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Serine Proteinases (serine + proteinase)
Terms modified by Serine Proteinases Selected AbstractsIDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARPJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004MIN-JIE CAO ABSTRACT Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55,60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as ,-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase. [source] Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx moriENTOMOLOGICAL RESEARCH, Issue 3 2002Doo-Sang PARK ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis. [source] Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)FEBS JOURNAL, Issue 3 2006Sanjida Ahmed Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1. Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface. [source] Thrombin induces cyclooxygenase-2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2- and p38 MAPK-dependent pathway in murine macrophagesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009Huey-Ming Lo Abstract Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti-thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in macrophages. Thrombin-induced COX-2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)-binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX-2 expression by thrombin was functionally linked to release of PGE2 and PGI2 but not thromboxane A2 into macrophage culture medium. Thrombin-induced COX-2 expression and PGE2 production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase-activated receptor 1 (PAR1)-activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist-SCH79797 could attenuate thrombin-induced COX-2 expression and PGE2 release. Taken together, we provided evidence demonstrating that thrombin can induce COX-2 mRNA and protein expression and PGE2 production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK-dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions. J. Cell. Biochem. 108: 1143,1152, 2009. © 2009 Wiley-Liss, Inc. [source] PROTEINASES IN HYBRID CATFISH VISCERA: CHARACTERIZATION AND EFFECT OF EXTRACTION MEDIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT Proteolytic activity from viscera extract of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was investigated. Optimal pH and temperature for casein hydrolysis were 9.0 and 50C, respectively. The enzyme was stable to heat treatment up to 40C and over a pH range of 7,11 for 30,120 min. The proteolytic activity was effectively inhibited by soybean trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride and N -p-tosyl-L-lysine chloromethyl ketone. Activities of the viscera extract continuously decreased as NaCl concentration increased, while activities increased as CaCl2 concentration increased. Based on the proteinase activity of zones separated by electrophoresis, the molecular mass of the major proteinases in hybrid catfish viscera was 23 and 20 kDa. The effect of extraction media on recovery of proteinases was also studied. Extraction of the viscera powder with 50 mM Tris-HCl, pH 7.0 containing 0.5 M NaCl and 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in hybrid catfish viscera were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. PRACTICAL APPLICATIONS Hybrid catfish viscera is an abundant and underutilized resource that can be used as a unique proteinase source. Proteinase from various sources catalyzes the hydrolysis of peptide bonds. Thus, it is expected that like other proteinases, hybrid catfish proteinase would be useful in biomedical, food and beverage application. Moreover, the presented extraction media could be adopted to recover the trypsin-like serine proteinase from hybrid catfish viscera, which is currently a solid waste of Pa-duk-ra industry. [source] IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARPJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004MIN-JIE CAO ABSTRACT Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55,60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as ,-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase. [source] Changes in Wheat Leaf Extracellular Proteolytic Activity after Infection with Septoria triticiJOURNAL OF PHYTOPATHOLOGY, Issue 3 2002C. I. SEGGARRA The proteolytic activity of the leaf extracellular space of wheat cultivars Pigüé and Isla Verde was estimated after inoculation of either detached leaves or plants with the fungus Septoria tritici. Pigüé is resistant, whereas Isla Verde is susceptible to the disease caused by S. tritici. Viable conidiospores of the fungus caused similar increases in both hydrogen peroxide production and chitinase activity of the cultivars studied. In contrast, they caused a decrease in the extracellular serine proteinase activity of Isla Verde and a significant increase in that of Pigüé. Independently of the cultivar from which it was extracted, the extracellular serine proteinase inhibited the germination of Septoria tritici conidiospores. These results suggest that the proteolytic activity of the leaf extracellular space can participate in the defence of wheat plants against Septoria tritici. Its regulation may be controlled by specific defence components of each cultivar. [source] Uterine secretion of ISP1 & 2 tryptases is regulated by progesterone and estrogen during pregnancy and the endometrial cycleMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004Colleen M. O'Sullivan Abstract We have described two novel implantation serine proteinase (ISP) genes that are expressed during the implantation period. The ISP1 gene may encode the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The ISP2 gene, which encodes a related tryptase, is expressed in endometrial glands and is regulated by progesterone during the peri-implantation period. Based on similarities between