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Background: Cardiovascular Diseases (CVDs) such as stroke, high blood pressure, peripheral vascular disease, ischemic heart disease and acute myocardial infarction are some of the leading causes of death. To treat CVDs, commercially available thrombolytic agents are widely used. However, these thrombolytic agents have various side effects. Alternatively, fibrinolytic enzymes from bacterial sources are highly safe and have direct blood clot lytic activity., Methods: A fibrinolytic enzyme producing bacterial strain, Bacillus flexus BF12, was isolated from a solar saltpan in Kanyakumari District, Tamilnadu, India. Enzyme production was improved by optimizing physical factors and nutritional factors., Results: A novel fibrinolytic enzyme was isolated from a strain of the studied B. flexus BF12. Enzyme production was enhanced significantly by optimizing process parameters. The critical physical factors (pH and salinity) and influencing nutritional factors (carbon, nitrogen and ions) were optimized by one variable at a time approach, followed by the statistical method. The strain BF12 was highly active at alkaline pH (>7.0) and between 4 and 6% NaCl concentration. The nutrients such as fructose (carbon source), beef extract (nitrogen source) and CaCl 2 significantly influenced enzyme production. Central composite design and response surface methodology improved 3.2-fold enzyme yield than unoptimized culture medium. Fibrinolytic protease was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography., Discussion: The molecular weight of an enzyme was found to be 23 kDa. It was active at a broad temperature (40-60 °C) and pH (7.0-9.0) ranges. Enzyme activity was enhanced by Ca 2+ and Co 2+ ions. The purified protease retained 100% enzyme activity in the presence of ethanol and acetone. Acetonitrile, butanol, DMSO, methanol and chloroform showed enzyme activity of 63%, 92.5%, 94.7%, 92.3% and 90.4%, respectively. The purified enzyme degraded 100% of human blood clot., Conclusion: The Bacillus flexus BF12 fibrinolytic enzyme shows promising potentials in nutraceutical and food fortification applications. The application of fibrinolytic enzymes could prevent CVDs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda , Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus , in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Natural products and their semisynthetic derivatives are an important source of drugs for the pharmaceutical industry. Bacteria are prolific producers of natural products and encode a vast diversity of natural product biosynthetic gene clusters. However, much of this diversity is inaccessible to natural product discovery. Here, we use a combination of phylogenomic analysis of the microviridin biosynthetic pathway and chemo-enzymatic synthesis of bioinformatically predicted microviridins to yield new protease inhibitors. Phylogenomic analysis demonstrated that microviridin biosynthetic gene clusters occur across the bacterial domain and encode three distinct subtypes of precursor peptides. Our analysis shed light on the evolution of microviridin biosynthesis and enabled prioritization of their chemo-enzymatic production. Targeted one-pot synthesis of four microviridins encoded by the cyanobacterium Cyanothece sp. PCC 7822 identified a set of novel and potent serine protease inhibitors, the most active of which had an IC 50 value of 21.5 nM. This study advances the genome mining techniques available for natural product discovery and obviates the need to culture bacteria.

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules. Previously, we demonstrated that functional regulatory molecules against interleukin-6 receptor (IL-6R) could be obtained by directed evolution of the three-finger toxin (3F) scaffold. In the present study, trypsin was selected as a target for directed evolution to further explore the potential use of the 3F cDNA display library. After seven rounds of selection, the DNA sequences converged. The recombinant proteins produced by the selected candidates had inhibitory activity against trypsin (Ki of 33-450 nM). Three of the six groups had Ki values that were comparable to bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor. Two of the candidates also had inhibitory effects against chymotrypsin and kallikrein. This study suggests that 3F protein is suitable for the preparation of high-diversity libraries that can be utilized to obtain protease inhibitors. In addition to our previous successful targeting of IL-6R, the technique developed in our studies may have wide applications in the generation of regulatory molecules for targets of interest, such as receptors, enzymes for research, diagnostic applications, and therapeutic uses.

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.

