JOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2000
G. Chilosi
A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC50) values ranging from 111.7 to above 500 ,g/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens. Zusammenfassung Ein Trypsinhemmer aus Weizenkörnern (WTI) zeigte eine starke antifungale Aktivität gegenüber verschiedenen pathogenen Pilzen und hemmte deren trypsinähnliche Aktivität. WTI hemmte in vitro die Sporenkeimung und das Hyphenwachstum der Pathogene, wobei die IC50 -Werte zwischen 111,7 und mehr als 500 ,g/ml lagen. Elektronenmikroskopische Untersuchungen zeigten, dai WTI morphologische Veränderungen bewirkte, die aus einer Hemmung des Hyphenwachstums und einer veränderten Verzweigung bestanden. Eine der untersuchten Pilzarten, Botrytis cinerea, bildete eine trypsinähnliche Protease, die durch den Trypsininhibitor gehemmt wurde. Ebenso wie andere sameneigene Abwehrproteine scheint WTI während der Keimruhe und in den frühen Stadien der Keimung, wenn die Pflanzen gegenüber möglichen bodenbürtigen Pathogenen besonders exponiert sind, ein wichtiger Resistenzfaktor in Weizenkörnern zu sein. [source]

Vascular and dendritic cell coagulation signaling in sepsis progression

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009
W. RUF
Summary., The intrinsic signaling networks of the coagulation pathways have recently emerged as crucial determinants for survival in sepsis and systemic inflammatory response syndromes. Protease activated receptor (PAR) 1 is central to both lethality promoting and vascular protective signaling. In the vascular anticoagulant pathway, EPCR/aPC-PAR1 signaling prevents vascular leakage and genetic or acute deficiencies in this pathway promote lethality. In addition, coagulation signaling acts directly on cells of the innate immune system. Dendritic cell (DC) thrombin-PAR1 signaling is coupled to the migration promoting sphingosine 1 phosphate receptor 3 (S1P3). Thrombin generated in the lymphatic compartment perturbs DCs to promote systemic inflammation and disseminated intravascular coagulation in severe sepsis. Signaling-selective aPC variants and selective modulators of the S1P receptor system attenuate sepsis lethality, suggesting novel therapeutic approaches that can be employed to rebalance alterations in the coagulation signaling pathways in severe inflammatory disorders. [source]

Role of Protease Activated Receptor 2 in Experimental Acute Lung Injury and Lung Fibrosis

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 4 2009
Xiao Su
Abstract Protease activated receptor 2 (PAR2) is widely-distributed (lung, liver, kidney, etc.) and expressed by variety of cells (i.e. leukocytes, epithelial cells, endothelial cells, and fibroblast). PAR2 may participate in many pathological processes, such as, inflammation, injury, as well as fibrosis. Therefore, in this study, we tested whether PAR2 would exert a role in acid-induced acute lung injury, E. coli pneumonia, bleomycin-induced acute lung injury and fibrosis. Acid, E. coli, or bleomycin were intratracheally instilled into the lungs of both wildtype and PAR2 knockout mice to detect differences in pulmonary edema, lung vascular permeability, lung fibrosis, and other parameters. Knockout of PAR2 did not affect the extent of pulmonary edema and lung vascular permeability in acid-induced acute lung injury. Also, both activation of PAR2 in the airspaces of the lung and deletion of PAR2 did not alter the magnitude of pulmonary edema and lung vascular permeability in E. coli pneumonia. Finally, PAR2 deficiency did not affect the severity of lung inflammation and lung fibrosis in bleomycin-induced acute lung injury and lung fibrosis models. Thus, PAR2 does not appear to play a critical role in the pathogeneses of experimental acid-induced acute lung injury, E. coli pneumonia, and bleomycin-induced acute lung injury and pulmonary fibrosis in mice. Anat Rec, 2009. © 2009 Wiley-Liss, Inc. [source]