ISP2 gene expression and that of a progesterone-regulated lumenal serine proteinase activity associated with lysis of the zona pellucida, we have suggested that the strypsin related protein, ISP2, may encode a zona lysin proteinase. Recently strypsin has also been found within uterine fluid, suggesting a second potential role in hatching. Consistently, we have discovered that ISP1 is also expressed in the uterine secretory gland at the time of hatching. In this study we demonstrate that both ISP1 and ISP2 are secreted together into the uterine lumen at peri-implantation, and that the appearance of ISP protein is regulated positively at the transcriptional level by progesterone and negatively at the posttranscriptional level by estrogen. This negative regulation by estrogen may be overridden in pregnancy as ISP protein expression is restored during oil-induced decidualization. ISP1 and ISP2 proteins are also expressed in proestrous suggesting additional roles in the endometrial cycle. Mol. Reprod. Dev. 69: 252,259, 2004. © 2004 Wiley-Liss, Inc. [source] Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritisARTHRITIS & RHEUMATISM, Issue 7 2010Jennifer M. Milner Objective Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). Methods Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate,polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. Results Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. Conclusion Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment. [source] Crystallization and preliminary X-ray crystallographic analysis of blood coagulation factor V-activating proteinase (RVV-V) from Russell's viper venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Daisuke Nakayama Russell's viper venom blood coagulation factor V activator (RVV-V) is a thrombin-like serine proteinase that specifically activates factor V by cleaving a single peptide bond between Arg1545 and Ser1546. Activated factor V combines with activated factor X produced by the enzyme RVV-X in the venom to form the prothombinase complex, which can induce disseminated intravascular coagulopathy in envenomated animals. In the current study, RVV-V was crystallized in order to attempt to understand its substrate specificity for factor V. Four distinct crystal forms of RVV-V were obtained using the sitting-drop vapour-diffusion method and diffraction data sets were collected on SPring-8 beamlines. The best crystal of RVV-V generated data sets to 1.9,Å resolution. [source] Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus speciesCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000W.-L. Lin Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts. [source] Expression of prostate-specific antigen and androgen receptor in extramammary Paget's disease and carcinomaCLINICAL & EXPERIMENTAL DERMATOLOGY, Issue 1 2007N. Inoguchi Summary Prostate-specific antigen (PSA) is a kallikrein-like serine proteinase (human kallikrein 3) produced by epithelial cells of both benign and malignant prostate tissue. In this study, PSA expression was histologically examined in tissue specimens from 34 patients with extramammary Paget's disease (EPD; 31 cases) and extramammary Paget's carcinoma (EPC; three cases), but no associated prostate carcinoma. Tumour cells positive for PSA were found in 17 of the 34 cases. Based on this finding, we examined serum PSA level in the three EPC cases. A high level of serum PSA was observed in one case of EPC, which was correlated with disease progression. Because some reports suggest that 50,80% cases of EPD/EPC express androgen receptor (AR), we also examined expression of AR. Immunohistological staining showed correlation of PSA and AR in expression. These results suggest that PSA and the androgen signalling pathway may be involved in the pathogenesis of EPD. [source] Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)INSECT MOLECULAR BIOLOGY, Issue 6 2008B. S. Coates Abstract Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full-length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di-, tri- or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C-termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development. [source] pKa optimized catalysis in serine proteinases, an ab initio study on the catalaytic HisINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 11 2007Péter Hudáky Abstract First principle models of catalytic apparatus of enzymes can be used for studying stability as well as the atomic details of a catalytic mechanism. For example, the catalytic triad of chymotrypsin was recently investigated by using an ab initio geometry optimized (Hudáky and Perczel, Proteins: Struct Funct Genet, 2006, 62, 749) self-stabilizing molecule ensemble without the presence of the complete enzyme and substrate. Several parameters of the above catalytic reaction turned out to be the same within the model and the in vitro enzymatic reaction. Among the numerous parameters of the catalytic process geometrical changes of the catalytic histidine was investigated here and the variation of its pKa value was determined. A relatively large range, 3.5 unit, was determined as the variation of pKa as function of the conformational subspace available in serine proteases. Comparing PDB structures of the free and the complex enzymes it was shown, that histidine, after accepting the proton from the OH group of the catalytic serine, undergoes a minor conformational change accompanied by a 2.5 unit decrease in pKa. We conclude that the changes of pKa during catalysis are predominantly determined by the geometrical arrangement of the histidine moiety and this change serves as a significant driving force in the catalytic process. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007 [source] The Bone Lining Cell: Its Role in Cleaning Howship's Lacunae and Initiating Bone FormationJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2002V. Everts Abstract In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell "rescues" bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process. [source] PROTEINASES IN HYBRID CATFISH VISCERA: CHARACTERIZATION AND EFFECT OF EXTRACTION MEDIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT Proteolytic activity from viscera extract of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was investigated. Optimal pH and temperature for casein hydrolysis were 9.0 and 50C, respectively. The enzyme was stable to heat treatment up to 40C and over a pH range of 7,11 for 30,120 min. The proteolytic activity was effectively inhibited by soybean trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride and N -p-tosyl-L-lysine chloromethyl ketone. Activities of the viscera extract continuously decreased as NaCl concentration increased, while activities increased as CaCl2 concentration increased. Based on the proteinase activity of zones separated by electrophoresis, the molecular mass of the major proteinases in hybrid catfish viscera was 23 and 20 kDa. The effect of extraction media on recovery of proteinases was also studied. Extraction of the viscera powder with 50 mM Tris-HCl, pH 7.0 containing 0.5 M NaCl and 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in hybrid catfish viscera were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. PRACTICAL APPLICATIONS Hybrid catfish viscera is an abundant and underutilized resource that can be used as a unique proteinase source. Proteinase from various sources catalyzes the hydrolysis of peptide bonds. Thus, it is expected that like other proteinases, hybrid catfish proteinase would be useful in biomedical, food and beverage application. Moreover, the presented extraction media could be adopted to recover the trypsin-like serine proteinase from hybrid catfish viscera, which is currently a solid waste of Pa-duk-ra industry. [source] COMPARATIVE STUDIES ON PROTEOLYTIC ACTIVITY OF SPLENIC EXTRACT FROM THREE TUNA SPECIES COMMONLY USED IN THAILANDJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004SUPPASITH KLOMKLAO ABSTRACT Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl2 on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl2 concentration increased. The highest activity was obtained in the presence of 1 mM CaCl2. SDS-substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin-like serine proteinases. [source] The defensive role of lysozyme in human gingiva in inflammatory periodontal diseaseJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2009R. Younes Background and Objective:, The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G. Materials and Methods:, Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study. Results:, Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients. Conclusion:, Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva. [source] Origin of the murine implantation serine proteinase subfamily,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Colleen M. O'Sullivan Abstract The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5,-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo. Mol. Reprod. Dev. 69: 126,136, 2004. © 2004 Wiley-Liss, Inc. [source] Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibitionPROTEIN SCIENCE, Issue 8 2000Miljan Simonovic Abstract CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 Å resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for , sheet A, whose sequence and length are unique to crmA. [source] REVIEW ARTICLE: Evolution and Function of the Uterine Serpins (SERPINA14)AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2010Maria B. Padua Citation Padua MB, Hansen PJ. Evolution and function of the uterine serpins (SERPINA14). Am J Reprod Immunol 2010 Uterine serpins (recently designated as SERPINA14) are hormonally induced proteins secreted in large quantities by the endometrial epithelium during pregnancy. The SERPINA14 proteins belong to the serine proteinase inhibitor (serpin) superfamily, but their apparent lack of inhibitory activity toward serine proteinases suggests that these proteins evolved a different function from the anti-proteinase activity typically found in most members of the serpin superfamily. The gene is present in a limited group of mammals in the Laurasiatheria superorder (ruminants, horses, pigs, dolphins and some carnivores) while being absent in primates, rodents, lagomorphs and marsupials. Thus, the gene is likely to have evolved by gene duplication after divergence of Laurasiatheria and to play an important role in pregnancy. That role may vary between species. In sheep, SERPINA14 probably serves an immunoregulatory role to prevent rejection of the fetal allograft. It is inhibitory to lymphocyte proliferation and natural killer cell function. In the pig, SERPINA14 is involved in iron transport to the fetus by binding to and stabilizing the iron-binding protein uteroferrin. It is possible that SERPINA14 has undergone divergence in function since the original emergence of the gene in a common ancestor of species possessing SERPINA14. [source] Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritisARTHRITIS & RHEUMATISM, Issue 7 2010Jennifer M. Milner Objective Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). Methods Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate,polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. Results Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. Conclusion Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment. [source] Purification, N-terminal sequencing, partial characterization, crystallization and preliminary crystallographic analysis of two glycosylated serine proteinases from Agkistrodon acutus venomACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Zhongliang