Plant protease inhibitors (PIs) are among the most well-studied and widely distributed resistance traits that plants use against their herbivore attackers. There are different types of plant PIs which putatively function against the different types of proteases expressed in insect guts. Serine protease inhibitors (SPIs) and cysteine protease inhibitors (CPIs) are hypothesized to differentially function against the predominant gut proteases in lepidopteran and coleopteran herbivores, respectively. Here, we test the hypothesis that tall goldenrod, Solidago altissima, can specifically respond to damage by different herbivores and differentially induce SPIs and CPIs in response to damage by lepidopteran and coleopteran herbivores. Moreover, we ask if the concerted induction of different types of PIs accounts for variation in induced resistance to herbivory. We altered and optimized a rapid and effective existing methodology to quantitatively analyze both SPI and CPI activity simultaneously from a single tissue sample and to use the same plant extracts directly for characterization of inhibitory effects on insect gut protease activity. We found that both SPIs and CPIs are induced in S. altissima in response to damage, regardless of the damaging herbivore species. However, only SPIs were effective against Spodoptera exigua gut proteases. Our data suggest that plant PI responses are not necessarily specific to the identity of the attacking organism but that different components of generally induced defense traits can specifically affect different herbivore species. While providing an efficient and broadly applicable methodology to analyze multiple PIs extracted from the same tissue, this study furthers our understanding of specificity in induced plant resistance.

Hepatocarcinoma represents one of the most malignant cancer types. Esophageal cancer-related gene 2 (ECRG2) is found to be critical in the process of carcinogenesis. It regulates urokinase-type plasmin activator receptor and extracellular matrix function and its polymorphism in exon 4 is associated with cancer relapse. To explore new strategies to fight against cancer, here we first systematically evaluated the therapeutic potential as a biological tool using adenoviral vector (Ad-ECRG2). Ad-ECRG2 is exogenously expressed in cytoplasm and is potent to suppress the growth of cancer cell by inducing apoptosis as effective as Ad-p53. Ad-ECRG2 is able to suppress the invasion and adhesion of cancer cells at low titers. It alters the expression of a panel of cancer-related molecules, including nuclear factor-kB, matrix metalloproteinase 2 and E-cadherin, contributing to reverse malignancy phenotype of cancer cells. In vivo experiments show a significant inhibition of cancer growth by intratumoral Ad-ECRG2 administration. No evident toxicity was observed in the model animal during the study. We concluded that ECRG2 is a potential molecular target in biological therapy strategies for cancer treatment.

PC4 or PCSK4 belongs to the 9-member superfamily of mammalian subtilases collectively called Proprotein Convertases or Proprotein Convertase Subtilisin/Kexins that convert inactive precursor proteins into their active mature forms by endoproteolytic cleavage. PC4-activity plays a crucial role in mammalian fertilization via activation of sperm surface proteins. PC4 knockout mice exhibit severely impaired male fertility due to premature sperm acrosome reaction. Regulation of sperm-PC4 activity during its storage and transport through epididymis is an important determinant for ultimate egg-binding and fertilizing capacities of sperms. Herein we show that epididymal serpin CRES (cystatin related epididymal spermatogenic) recombinant protein inhibits PC4 activity in vitro in a differential manner when measured against the fluorogenic substrate Boc- RVRR-MCA depending on its oligomeric state. Thus while CRES-dimer exhibits K(i) ∼8 μM, the corresponding monomer showed K(i) > 100 μM. Both forms also blocked PC4-mediated processing of human proIGF-2 in human placenta tropoblast cell line with dimer being more efficient. Using specific inhibitors and substrates, we also demonstrated the presence of PC4-like activity and CRES protein in varying levels in the fluids of various epididymal compartments. Our observations suggest a potential function of CRES as a regulator of PC4 in sperm-egg interaction and fertilization.

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A five-domain Kazal-type serine proteinase inhibitor, SPIPm2, from Penaeus monodon has recently been implicated in antiviral responses for it is up-regulated upon viral infection and needs further studies. The SPIPm2 genomic gene was composed of seven exons and six introns. The genomic DNA segments coding for each Kazal domain were separated by introns of variable lengths supporting the hypothesis of gene duplication in the Kazal-type gene family. RT-PCR and Western blot analysis revealed that the SPIPm2 transcript and its five-domain protein product were expressed mainly in the hemocytes and less in gill, heart and antennal gland. Upon white spot syndrome virus (WSSV) infection, the SPIPm2 was only detected in the hemocytes and plasma. Immunocytochemical study of P. monodon hemocytes showed that the percentage of SPIPm2-producing hemocytes was reduced by about half after WSSV infection. Quantitative RT-PCR revealed further that the SPIPm2 was up-regulated early in the hemocytes of WSSV-infected shrimp and gradually reduced as the infection progressed. Injection of the recombinant SPIPm2 (rSPIPm2) prior to WSSV injection resulted in a significant inhibition of WSSV replication. The rSPIPm2 injection also prolonged the mortality rate of WSSV-infected shrimp. Therefore, the SPIPm2 was involved in the innate immunity against WSSV infection in shrimp., (2010 Elsevier Ltd. All rights reserved.)