A Protease-Based Strategy for the Controlled Release of Therapeutic Peptides,

ANGEWANDTE CHEMIE, Issue 29 2010
Hongjian Li Dr.
Ein Polypeptid bestehend aus einer Albumin-bindenden Domäne (ABD), einer Aminosäurekette und dem Glucagon-artigen Peptid-1 (GLP-1), einem potenziellen Antidiabetikum, wurde so entworfen, dass es langsam GLP-1 freisetzt, indem es durch native Plasmaproteasen schrittweise gespalten wird (siehe Bild). Durch die Anbindung an humanes Serumalbumin (HSA) wird die Halbwertszeit des Polypeptids im Blut verlängert. [source]

An Efficient Method for the Synthesis of Peptide Aldehyde Libraries Employed in the Discovery of Reversible SARS Coronavirus Main Protease (SARS-CoV Mpro) Inhibitors

CHEMBIOCHEM, Issue 7 2006
Samer I. Al-Gharabli Dr.
Abstract A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess,Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV Mpro(also known as 3CLpro), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV Mpro. Several potent inhibitors were found with IC50 values in the low micromolar range. An IC50 of 7.5 ,M was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV Mpro in a noncanonical binding mode. [source]

Design and Synthesis of Spiro-cyclopentenyl and Spiro-[1,3]-dithiolanyl Substituted Pyrrolidine-5,5-trans-lactams as Inhibitors of Hepatitis C Virus NS3/4A Protease.

CHEMINFORM, Issue 38 2003
David M. Andrews
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Pyrrolidine-5,5-trans-lactams as Novel Mechanism-Based Inhibitors of Human Cytomegalovirus Protease.

CHEMINFORM, Issue 43 2002
Part 3.
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Unexpected Novel Binding Mode of Pyrrolidine-Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV Protease

CHEMMEDCHEM, Issue 1 2006
Edgar Specker Dr.
Abstract At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp,25 and 25,. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin,D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin,D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus. [source]

A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone subunit TCP-1, in maize

CYTOSKELETON, Issue 4 2004
Zhengyuan Wang
Abstract The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1, (TCP-1,) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1, isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1, co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1, for regulating folding or providing protection against denaturation. Cell Motil. Cytoskeleton 57:218,232, 2004. © 2004 Wiley-Liss, Inc. [source]

Liposome-mediated transfection of mature taste cells

DEVELOPMENTAL NEUROBIOLOGY, Issue 1 2005
Ana Marie Landin
Abstract The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used ,-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005 [source]

Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca2+ mobilization, and apoptosis ,

DEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001
Irina V. Smirnova
Abstract Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca2+]i flux, receptor cleavage, and elevation of rest [Ca2+]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87,100, 2001 [source]

Structure and function studies of glucagon-like peptide-1 (GLP-1): the designing of a novel pharmacological agent for the treatment of diabetes

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 4 2005
Hongxiang Hui
Abstract Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived peptide secreted from gut endocrine cells in response to nutrient ingestion. The multifaceted actions of GLP-1 include the following: (1) the stimulation of insulin secretion and of its gene expression, (2) the inhibition of glucagon secretion, (3) the inhibition of food intake, (4) the proliferation and differentiation of beta cells, and (5) the protection of beta-cells from apoptosis. The therapeutic utility of the native GLP-1 molecule is limited by its rapid enzymatic degradation by a serine protease termed dipeptidyl peptidase-IV (DPP-IV). The present article reviews the research studies aimed at elucidating the biosynthesis, metabolism, and molecular characteristics of GLP-1 since it is from these studies that the development of a GLP-1-like pharmacological agent may be derived. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Expression of EMMPRIN and matriptase in esophageal squamous cell carcinoma: Correlation with clinicopathological parameters

