Trypsin-like Protease Activity Research Articles

Decreased Cry1Ac activation by midgut proteases associated with Cry1Ac resistance in Helicoverpa zea.

Field-evolved resistance of Helicoverpa zea to Bacillus thuringiensis (Bt) toxin Cry1Ac was first reported more than a decade ago, yet the underlying mechanisms remain elusive. Towards understanding the mechanisms of resistance to Cry1Ac, we analyzed a susceptible (LAB-S) and two resistant (GA and GA-R) strains of H. zea. The GA strain was derived from Georgia and exposed to Bt toxins only in the field. The GA-R strain was derived from the GA strain and selected for increased resistance to Cry1Ac in the laboratory. Resistance to MVPII, a liquid formulation containing a hybrid protoxin similar to Cry1Ac, was 110-fold for GA-R and 7.8-fold for GA relative to LAB-S. In midgut brush border membrane vesicles, activity of alkaline phosphatase and aminopeptidase N did not vary significantly among strains. The activity of total proteases, trypsin-like proteases and chymotrypsin-like proteases was significantly lower for GA-R and GA than LAB-S, but did not differ between GA-R and GA. When H. zea midgut cells were exposed to Cry1Ac protoxin that had been digested with midgut extracts, toxicity was significantly lower for extracts from GA-R and GA relative to extracts from LAB-S, but did not differ between GA-R and GA. Transcriptional analysis showed that none of the five protease genes examined was associated with the decline in Cry1Ac activation in GA-R and GA relative to LAB-S. The results suggest that decreased Cry1Ac activation is a contributing field-selected mechanism of resistance that helps explain the reduced susceptibility of the GA-R and GA strains. Relative to the LAB-S strain, the two Cry1Ac-resistant strains had lower total protease, trypsin and chymotrypsin activities, a lower Cry1Ac activation rate, and Cry1Ac protoxin incubated with their midgut extracts was less toxic to H. zea midgut cells. © 2018 Society of Chemical Industry.

Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands.

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin-like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin-like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate Na -benzoyl-L-arginine-ethyl-ester-hydrochloride (BAEE), trypsin-like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) -agarose affinity column was used to independently identify trypsin-like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS-PAGE gels. Taken together, these data demonstrate the presence of trypsin-like activity and several trypsin-like proteins in the Culex male accessory glands.

Ligand binding modulates the structural dynamics and activity of urokinase-type plasminogen activator: A possible mechanism of plasminogen activation.

The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.

Protease/protease inhibitor balance in blood plasma to predict postoperative complications in operated patients with pancreatic head cancer.

258 Background: Our purpose was to study the protease/protease inhibitor (P/PI) balance in the blood plasma of patients with cancer of the head of the pancreas before and after pancreatoduodenal resection (PDR) with postoperative complications. Methods: The study was performed using clinical observation, biochemical examinations and statistical analysis in Microsoft Office Excel 2010. The blood plasma of 92 patients with pancreatic head cancer (53 men and 39 women aged 45-76 years, Т2-4N0M0) was studied before the surgery (b/s) and on days 1, 7, 14 and 17 after PDR. The patients were divided into two groups: g1 – 69 patients without postoperative (p/o) complications and g2 – 23 patients with p/o complications: generalization – 7, thrombosis - 8, acute postoperative pancreatitis - 2, gastrostasis - 2, anastomotic leakage - 4 patients. Kinetics of trypsin-like proteases (TLP) and α-1-proteinase inhibitor (α1PI) was studied by spectrophotometry. The data were compared with the blood plasma of 39 healthy donors (N). Results: TLP activity b/s exceeded N in g1 and g2 by 4.1 and 10.6 times; TLP in g2 was 2.6 times higher than in g1. The α1PI activity b/s was higher than N by 1.2 times (p < 0.05) in g1 and lower than N by 2.0 times in g2; α1PI in g2 was 2.4 times lower than in g1. After PDR, activity of TLP increased in all patients on day 1 but decreased on days 7-14 in g1 remaining 2.7 times higher than N by the discharge. The TLP activity in g2 by the discharge was similar to levels b/s and exceeded g1 by 4.4 and N by 12.1 times. The α1PI activity after PDR increased in all patients on days 1-17, but in g1 by the discharge it was similar to N and in g2 it was 1.5 times lower than N. The TLP/α1PI ratio was higher in g2 than in g1 at all times. Conclusions: A high TLP activity and a low α1PI activity, compared to N, were maintained in the blood plasma of all patients with p/o complications, despite their types. The P/PI balance in g2 was shifted to the left being 5.5-9.0 times higher than in g1 at all times which allowed the prognosis of postoperative complications before the surgery, perioperatively or on day 1 after the surgery.