Unlabelled: Ovarian cancer (OC) is a disease with poor prognosis, and molecular markers are needed to improve understanding of disease progression and resultant treatment. Only limited data concerning the expression of maspin, a serine protease inhibitor, in ovarian cancer (OC) are available. This study investigates the prognostic value of maspin expression (ME) in various OC cell lines and clinical tissue specimens from OC patients., Patients and Methods: Tumour purified mouse anti-human maspin monoclonal antibody was applied to tissue specimens from 87 OC patients. ME was recorded by an immunoreactive score, which was correlated with grading, stage, histopathological subtypes and overall survival. Additionally ME was evaluated in established ovarian cancer cell lines (HEY, SKOV3, OVCAR3/8) and paclitaxel- and docetaxel-resistant HEY cells by QRT-PCR., Results: There was significant correlation between cytoplasmatic ME and overall survival (p<0.05). OC patients with high levels of ME had a median survival of 28 vs. 57 months for those with low levels. Significant differential ME was detected between benign, borderline ovarian lesions and OC, as well as among different tumour gradings. Normal ovarian epithelial cells expressed less maspin than ovarian cancer cells as measured by QRT-PCR. Docetaxel- and paclitaxel-resistant ovarian cell lines showed an even higher level of ME, suggesting an unfavourable role of ME in OC cell lines., Conclusion: Maspin is expressed differentially in OC, and low expression levels of maspin are correlated with a longer survival.

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena. We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.

Serine proteinase inhibitor plays an essential role in arthropods by restraining the activities of endogenic or exogenic serine proteinases. Four Kazal-type serine proteinase inhibitors, Fcspi-1-4, from the hepatopancreas of Chinese white shrimp, Fenneropenaeus chinensis, were cloned and identified. The open reading frames (ORFs) of Fcspis are 1389, 1236, 1080, and 939 base pairs, encode the pre-proteins of 462, 411, 359, and 312 amino acids and form the 9, 8, 7, and 6 typical Kazal domains, respectively. When analyzing the amino acid sequences of the four inhibitors, it was found that they might have been derived from the same transcript, which was subjected to alternative splicing, and none of the Kazal domains were identical within each inhibitor. Multiple alignments showed that the Kazal inhibitors were homologous with a conserved motif of Cx(3)Cx(6)VCGSDGxTYx(3)CxLx(5)Cx(5)ITx(6)GC. The results from RT-PCR indicated that the expression of Fcspis as a whole was upregulated by bacterial challenge, no obvious change was noticed after viral challenge, and Fcspi-1 had a similar expression pattern with that of Fcspis. Recombinant FcSPIs were successfully expressed in bacteria and purified for further study. Recombinant FcSPI-1 was sensitive to DTT and had thermal stability. The inhibitory kinetics assay suggested that rFcSPI-1 was a mixed-type fast tight binding inhibitor with inhibitory activities against subtilisin A at a molar ratio of 1:1, 1:2 against proteinase K, and 2:1 against elastase. It can firmly bound to two Gram-positive and one Gram-negative bacteria but without anti-bacterial ability. In addition, it inhibited the activities of both bacterial-secreted proteinases and natural chymotrypsin of Chinese white shrimp, suggesting that FcSPI-1 may participate in the immune defence response by inhibition of bacterial pathogen proteinases and possibly be involved in the regulation of shrimp proteinase activity.

The liver has the ability to restore its functional capacity following injury or resection and the priming of liver regeneration is a complex process that has not been completely elucidated. In the current research, to further reveal the priming mechanism of liver regeneration, hepatocyte total protein and hepatocyte cytosol of the rats at 4 h after 2/3 partial hepatectomy (PHx) were studied, respectively, by 2-DE and 2-D blue native gel electrophoresis. Seventeen unique differential proteins were identified in hepatocyte total protein samples. Nine differential protein complexes containing 41 protein components were identified in hepatocyte cytosol samples. For the first time, at the priming stage of liver regeneration, the variations of serine protease inhibitor 2c, sulfite oxidase and valosin-containing protein (VCP) were presented and validated by Western blotting, and the VCP complex was further validated by antibody super-shift experiments. The current results suggested that at 4 h after PHx, VCP complex was down-regulated in hepatocyte cytosol, apoptosis pathways were inhibited, nuclear factor-kappaB and interleukin 6 pathways worked together and triggered the liver regeneration.