DISEASES OF THE ESOPHAGUS, Issue 6 2006
M.-F. Cheng
SUMMARY., Extracellular matrix metalloproteinase inducer (EMMPRIN) and the type II transmembrane serine protease, matriptase, are expressed in several human cancers and play an important role in tumor progression. The aim of the present study was to investigate the immuno-staining patterns of EMMPRIN and matriptase in patients with esophageal squamous cell carcinomas (SCC) and correlate the percentage tumor staining with tumor differentiation and clinical parameters. EMMPRIN and matriptase immunoreactivity was seen on the cell membrane and in the cytoplasm of tumor cells in all 41 cases of esophageal SCC evaluated. The percentage tumor staining of EMMPRIN was 48 ± 3% for well differentiated, 73 ± 3% for moderately differentiated, and 92 ± 3% for poorly differentiated esophageal SCC. Higher percentage tumor staining with EMMPRIN correlated significantly with poorly differentiated esophageal SCC (P < 0.05). The percentage tumor staining with matriptase correlated significantly with tumor differentiation (52 ± 3% for well differentiated, 85 ± 2% for moderately differentiated, and 88 ± 3% for poorly differentiated esophageal SCC). Additionally, higher percentage tumor staining with matriptase was significantly correlated with the advanced N and M stages (P < 0.05). Our results demonstrate that EMMPRIN and matriptase are over-expressed in esophageal SCC and are correlated with advanced clinicopathological stages. Pharmacological agents targeting EMMPRIN and matriptase expressions may be beneficial in the treatment of esophageal SCC. [source]

CE-ESI-MS/MS as a rapid screening tool for the comparison of protein,ligand interactions

ELECTROPHORESIS, Issue 7 2010
Thomas Hoffmann
Abstract In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug,target interactions. CE enables the separation of proteins and drug,enzyme complexes preserving their physiological activity in aqueous media. By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug,target complex after CID. In this study, ,-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of ,-CT and a chymotrypsin,chymostatin complex under MS-compatible conditions. The use of neutral-coated CE capillaries was mandatory to reduce analyte,wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID. Fragmentation of the drug,enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V50 voltage that enables 50% complex fragmentation as a reference value for chymotrypsin,chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug,enzyme complexes during ionization,desolvation processes. With this optimized setup, different CT inhibitors could be investigated and compared. [source]

"Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction products

ELECTROPHORESIS, Issue 13 2009
Helene Piccard
Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source]

Simultaneous separation of fifteen approved protease and reverse transcriptase inhibitors for human immunodeficiency virus therapy by capillary electrophoresis

ELECTROPHORESIS, Issue 4 2003
Nguyen Duc Tuan
Abstract In the present investigation, a novel approach towards a complete separation of all 15 protease and reverse transcriptase inhibitors which are currently approved for use in highly active antiretroviral therapy in a single analytical run is presented. The developed method employs an acidic background electrolyte with sodium polyanethol sulfonate (SPAS) as polyanionic electroosmotic flow (EOF) modifier to establish a strong cathodic EOF, sodium dodecyl sulfate (SDS) as pseudostationary selector, and acetonitrile and ethanol as organic modifiers. Separation of the analytes is based on two different mechanisms. The more basic analytes are protonated at the prevailing pH conditions and thus migrate in front of the cathodic EOF, whereas the less basic and neutral analytes interact with the SDS and are retained after the EOF. By optimizing electrolyte pH, the amount of solvents and SDS concentrations in the background electrolyte it is possible to completely separate all compounds of interest. [source]

The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease

EMBO MOLECULAR MEDICINE, Issue 1 2009
Janine Kirstein
Abstract A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp,protease complex. [source]

Utilization of tannery solid waste for protease production by Synergistes sp. in solid-state fermentation and partial protease characterization