37 Trypsin-like protease activity predicts disease severity and patient mortality in adults with cystic fibrosis

37 Trypsin-like protease activity predicts disease severity and patient mortality in adults with cystic fibrosis

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.

Регуляція тіаміном активності трипсиноподібних ферментів в тканинах білих щурів

The effect of intramuscular thiamine injection on the activity of trypsin-like enzymes in the liver, kidneys, stomach and small intestine of white rats has been researched. In the organs of intact rats, the maximum activity of trypsin-like proteases has been established in the small intestine, and the minimum one in the liver. Thiamine parenteral administration leads to the decrease of trypsin-like protease activity in the liver, kidney and stomach, and the increase of the enzyme activity in the small intestine that suggests the possibility of non-coenzyme thiamine effect on the trypsin-like enzyme activity.

Targeting NF-κB with First in Class N-Quinoline Benzenesulfonamides (NQBS) Reveals Potent Activity in Models of Diffuse Large B-Cell Lymphoma (DLBCL)

The first two authors contributed equally to this workIdentifying pharmacologic strategies to inhibit the activation of NF-κB and its target genes has been a major research pursuit. To date, no direct inhibitors of the NF-κB subunits have been explored in the clinic. Based on the constitutive activation of NF-κB in diffuse large B-cell lymphoma (DLBCL), we used this disease model to develop drugs targeting NF-κB. Using a fluorescence-based high throughput screening (HTC) approach, a unique N-quinoline-benzenesulfonamide (NQBS) scaffold was identified as potential small molecule inhibitor of the NF-κB pathway.A confocal microscopy based HTC assay performed in human umbilical vein endothelial cells (HUVEC) identified hit compounds that contained a unique NQBS core structure. The assay screened for compounds that inhibited nuclear translocation of NF-κB subunits in TNFα-induced HUVEC cells. To date over 100 NQBS analogs have been synthesized with varying potency and cytotoxicity in inhibiting growth of DLBCL lines (OCI-Ly10, RIVA, HBL-1 and OCI-Ly3).Cytotoxicity assays demonstrated that the most potent compounds exhibit IC50s in the 0.5 to 1.5 µM range. These most potent NQBS analogs identified as CU-O42 CU-O47 and CU-O75 were also able to induce apoptosis and caspase activation. Apoptosis was preceded by exclusion of the NF-κB proteins from the nucleus.To analyze the localization of NF-κB proteins within the cell compartments before and after the treatment with CU-O42, CU-O47 and CU-O75, we used confocal microscopy, electromobility shift (EMSA) and ELISA assays. Control cells tested positive for p50/p65 both within the cytoplasm and the nucleus. Following treatment with CU-O42 NF-κB was sequestered within the cytoplasm of the cell which occurred as early as 3h after exposure. In addition, all three analogs reduced the nuclear levels of NF-κB in a concentration-dependent manner when measured by EMSA and ELISA.Furthermore, CU-O47 and CU-O75 were able to inhibit TNFα induced luciferase expression in a HEK293T cell model where luciferase is controlled by an NF-κB promoter. A KINOMEscan platform (examining the activity of over 450 different kinases) showed that no NQBS analog screened (CU-O42 and CU-O75) inhibited any of the kinases in the assay. In addition, a proteasome inhibition assay tested negative for trypsin-like and chromotrypsin-like protease activity (CU-O42, CU-O47 and CU-O75).Stabilization of the inactive trimer of p50, p65 and IκBα was hypothesized as a potential mechanism of action of CU-O42 and CU-O75 through Internal Coordinate Mechanics (ICM) software. This binding hypothesis was further corroborated by cellular thermal shift assays (CETSA) with an increase of the IκBα melting temperatures (2.5-3°C) in whole cell lysates following rapid (30min) exposure to CU-O42 and CU-O75. Using a genome-wide regulatory network perturbation analysis (DeMAND) based on the RNA-Seq data collected from OCI-Ly10 cells treated with CU-O75, we identified IκBα as one of the potential targets of the compounds. Gene set enrichment analysis demonstrated NF-κB target gene downregulation using IC20 of CU-O75 at 24h (p=0.045).In vivo experiments were conducted in two models: (1) xenografts with human DLBCL cell lines of both ABC and GC subtype; and (2) myc cherry luciferase mouse model where mice spontaneously develop aggressive lymphomas. In both models, CU-O42 was able to inhibit tumor growth. Interestingly, in the xenograft model, malignant cell growth was inhibited in both ABC (HBL-1) and GC (OCI-Ly1) cells when compared to controls (p=0.01 and p=0.02). However, overall survival of mice with ABC xenografts treated with CU-042 significantly exceeded the survival of mice with GC xenografts (p<0.01) suggesting a more sustainable response in this subtype of disease, consistent with its dependency on NF-κB.Identification of a unique NQBS scaffold has led to the chemical synthesis of over 100 structural analogs with a potent inhibition on NF-κB nuclear translocation. They display potent activity across a panel of lymphoma cell lines, producing a survival benefit in mice implanted with an ABC-subtype of lymphoma. ICM, CETSA and DeMAND suggest that this is a direct effect mediated on the proteins within the p65/p50/IκBα complex. These findings point to a novel mechanism of action and warrant further research into potential clinical translation of this class of small molecules. DisclosuresCalifano:Thermo Fischer Scientific: Consultancy; Ipsen pharmaceuticals: Consultancy; Cancer Genetics Inc: Consultancy; Therasis Inc: Employment. O'Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria, Research Funding; Acetylon Pharmaceuticals, INC: Consultancy.