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

SKPI (shrimp Kunitz-type protease inhibitor) from Marsupenaeus japonicus is a member of serine protease inhibitors which play an important role in the arthropod immunity. To fully understand its function in the innate immunity of shrimp, the skpi gene was cloned into a modified pPIC9K vector with a 6-His tag and expressed by Pichia pastoris GS115. The secretory SKPI was purified from the medium with high purity by using Ni Sepharose High Performance. This results also indicated that the purified SKPI could inhibit the activity of trypsin specifically.

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.

Various studies suggest that proteolytic activity may be involved in a number of neurodegenerative disorders, including stroke and seizure. In this report, we examined the role of tryptic serine proteases, plasminogen activators (PAs), in the evolution of a neurodegenerative disease. Transgenic mice overexpressing an axonally secreted inhibitor of serine proteases (neuroserpin) were crossed with mice characterized by a "dying-back" motor neuron disease [progressive motor neuronopathy (pmn/pmn)]. Compared with pmn/pmn mice that showed an increase in PA activity, double mutant mice had decreased PA activity in sciatic nerves and spinal cord; their lifespan was increased by 50%, their motor behavior was stabilized, and histological analysis revealed increased numbers of myelinated axons and rescue of motoneuron number and size. This is the first report showing that a class of serine proteases (PAs) may be involved in the pathogenesis of a motor neuron disease and more specifically in axonal degeneration. Inhibiting serine proteases could offer a new strategy for delaying these disorders.

The analysis of EpiDerm cultures treated with the known skin irritant sodium lauryl sulphate (SLS) was performed using 2D-gel electrophoresis in order to understand the mechanism of action and thereby identify novel markers of skin irritation. A range of both broad and narrow pH gradient first-dimension gels were run (pH 4-7, 6-11, 4-5, 5-6 and 6-9) consistently followed by 12% SDS-PAGE in the second-dimension. Following treatment of EpiDerm with SLS, 67 proteins of interest were identified, of which 8 were selected as interesting: calmodulin-like skin protein, involucrin, epithelial cell marker protein, HS1, peroxiredoxin 1, serine protease inhibitor, KIAA0117 and ribosomal protein L17. Involucrin was confirmed as being up-regulated by both ELISA and Western blotting. The use of proteomics has identified a number of proteins which could be used as general markers for skin irritation and which may in particular be of value for the development of in vitro predictive models.

Proteinase inhibitor 9 (PI-9) is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. It is the only known endogenous natural antagonist of granzyme B (GrB), and its proposed function is protection of cells from misdirected GrB. We have studied the regulation of PI-9 in primary peripheral blood mononuclear cells (PBMCs) following ex-vivo stimulation, and in PBMCs from patients suffering from viral or bacterial infections. By intracellular flow cytometry, we found identical PI-9 expression in all lymphocyte subsets, lower levels in monocytes and none in granulocytes. PI-9 was stable for 48 h in the presence of cycloheximide, indicating slow protein turnover. Incubation of PBMCs with several stimuli including lipopolysaccharide (LPS) led to up-regulation in the monocyte, but not the lymphocyte fraction, within 48 h, inhibitable by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Up-regulation of PI-9 was observed in lymphocytes and monocytes of patients with acute Epstein-Barr virus (EBV), but not bacterial infection. Preterm infants had similar PI-9 expression as adults in monocytes, but lower in lymphocytes, decreasing during bacterial infection. Taken together, our data indicate that PI-9 is rapidly up-regulated upon stimulation of monocytes, but not lymphocytes. By protecting monocytes and macrophages from misdirected GrB in the inflammatory process, PI-9 might be involved in the regulation of antigen presentation.

A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347-355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.