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2009
Arumugam Ganesh Kumar
Abstract Synergistes sp. DQ560074 produced a protease in submerged fermentation (SmF) at 400,420,U/mL and in solid-state fermentation (SSF) at 745,755,U/g. The protease, which belongs to the aspartic protease class, was active over a wide range of pH (5,7) and at high temperatures (25,45°C). The protease is stable and active in various polar protic solvents (50%,v/v) like ethanol, isopropanol, n,butanol, in polar aprotic solvents (50%,v/v) like acetonitrile, and in non-polar solvents (50%,v/v) such as ethylacetate and toluene, but not in hydrophilic organic solvents (methyl alcohol and acetone). As far as we know, this is the first contribution to the production of a mesophilic protease with solvent stability in SSF using a proteinaceous solid waste. [source]

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus,MTTC511

Process Development in Biotechnology , A Re-Evaluation

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2005
K. Schügerl
Abstract This review considers some process development problems in biotechnology and presents examples of solutions, which were developed in cooperation with industrial partners. These processes include the production of restriction endonuclease EcoRI by recombinant Escherichia coli, which is toxic to the cell, penicillin V by Penicillium chrysogenum, xylanase by Aspergillus awamori, cephalosporin C by Acremonium chrysogenum, erythritol by Moniliella tomentosa var pollinis, and alkaline serine protease by Bacillus licheniformis. Special attention is given to the practical aspects of product development. [source]

Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environment

ENVIRONMENTAL MICROBIOLOGY, Issue 6 2009
Lilia Macovei
Summary The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd,Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Enterococcus casseliflavus (10.4%), Enterococcus gallinarum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelatinase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelatinase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4,8°C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies. [source]

Coexistence of Unverricht-Lundborg disease and congenital deafness: Molecular resolution of a complex comorbidity

EPILEPSIA, Issue 6 2009
Miljana Kecmanovi
Summary Purpose:, We report on genetic analysis of a complex condition in a Serbian family of four siblings, wherein two had progressive myoclonic epilepsy (PME) and congenital deafness (CD), one had isolated congenital deafness (ICD), and one was healthy. Methods and Results:, Molecular diagnosis performed by Southern blotting confirmed Unverricht-Lundborg disease in the available sibling with PME/CD. In the sibling with ICD (heterozygote for expansion mutation in CSTB) we demonstrated recombination event between the D21S2040 marker and the CSTB gene and identified c.207delC (p.T70Xfs) mutation in the fourth exon of the transmembrane protease, serine-3 (TMPRSS3) gene (maps in close proximity to CSTB), responsible for nonsyndromic deafness in the sibling with PME/CD as well. Discussion:, To the best of our knowledge this is the first genetic confirmation of the coexistence of these two mutations. [source]

Molecular mechanisms activating muscle protein degradation in chronic kidney disease and other catabolic conditions

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2005
J. Du
Abstract Muscle atrophy is a prominent feature of chronic kidney disease (CKD) and is frequent in other catabolic conditions. Results from animal models of these conditions as well as patients indicate that atrophy is mainly owing to accelerated muscle proteolysis in the ubiquitin-proteasome (Ub-P'some) proteolytic system. The Ub-P'some system, however, rapidly degrades actin or myosin but cannot breakdown actomyosin or myofibrils. Consequently, another protease must initially cleave the complex structure of muscle. We identified caspase-3 as an initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils to produce substrates degraded by the Ub-P'some system. In rodent models of CKD and other catabolic conditions, we find that caspase-3 is activated and cleaves actomyosin to actin, myosin and their fragments. This initial proteolytic step in muscle leaves a characteristic footprint, a 14-kDa actin band, providing a potential diagnostic tool to detect muscle catabolism. We also found that stimulation of caspase-3 activity depends on inhibition of IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity; inhibiting PI3K in muscle cells also leads to expression of a critical E3-ubiquitin-conjugating enzyme involved in muscle protein breakdown: atrogin-1/MAFbx. Thus, protein breakdown by caspase-3 and the ubiquitin-proteasome system in muscle are stimulated by the same signal: a low PI3K activity. These responses could yield therapeutic strategies to block muscle atrophy. [source]