Soybean Trypsin-chymotrypsin Inhibitor Research Articles

Assessment of soybean inhibitor as a biopesticide against melon fruit fly, Bactrocera cucurbitae (Coquillett)

In the current study, the soybean trypsin–chymotrypsin inhibitor (Bowman–Birk Inhibitor, SBBI) was tested against Bactrocera cucurbitae (Coquillett), a major pest of cucurbit crops. Bioassays conducted using different concentrations (12.5, 25, 50, 100 and 200 ppm) revealed a detrimental effect of the inhibitor on the growth and development of the second instar larvae of the melon fruit fly. SBBI prolonged the larval and total development period and reduced the percentage pupation and emergence. Enzymatic assays of proteases conducted at three time intervals using the LC40 (59 ppm) concentration of SBBI showed an inhibitory effect on trypsin activity, whereas an increase was observed in the activity of chymotrypsin, elastase and leucine aminopeptidase. Among the enzymes involved in detoxification, antioxidant and general metabolism, an increase was observed in the activity of catalases, and acid and alkaline phosphatases at most treatment intervals. The activity of esterases was induced only with prolonged treatment whereas that of glutathione S-transferases was suppressed in larvae treated with SBBI. The findings revealed the potential of SBBI to disrupt the growth of the melon fruit fly.

Induction of multicellular 3‐D spheroids of MCF‐7 breast carcinoma cells by neutrophil‐derived cathepsin G and elastase

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF-7 human breast carcinoma cells adhering to culture substrates. When MCF-7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N-formyl-Met-Leu-Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3-D spheroids formed when the cultures were prolonged. The spheroid-inducing reaction was reversible and energy-dependent. The MCF-7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E-cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti-E-cadherin antibody following pipetting. The aggregation-inducing activity in neutrophils was completely inhibited by soybean trypsin-chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF-7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites.

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula)

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1–3), LHRH (1–4) and LHRH (1–5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn(2+) or Ni(2+)) the role of which is unknown. In the present work we find that neither Zn(2+) nor Ni(2+) is required for DNase activity, which instead requires Mg(2+) ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn(2+), Ni(2+), or Co(2+), the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn(2+) was less than or equal to nanomolar, while that for Co(2+) and Ni (2+) was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.

Identification of factors from rat neutrophils responsible for cytotoxicity to isolated hepatocytes

Activated polymorphonuclear neutrophils (PMNs) have been shown to be cytotoxic to rat hepatic parenchymal cells in vitro. This cytotoxicity could be observed without direct cell-cell contact, since the conditioned medium from PMNs activated with formyl-Met-Leu-Phe (fMLP) was effective in hepatocyte killing. To identify the toxic factor(s) released by PMNs, degranulation was induced by fMLP in PMNs pretreated with cytochalasin B. The contents released from the phagocytes were subjected to gel filtration on a Sephadex G-100 column. Resulting fractions were tested for cytotoxicity to isolated hepatocytes by using release of alanine aminotransferase as a marker for hepatocyte injury. Cytotoxicity was associated with fractions containing cathepsin G and elastase and not with other fractions, including those containing myeloperoxidase. The former two enzymes were purified to homogeneity with a carboxymethyl cellulose column. Each of these enzymes demonstrated concentration-dependent cytotoxicity to hepatocytes at concentrations > 2 microgram/mL. Moreover, they exhibited an additive cytotoxic effect. Effective concentrations for the combined cathepsin G and elastase in the incubation mixture were similar to the concentrations of these enzymes in PMN-conditioned medium that produced cytotoxicity to hepatocytes. Cytotoxicity of either purified enzyme or of conditioned medium could be prevented by plasma alpha-1-antitrypsin or soybean trypsin-chymotrypsin inhibitor, which were also potent inhibitors of enzymic activity of both cathepsin G and elastase. By contrast, the serine protease inhibitors, aprotinin and 4-(2-aminoethyl)-benzene-sulfonyl fluoride, were less effective in inhibiting cathepsin G and elastase activities as well as cytotoxicity caused by the purified proteases or PMN-conditioned medium. These results support the hypothesis that cathepsin G and elastase are important mediators of hepatic parenchymal cell killing produced by activated PMNs in vitro.

Mechanisms Involved in the Induction of Human Endothelial Cell Necrosis

The effects of the inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF) and unstimulated and activated neutrophils (PMNs) on endothelial cell (EC) necrosis were studied using the cultured human EC line (ECV-304) and human PMNsin vitro.LPS and TNF alone or their combination failed to induce EC necrosis. Activated PMNs, as evidenced by augmentations in CD11b expression and respiratory burst, induced significant EC necrosis commencing at 12 hr of coculture, which was strongly dependent on the ratio of PMN:ECs and the duration of PMN:EC coculture. In contrast, unstimulated PMNs induced no significant increases in EC necrosis. To examine the mechanisms of activated PMN-mediated EC necrosis, the oxygen radical scavengers superoxide dismutase (SOD) and catalase, as well as the protease inhibitors phenylmethysulfonyl fluoride (PMSF), α1-antitrypsin (α1-AT), soybean trypsin–chymotrypsin inhibitor (TCI), and aprotinin, were studied in coculture experiments. EC necrosis induced by activated PMNs could be markedly attenuated by SOD, PMSF, α1-AT, TCI, aprotinin, or their combinations. Although aprotinin enhanced respiratory burst, this agent inhibited necrosis by downregulating PMN CD11b and PMN–EC adhesion. These results demonstrate that the inflammatory mediators LPS and TNF and quiescent PMNs fail to induce EC necrosis. However, PMNs activated by inflammatory mediators can induce EC necrosis through oxidative and nonoxidative mechanisms and this process is dependent on PMN–EC adhesion.

Phosphorylation of the Transcription Factor NFATp Inhibits Its DNA Binding Activity in Cyclosporin A-treated Human B and T Cells

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the α subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).