Dipetalogastin is a potent thrombin inhibitor from Dipetalogaster maximus. The cDNA of dipetalogastin codes for a large protein which consists of six Kazal-type domains. There are three tandem, homologous regions each including two domains. Three biologically active recombinant proteins rDI, rDII and rDIII each corresponding to one region of the dipetalogastin cDNA were expressed, purified and investigated with regard to their biological activities. rDI and rDII with molecular masses of 12,660 and 12,911 Da, respectively, proved to be potent thrombin inhibitors. The investigation of their influences on amidolytic activities of different serine proteases showed no inhibition of factor Xa (FXa) and alpha-chymotrypsin. At a large molar excess of rDI and rDII over the enzymes only low effects on the activities of trypsin and plasmin were observed. rDIII differs much from the both others. An inhibition of thrombin was found only at a molar excess of rDIII over the enzyme. Furthermore, an inhibition of trypsin and low effects on plasmin were detected at a molar excess of inhibitor over these enzymes. These results indicate that rDIII is active against thrombin, trypsin and plasmin, and finally possesses no specificity for only one serine proteinase., (Copyright 2004 Elsevier Ltd.)

Myoepithelial cells (MEs), which surround ducts and acini of the breast glands, exhibit an anti-invasive phenotype and form a natural border separating proliferating tumour cells of ductal carcinoma in situ (DCIS) from basement membrane (bm) and underlying stroma. Invasion requires penetration of these host cellular and extracellular matrix barriers. This destruction is caused by proteolytic activity of tumour cells and host bystander cells. There is substantial evidence that high concentrations of the urokinase plasminogen-activating system are conducive to tumour cell spread and metastasis. Prompted by the conspicuous absence of studies examining the role of the ME in breast cancer progression, we studied the expression of the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1) in MEs of 60 DCIS samples. Our results show that nearly all MEs of DCIS and normal breast glands exhibit the uPAR antigen, whereas the PAI-1 antigen was mainly expressed in MEs of high-grade DCIS. In one intermediate DCIS numerous ducts showed an incomplete myoepithelial layer expressing uPAR and PAI-1. We conclude that uPAR in MEs may be necessary to attach them to the bm by uPAR/vitronectin (Vn) interaction. The strong expression of PAI-1, which is known to resolve the uPAR/Vn binding, may be involved in the detachment of MEs of DCIS. Although the role of PAI-1 acting as cell detachment factor could not be demonstrated in our study, we speculate that the loss of the anti-invasive ME layer in DCIS may be triggered by PAI-1 and could be an early sign of subsequent tumour cell infiltration.

Background: Maspin inhibits cell motility, invasion and metastasis. Loss or reduction in maspin expression has been associated with tumoral progression., Methods: The presence of maspin was studied immunohistochemically in salivary gland tumours presenting cells with myoepithelial differentiation in their composition, and in normal salivary gland., Results: Pleomorphic adenoma (PA) presented high expression of maspin, except in the spindle cells and occasional luminal cells. Epithelial-myoepithelial carcinoma and tubular adenoid cystic carcinoma (ACC) showed intense expression in all cells. Cribriform ACC evidenced only few positive cells of the luminal type, while solid subtype showed rare positive cells. Normal salivary gland tissue has shown low levels of maspin positivity., Conclusions: Maspin has small participation in normal salivary gland, is increased in PA, and decreases as the histological malignancy raises. Hence, in salivary gland, its expression is not exclusive of myoepithelial cells; thus, it should not be used as a marker for this cell. Nevertheless, we believe it is an important marker of biological behaviour in these tumours.

SGCI (Schistocerca gregaria chymotrypsin inhibitor) and SGTI (Sch. gregaria trypsin inhibitor) are small, 35-residue serine protease inhibitors with intriguing taxon specificity: SGTI is specific for arthropod proteases while SGCI is an excellent inhibitor on both mammalian and arthropodal enzymes. Here we report the cloning, expression, and (15)N backbone dynamics investigations of these peptides. Successful expression could be achieved by a "dimeric" construct similar to the natural precursor of the inhibitors. An engineered methionine residue between the two modules served as a unique cyanogen bromide cleavage site to cleave the precursor and physically separate SGCI and SGTI. The overall correlation time of the precursor (5.29 ns) as well as the resulted SGCI (3.14 ns) and SGTI (2.96 ns) are as expected for proteins of this size. General order parameters (S(2)) for the inhibitors are lower than those characteristic of well-folded proteins. Values in the binding loop region are even lower. Interestingly, the distribution of residues for which a chemical exchange (R(ex)) term should be considered is strikingly different in SGCI and SGTI. Together with H-D exchange studies, this indicates that the internal dynamics of the two closely related molecules differ. We suggest that the dynamic properties of these inhibitors is one of the factors that determine their specificity.