Identification of Novel Proteasome Inhibitors from an Enaminone Library.

A library of structurally distinct enaminones was synthesized using sonication or Ru(II) catalysis to couple primary, secondary, and tertiary thioamides with α-halocarbonyls or α-diazocarbonyls. Screening the library for proteasome inhibition using a luciferase-based assay identified seven structurally diverse compounds. Two of these molecules targeted luciferase, while the remaining five exhibited varying potency and specificity for the trypsin-like, chymotrypsin-like, or caspase-like protease activities of the proteasome. Physiological relevance was confirmed by showing these molecules inhibited proteasomal degradation of the full-length protein substrate p21cip1 expressed in tissue culture cells. A cell viability analysis revealed that the proteasome inhibitors differentially affected cell survival. Results indicate a subset of enaminones and precursor molecules identified in this study are good candidates for further development into novel proteasome inhibitors with potential therapeutic value.

Hepatocyte growth factor activator inhibitor type-1 in cancer: Advances and perspectives (Review)

Cancer is one of the most common diseases, with high morbidity and mortality rates. Large‑scale efforts have been made to understand the pathogenesis of the disease, particularly in the advanced stages, in order to develop effective therapeutic approaches. Hepatocyte growth factor activator inhibitor type-1 (HAI-1), also known as serine protease inhibitor Kunitz type 1, inhibits the activity of several trypsin-like serine proteases. In particular, HAI-1 suppresses hepatocyte growth factor (HGF) activator and matriptase, resulting in subsequent inhibition of HGF/scatter factor and macrophage‑stimulating protein (MSP). HGF and MSP are involved in cancer development and progression, via the receptors Met receptor tyrosine kinase (RTK) and Ron RTK, respectively. Therefore, HAI-1-mediated downregulation of HGF and MSP signaling may suppress tumorigenesis and progression in certain types of cancers. Abnormal HAI-1 expression levels have been observed in various types of human cancer. The exact function of HAI-1 in cancer pathogenesis, however, has not been fully elucidated. In this review, the focus is on the potential impact of aberrant HAI-1 expression levels on tumorigenesis and progression, the underlying mechanisms, and areas that require further investigation to clarify the precise role of HAI-1 in cancer.

The Inhibition of Acetylshikonin on Bacteria and its Trypsin-like Protease and Glycosidic Enzyme may be Essential to Conquer Periodontal Ecological Niche

Background/Purposes: The objective of this study was to evaluate the inhibitory effects of acetylshikonin on bacteria in periodontal disease and the activities of trypsin-like protease and glycosidic enzyme of P. gingivalis in vitro. Methods: The inhibition activity of acetylshikonin against bacteria was identified by microbial sensitivity tests of broth dilution method on 96-microwell plate. Trypsin-like protease and glycosidic enzyme activities were measured by a fluorescence spectrophotometer. Results: The minimum inhibitory concentration of acetylshikonin on gram-negative were found to be 0.0625mg/ml, 0.125mg/ml, 0.125mg/ml, 0.25mg/ml, 0.25mg/ml and 0.5mg/ml, for S. intermedius, P. micros, P. acnes, L. acidophilus, A. viscosus and E. lentum respectively. Acetylshikonin showed extensive antibacterial activity against gram-negative bacteria, and it was more active against gram-positive bacteria. The acetylshikonin inhibited the activities of both trypsin-like protease and glycosidic enzyme by 45% and 95% with the final concentration of acetylshikonin from 0.0195 to 2.5 mg/ml. Conclusion: The inhibition activity of acetylshikonin on bacteria and its enzyme of trypsin-like protease and glycosidic may be essential to conquer periodontal ecological niche for treating periodontal disease.