Zhu AaV-SP-I and AaV-SP-II, two glycosylated serine proteinases from Agkistrodon acutus venom with fibrinogenolysis and esterolysis activities, have been purified to homogeneity by three-step ion-exchange chromatography. Estimated by SDS,PAGE, the molecular weights of AaV-SP-I and AaV-SP-II are about 32 and 31,kDa under reducing conditions and 26 and 25,kDa under non-reducing conditions, respectively. The first 24 N-terminal amino-acid residues are the same in both sequences and display a high homology with those of several snake-venom serine proteinases. However, the proteins possess obviously distinct carbohydrate contents. Using the conventional hanging-drop vapour-diffusion method, single crystals of both enzymes were grown that were suitable for X-ray diffraction analysis. The crystals of AaV-SP-I and AaV-SP-II belong to space groups P212121 and C2, respectively. In each case there is only one molecule in the asymmetric unit. [source] Crystallization and preliminary X-ray diffraction analysis of a recombinant cysteine-free mutant of crmAACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Miljan Simonovic CrmA is an unusual serpin that has a reactive-center loop one residue shorter than other members of the superfamily. Most interestingly, crmA has inhibitory activity against both cysteine and serine proteinases involved in the regulation of cell apoptosis. The three-dimensional structure of crmA will give insight into the mechanism that this serpin employs to inhibit both cysteine and the serine proteinases, as well as help to explain the significance of the shorter reactive-center loop. The monodisperse cysteine-free mutant of crmA was crystallized in the presence of phosphate salts. Crystals diffract to 2.90,Å and belong to space group P212121, with unit-cell parameters a = 42.67, b = 93.15, c = 101.63,Å. [source] Vitamin D and systemic cancer: is this relevant to malignant melanoma?BRITISH JOURNAL OF DERMATOLOGY, Issue 2 2002J.E. Osborne Summary 1,25-dihydroxyvitamin D3[1,25(OH)2D3] is a well-known potent regulator of cell growth and differentiation and there is recent evidence of an effect on cell death, tumour invasion and angiogenesis, which makes it a candidate agent for cancer regulation. The classical synthetic pathway of 1,25(OH)2D3 involves 25- and 1,-hydroxylation of vitamin D3, in the liver and kidney, respectively, of absorbed or skin-synthesized vitamin D3. There is recent focus on the importance in growth control of local metabolism of 1,25(OH)2D3, which is a function of local tissue synthetic hydroxylases and particularly the principal catabolizing enzyme, 24-hydroxylase. The classical signalling pathway of 1,25(OH)2D3 employs the vitamin D nuclear receptor (VDR), which is a transcription factor for 1,25(OH)2D3 target genes. Effects of this pathway include inhibition of cellular growth and invasion. Cytoplasmic signalling pathways are increasingly being recognized, which similarly may regulate growth and differentiation but also apoptosis. 1,25(OH)2D3 has a major inhibitory effect on the G1/S checkpoint of the cell cycle by upregulating the cyclin dependent kinase inhibitors p27 and p21, and by inhibiting cyclin D1. Indirect mechanisms include upregulation of transforming growth factor-, and downregulation of the epidermal growth factor receptor. 1,25(OH)2D3 may induce apoptosis either indirectly through effects on the insulin-like growth receptor and tumour necrosis factor-, or more directly via the Bcl-2 family system, the ceramide pathway, the death receptors (e.g. Fas) and the stress-activated protein kinase pathways (Jun N terminal kinase and p38). Inhibition of tumour invasion and metastasis potential has been demonstrated and mechanisms include inhibition of serine proteinases, metalloproteinases and angiogenesis. The lines of evidence for an effect of vitamin D3 in systemic cancer are the laboratory demonstration of relevant effects on cellular growth, differentiation, apoptosis, malignant cell invasion and metastasis; epidemiological findings of an association of the occurrence and outcome of cancers with derangements of vitamin D3/1,25(OH)2D3 and the association of functional polymorphisms of the VDR with the occurrence of certain cancers. In addition, vitamin D3 analogues are being developed as cancer chemotherapy agents. There is accumulating evidence that the vitamin D3/1,25(OH)2D3/VDR axis is similarly important in malignant melanoma (MM). MM cells express the VDR, and the antiproliferative and prodifferentiation effects of 1,25(OH)2D3 have been shown in cultured melanocytes, MM cells and MM xenografts. Recently, an inhibitory effect on the spread of MM cells has been demonstrated, low serum levels of 1,25(OH)2D3 have been reported in MM patients and the VDR polymorphisms have been shown to be associated with both the occurrence and outcome of MM. The relationship between solar irradiation and MM is more complex than for the systemic cancers. As in other cancers, there is evidence of a protective effect of vitamin D3 in MM, but ultraviolet radiation, which is a principal source of vitamin D3, is mutagenic. Further work is necessary on the influence of serum vitamin D3 levels on the occurrence and prognosis of MM, the effects of sun protection measures on serum vitamin D3 levels in temperate climates and epidemiological studies on geographical factors and skin type on the prognosis of MM. Meanwhile, it would seem mandatory to ensure an adequate vitamin D3 status if sun exposure were seriously curtailed, certainly in relation to carcinoma of breast, prostate and colon and probably also MM. [source] Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus speciesCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000W.-L. Lin Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts. [source] | ||||||||||||||||