Background: The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PAs) and their inhibitors may be important for understanding the pathogenesis of radicular cysts. The purpose of this study was to investigate the in situ localization of tissue-type PA (t-PA) and type I PA inhibitor (PAI-1) in radicular cysts., Methods: Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were examined using immunohistochemistry. In addition, another section from each radicular cyst specimen was stained with hematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in t-PA and PAI-1 expression between tissues with low and high levels of inflammation were subsequently analyzed using Fisher's exact test., Results: Both t-PA- and PAI-1-positive cells were detected in the lining epithelium, connective tissue, inflammatory infiltrates, and endothelium. In addition, the t-PA signal was mainly expressed in epithelial cells. However, the PAI-1 signal was mainly expressed in fibroblasts. Moreover, significantly greater t-PA as well as PAI-1 expression was noted in radicular cysts with high levels of inflammation as compared to tissues with low levels of inflammatory cell infiltrates (P < 0.05)., Conclusions: The present study confirms earlier indications of local production of PA and its inhibitor in radicular cysts. In addition, this study further shows the tissue localization of the antigens for t-PA as well as PAI-1, and demonstrates that the expression of both t-PA and PAI-1 increases with the grade of inflammation in radicular cysts.

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.

Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.

The neuronal serine protease inhibitor neuroserpin is widely expressed in the developing and adult brain. In the neocortex, neuroserpin is displayed particularly during the period of synaptic specification and refinement, indicating a role as modulator of extracellular proteolytic processes. The synaptic connections of the visual system of the mouse are shaped during early postnatal life by an activity-dependent process. We have studied the expression of the neuronal serine protease inhibitor neuroserpin in the primary visual cortex of mice from birth until the end of the critical period by means of reverse transcription polymerase chain reaction and in situ hybridization. The localization and the level of expression were constant throughout this period. Monocular deprivation with an eyelid sutured induced a decrease in neuroserpin expression in neurons of area 17 after 1 week of deprivation, the decrease being more pronounced on the side contralateral to the closed eye. The expression of neuroserpin in the visual cortex during the critical period and its decrease in parallel to the refinement of synaptic contacts after visual deprivation suggests a regulative role of neuroserpin on these processes.

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.

The objective was to analyze gene expression in bovine granulosa cells of the dominant follicle by mRNA differential display. Total RNA was extracted from granulosa cells of

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.

Background: Plasminogen activator inhibitor-1 (PAI-1) is a main regulator of the fibrinolytic system. PAI-1 inhibits tissue plasminogen activator and urokinase-type plasminogen activator, resulting in reduced plasminogen activity and attenuated fibrinolysis and proteolysis. The present study was performed to determine the gene expression encoding for PAI-1 in cultured pigmented ciliary epithelial cells of the porcine eye and to detect PAI-1 activity in cell culture supernatants., Methods: Total mRNA of respective confluent primary cultures of porcine ciliary epithelial cells, porcine liver cells and porcine kidney cells was isolated. Reverse transcribed PAI-1 mRNA was measured by real-time polymerase chain reaction (TaqMan PCR) with PAI-1 primers and probes deduced from the human PAI-1 gene. PAI-1 activity in supernatants of the cell cultures was determined by a specific chromogenic test (Coatest PAI)., Results: PAI-1 mRNA was localized in all samples of primary cultures of porcine pigmented ciliary epithelial cells. As a negative control we analyzed total mRNA of porcine kidney cells. PAI-1 mRNA was not detectable in these cells. On the other hand, we established PAI-1 mRNA in porcine liver cells as a positive control. High levels of PAI-1 activity were found in all samples of cell culture supernatants., Conclusions: Our results indicate that PAI-1 is produced and secreted by the porcine ciliary epithelium. We suggest that PAI-1 together with components of the fibrin/fibrinolytic system may be involved in aqueous humor outflow. Overproduction of PAI-1 may induce less fibrinolysis and extracellular proteolysis in aqueous humor and trabecular meshwork, which could result in an elevated intraocular pressure by increasing the outflow obstruction. Therefore stimulation of PAI-1 production may perhaps contribute to the pathogenesis of primary open-angle glaucoma.