The effect of tacrolimus compared with betamethasone valerate on the skin barrier in volunteers with quiescent atopic dermatitis

Atopic dermatitis (AD) is an inflammatory skin disease arising as a result of immune system and skin barrier defects. Topical corticosteroids are safe and effective treatments for AD, when used in short courses. Prolonged use is associated with skin barrier damage. Topical calcineurin inhibitors are alternative immune-modulating treatments for AD purported to have no negative effects on the skin barrier. To compare the effects of betamethasone valerate 0·1% cream (BMVc) and tacrolimus 0·1% ointment (TACo) on the skin barrier. Twenty volunteers with quiescent AD (no active signs for 6months) participated in a randomized observer-blind study, wherein BMVc was applied to one forearm and TACo to the other, twice daily for 4weeks. The biophysical/biological properties of the stratum corneum were assessed before and after treatment. Nine volunteers with active disease and 10 with healthy skin were assessed at untreated sites. BMVc significantly reduced skin barrier function, integrity and cohesion, and the levels of pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA) towards the subclinical barrier defect observed in patients with AD (nonlesional sites). TACo preserved skin barrier function, integrity, cohesion and PCA and UCA levels, while significantly increasing skin hydration to levels comparable with healthy skin. Both treatments reduced skin surface pH and trypsin-like protease activity, with TACo doing so to a significantly greater degree. In quiescent AD, 4weeks of BMVc treatment adversely affected the biophysical properties of the skin and reduced the levels of natural moisturizing factor, whereas TACo improved the condition of the skin barrier.

Dysregulation of ubiquitin-proteasome pathway and apolipoprotein A metabolism in sickle cell disease-related pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a major complication of sickle cell disease (SCD). Low levels of apolipoprotein A1 (Apo-A1) have been implicated in the development of PAH in SCD. We speculate that lower levels of Apo-A1 are related to dysregulation of the ubiquitin-proteasome pathway (UPP). Of 36 recruited patients with SCD, 14 were found to have PAH on the basis of right heart catheterization. Levels of Apo-A1 and Apo-B, polyubiquitin, total protease, and specific and normalized activity of chymotrypsin-like, trypsin-like, and caspase-like proteases in plasma were measured. Levels of Apo-A1 were found to be lower and polyubiquitin levels were found to be significantly higher in the PAH group ([Formula: see text]) in SCD. Apo-A levels were inversely correlated with polyubiquitin levels ([Formula: see text], [Formula: see text]). These results indicate that lower levels of Apo-A1 in SCD patients with PAH are likely related to enhance degradation by UPP, potentially contributing to pulmonary vascular pathology. These findings may provide significant insight in identifying suitable therapeutic targets in these patients.

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity.

Impairment of the Ubiquitin-Proteasome Pathway by Methyl N-(6-Phenylsulfanyl-1H-benzimidazol-2-yl)carbamate Leads to a Potent Cytotoxic Effect in Tumor Cells: A NOVEL ANTIPROLIFERATIVE AGENT WITH A POTENTIAL THERAPEUTIC IMPLICATION

In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.

Role of Proteases in Extra-Oral Digestion of a Predatory Bug,Andrallus spinidens

Roles of salivary proteases in the extra-oral digestion of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) were studied by using 2% azocasein as a general substrate and specific protease substrates, as well as synthetic and endogenous inhibitors. It was found that salivary glands of A. spinidens have two anterior, two lateral, and two posterior lobes. Azocasein was used to measure the activity of general proteases in the salivary glands using different buffer solutions. The enzyme had the highest activity at pH 8. General protease activity was highest at 40 °C and was stable for 6–16 hours. The use of specific substrates showed that trypsin-like, chymotrypsin-like, aminopeptidase, and carboxypeptidase are the active proteases present in salivary glands, by the maximum activity of trypsin-like protease in addition to their optimal pH between 8–9. Ca2+ and Mg2+ increased proteolytic activity about 216%, while other ions decreased it. Specific inhibitors including SBTI, PMSF, TLCK, and TPCK significantly decreased enzyme activity, as well as the specific inhibitors of methalloproteases including phenanthroline, EGTA, and TTHA. Extracted endogenous trypsin inhibitors extracted from potential prey, Chilo suppressalis, Naranga aenescens, Pieris brassicae, Hyphantria cunea, and Ephestia kuhniella, had different effects on trypsin-like protease activity of A. spinidens salivary glands. With the exception of C. suppressalis, the endogenous inhibitors significantly decreased enzyme activity in A. spinidens.