Introduction: Plasminogen activator inhibitor-1 (PAI-1), a known inhibitor of plasminogen activators, may regulate smooth muscle cell migration (SMC) through alteration in matrix metalloproteinase (MMP) activity., Methods: To study the effect of endothelial cell (EC) PAI-1 overexpression on SMC migration, RT-PCR was used to clone the full length PAI-1 gene, which was ligated into the pCMV/myc/ER expression vector. With electroporation, bovine aortic ECs were transfected with either the PAI-1 construct or the empty vector as control. EC PAI-1 overexpression was shown with a specific PAI-1 activity assay and enzyme-linked immunosorbent assay. The effect of EC PAI-1 overexpression on SMC migration was measured with a modified Boyden-chamber assay. SMC MMP expression was measured with zymography., Results: Selected clones (EC9, EC21) had a three-fold to five-fold increase in PAI-1 activity compared with untransfected EC and empty vector EC (ECC). Similarly, enzyme-linked immunosorbent assay results showed a 3.5-fold to 5.5-fold increase in PAI-1 levels in EC9 and EC21 versus ECC. Untransfected EC and ECC had similar effects on SMC migratory patterns. Migration of SMC exposed to PAI-1 overexpressing EC was inhibited by 35% to 57% compared with ECC. This inhibitory effect was reversed with addition of exogenous urokinase-type plasminogen activator (uPA). Zymography showed downregulation of MMP-2 and MMP-9 in SMCs exposed to PAI-1 overexpressing EC., Conclusion: PAI-1 overexpression with transfected EC inhibits SMC migration. This effect may be mediated through decreased SMC MMP activity.

Objective: Monocyte fibrinolytic activity may influence thrombus resolution. The balance between uPA and PAI-2 could determine the fibrinolytic activity of the monocyte. Inhibiting PAI-2 production using specific antisense sequences might alter this balance. Selecting effective sequences is a problem as prediction of the secondary structure of target mRNA is difficult. This study reports the modification of a cell free system for rapid antisense screening., Methods: Five 18-19 mer oligodeoxynucleotides (ODN), sequences A, B, K, T and Q, and their matched scrambled controls were designed and screened using a modified rabbit reticulocyte lysate transcription and translation system (RRL). Intracellular uptake of ODNs was confirmed by fluorescence microscopy, scanning laser confocal microscopy and fluorimetry. Monocytes were transfected with a liposome/ODN complex using sequences A, B, A + B combined, or T and PAI-2 levels measured by ELISA. Inhibition of PAI-2 production was calculated as a percentage of control levels (baseline and scrambled)., Results: (i) RRL System--Sequence A was the most effective inhibitor of PAI-2 production in this system (median 63%) compared with sequences, B median 9%, K median 14%, T median 11% and Q median -8% respectively (n = 3). Sequence A was the only sequence, which always inhibited PAI-2. This was confirmed using fluorescently labelled protein (n = 2). (ii) Monocyte transfection--Fluorescence microscopy and fluorimetry showed that intracellular delivery of labelled antisense was only achieved when a liposome was used. Transfection of monocytes extracted from 5 subjects showed that sequence A significantly reduced PAI-2 production (mean % 41.4, sem 9.1) compared with sequences B (mean% 3.4, sem 8.9, p = 0.04), A + B (mean % 0.4, sem 7.8, p = 0.04), and T (mean % 5.4, sem 4.9, p = 0.01). Further studies using sequence A on cells from 10 subjects showed a significant reduction in monocyte PAI-2 production (27.6 ng/ml, sem 3.9) compared with matched scrambled controls (mean 38.3 ng/ml, sem 4.5, p = 0.0112) and baseline (mean 51.4 ng/ml, sem 6.7, p = 0.0009)., Conclusion: Use of the RRL screening system allowed the selection of a novel antisense sequence, which significantly reduced PAI-2 production in monocytes.