Artemia 공급 단계에서 은어(Plecoglossus altivelis), 조피볼락(Sebastes schlegeli ), 감성돔(Acanthopagrus schlegeli ) 및 넙치(Paralichthys olivaeus)의 소화효소 활성

We compared the nutritional requirements of whole larvae of the black seabream Acanthopagrus schlegeli, sweet fish Plecoglossus altivelis, olive flounder Paralichthys olivaeus and rock fish Sebastes schlegeli. The larvae were 20, 30, 14 and 5 DAH (or spawning) of black seabream, sweet fish, olive flounder and rock fish, respectively. Specific <TEX>${\alpha}$</TEX>-amylase activity (mU/mg protein) was highest (8,324.9 mU/mg protein) in rock fish larvae (P<0.05). Specific trypsin-like protease activity was highest (11,330.1 mU/mg protein) in black seabream larvae (P<0.05), which also exhibited the highest activity, 685.5 mU/mg dry weight (P<0.05). The specific activities per mg protein and mg dry weight of black seabream were the highest (187.4 mU/mg protein and 11.3 mU/mg dry weight, respectively) (P<0.05). A/P, P/L and A/L ratios of rock fish were 1.47, 90.3 and 133.1, respectively (P<0.05). We present here basic larval digestive enzymatic nutritional requirement data.

Trypsin‐like proteolytic contamination of commercially available psa purified from human seminal fluid

Prostate-Specific Antigen (PSA) is a serine protease whose expression is maintained in all stages of prostate cancer. A role for PSA in the pathobiology for prostate cancer has not been firmly established. Experimental studies to date support a role for PSA through mechanisms such as release or processing of growth factors and degradation of the extracellular matrix. Exposure of prostate cancer cells to exogenous PSA also results in gene expression changes. These in vitro and biochemical assays rely on the use of commercially available PSA. Contamination of these commercial preparations can significantly impact the results of these in vitro studies. We characterized PSA and trypsin-like activity of PSA preparations obtained from three commercial sources: Calbiochem, Fitzgerald, and AbD Serotec. Silver stained gels were used to compare the purity of each preparation and mass spectrometry was performed to characterize contaminating proteases. PSA activity varied between PSA preparations with AbD Serotec PSA having highest degree of activity. Significant trypsin-like activity, which was inhibited by aprotinin, was observed in PSA preparations from Calbiochem and Fitzgerald, but not AbD Serotec. These former two PSA preparations also contained the greatest degree of non-PSA contaminants by silver stain and mass spectrometry. Commercially available preparations of PSA contain contaminating proteins, including trypsin-like protease activity, that could potentially complicate the interpretation of results obtained from in vitro studies assessing PSA proteolysis of potential protein substrates and effects of PSA on gene expression.

Plasmin: Its Role in the Extracellular Processing of Progalanin in Tumor Tissue

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. In a previous study, we showed that a small cell lung carcinoma (SCLC) cell line, SBC-3A, released progalanin but not galanin, and that progalanin was then converted to galanin(1-20), the active form. Because the galanin(1-20) had undergone hydrolysis at Arg and Lys residues, the protease concerned was surmised to have a trypsin-like activity. The present study was performed to identify the trypsin-like protease which had previously been found to activate progalanin in this tumor tissue. The protease was isolated using chromatography and electrophoresis, and identified in tumor extracts from SBC-3A tumor-bearing mice; the major protease was found to be plasmin. We next confirmed that extracellular processing of progalanin occurs in SCLC tumor tissue (tumors produced by the implantation of SBC-3A cells into mice), and in two types of breast tumor tissue (obtained by implantation into mice of BT-549 and MDA-MB-436 cells). In cell culture, processed forms of progalanin were undetectable in SBC-3A, BT-549 or MDA-MB-436 cells. Conversely, gel filtration chromatography analysis of tumor extracts from SBC-3A, BT-549 and MDA-MB-436-bearing mice, revealed that galanin-like immunoreactivity (galanin-LI) in these tumor extracts was due to the presence of progalanin (14 kDa) and galanin(1-20) (2 kDa). Moreover, trypsin-like protease activity was elevated, and plasmin was expressed abundantly in SBC-3A, BT-549 and MDA-MB-436 tumors in mice. In addition, tranexamic acid, a plasmin inhibitor, inhibited progalanin conversion to galanin(1-20). The present study revealed that plasmin was present in tumor tissue, and that it was responsible for processing progalanin to galanin(1-20) in the extracellular environment.