The objective of this study was to determine whether disc cells could be cultured under serum-free conditions and whether they synthesized bovine pancreatic trypsin inhibitor (BPTI)-like serine proteinase inhibitory proteins (SPIs) previously demonstrated for ovine chondrocytes. Intervertebral discs from 1- to 2-year-old merino wether sheep were dissected into the annulus fibrosus and nucleus pulposus, and the cells isolated by sequential enzymatic digestion. The cells were grown encapsulated in calcium alginate microspheres under serum-free conditions for 10 days. They remained more than 92% viable as assessed using the vital fluorescent dyes chloromethyl fluorescein diacetate and ethidium homodimer-1 to delineate live/dead cells, respectively. Western and affinity blotting identified a 12-16-kDa media SPI and an additional 34-36-kDa BPTI-like species in solubilized bead samples. This study has demonstrated ovine disc cells synthesized BPTI-like SPIs in serum-free alginate bead culture similar to chondrocyte SPIs; however, the 58-kDa precursor SPI form was not detected suggesting differences in the endogenous processing of these SPIs.

Disruptions in TGF beta signaling have been implicated in various human cancers, including ovarian cancer. Our goal was to determine whether ovarian cancer cells isolated from patient ascites fluid were growth inhibited by TGF beta 1 treatment and further characterize the expression and activity profile of TGF beta/Smad signaling components in human ovarian cancer cells. We found that 9 of 10 primary cultures of ovarian cancer cells (OC2-10) were growth inhibited by 16 pM TGF beta 1. One primary ovarian cancer sample (OC1) and the established ovarian cancer cell lines CaOV3 and SkOV3 continued to grow in the presence of TGF beta 1. All cells expressed components of the TGF beta/Smad signaling pathway including TGF beta 1, T beta RI, T beta RII, Smad2, -3, -4, and Smad anchor for receptor activation. Although OC1, CaOV3, and SkOV3 are not growth inhibited by TGF beta 1, they can transmit the TGF beta 1 signal to turn on a transfected TGF beta/Smad reporter gene, p3TP.lux. In addition, all cells up-regulate the endogenous TGF beta target genes Smad7 and PAI-1. p15(Ink4B) mRNA is also up-regulated with TGF beta 1 treatment in OC2-9, whereas the p15(Ink4B) gene has been deleted in OC1, CaOV3, and SkOV3 cells. Homozygous deletion of p15(Ink4B) may account for TGF beta resistance in some populations of ovarian cancer cells. Our data demonstrate that the TGF beta/Smad signaling pathway remains functional in human ovarian cancer cells and suggest that if abnormalities exist in the cellular response of TGF beta signals, they must lie downstream of the Smad proteins.

The structure of propeptin, a new inhibitor of prolyl endopeptidase isolated from Microbispora sp. SNA-115, was determined. FAB/MS, Edman degradation and amino acid analysis revealed propeptin to be a cyclic polypeptide consisting of 19 common L-amino acids. By FAB/MS and protein chemical methods, the primary sequence of propeptin was determined to be Gly1-Tyr-Pro-Trp-Trp-Asp-Tyr-Arg-Asp9-Leu-Phe-Gly-Gly-His-Thr-Phe-Ile-Ser-Pro19, which cyclizes between the beta-carboxyl group of Asp9 and the a-amino group of Gly1.

During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.

Background: Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes., Results: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction., Conclusions: These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

Purpose: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules., Methods: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices., Results: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin., Conclusions: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.

Using an invasive technique, we studied the mean transit time, the net quantitative turnover rate, and the sites of synthesis and catabolism of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in healthy young volunteers in the fasting, steady state. Blood was sampled simultaneously from a large hepatic vein, an artery and the inferior caval vein, while measuring the splanchnic plasma flow rate and the plasma volume. We found that the catabolism of active t-PA and t-PA antigen took place in the splanchnic circulation with net rates of 7.2 and 6.3 pmol/min, respectively. The extraction fraction and the mean transit time in the splanchnic circulation were, respectively, 0.63 and 5.6 min for active t-PA and 0.17 and 21 min for t-PA antigen. Active PAI-1 was synthesized in the splanchnic circulation at a rate of 890 IU/min and had a mean transit time of about 9.8 min. No net extraction of PAI-1 antigen took place in the splanchnic circulation. In conclusion, we demonstrated that active t-PA and t-PA antigen are catabolized and active PAI-1 produced in the splanchnic circulation in young healthy subjects during steady state. Furthermore, our data show that active t-PA was also eliminated outside the splanchnic region with a catabolism rate of about 8.4 pmol/min. No net complex formation could be demonstrated in the peripheral circulation. We therefore suggest that active t-PA is eliminated by a re-uptake in the endothelium in the peripheral vessels or in the lung circulation.