Cell transformation induced by hepatitis C virus NS3 serine protease.

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356-4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.

Comparison of a vegetable-based (soya) and an animal-based low-protein diet in predialysis chronic renal failure patients.

There is some experimental evidence to suggest that progression of chronic renal failure (CRF) is slower on diets based on soya protein than on diets based on animal protein. We have compared the effect of a soya-based vegetarian low-protein diet (VPD) and an animal-based low-protein diet (APD) in 15 patients with CRF. 15 patients with CRF (51Cr-EDTA-measured glomerular filtration rate 15-50 ml/min/1.73 m2) were studied. In a randomized crossover trial, the patients were given each diet (each containing 0.75 g protein and 32 kcal per kilogram body weight) for a 6-month period. Nine patients completed the trial, 2 others dropped out because they could not tolerate the VPD, 3 because of unrelated medical complications, and 1 for technical reasons. The caloric intake was higher and the protein, phosphate and essential amino acid intake lower on the VPD than on the APD. The compliance with the suggested caloric intake was better with the VPD than with the APD (97 vs. 88% of recommended intake), as was the compliance with the suggested protein intake (94 vs. 112% of recommended intake) and with the suggested phosphate intake (102 vs. 116%). The mean glomerular filtration rate, as judged by 51Cr-EDTA, was similar after 6 months on each diet and remained unchanged throughout the entire year of the study. The rate of fall of glomerular filtration, as measured by the slope of 1/serum creatinine was slowed by 73% during the 1-year study period as compared with the prestudy period. Nutritional status (as measured by body mass index, midarm circumference, and lean body mass and percent body fat), serum transferrin, cholesterol and albumin, and total lymphocyte count were similar on the two diets. The serum albumin level on both diets, however, was significantly higher on the two diets than during the prediet period. Blood urea nitrogen, urine urea nitrogen, protein catabolic rate, and 24-hour urine creatinine and phosphate were lower on the VPD than on the APD. The 24-hour protein excretion was similar on the two diets. The two low-protein diets resulted in a slowing in the progression of CRF. A VPD is well tolerated in CRF and is associated with lower protein and phosphate intakes and a higher caloric intake than an APD and may, therefore, be used as a safe alternative or partial substitute for the usual APD in CRF.

Protein proteinase inhibitors in legume seeds--overview.

Protein proteinase inhibitors are widely distributed in plant seeds, particularly in legumes. The specificity and potency of inhibition depend on defined inhibitory sites and on the animal species of the target proteinase. Feeding experiments on diets containing isolated soybean trypsin inhibitors (the Kunitz soybean trypsin inhibitor STI and the Bowman-Birk trypsinchymotrypsin inhibitor BBI) caused insignificant growth depression in rats and chicks, but induced enlargement of the pancreas in rats, chicks and mice but not in pigs, dogs, calves, monkeys and presumably humans. The trypsin-inhibitory site has been responsible for induction of the pancreatic enlargement. The trypsin-chymotrypsin inhibitors from soybeans and from chickpeas inhibit insect midgut proteinases, supporting the hypothesis that proteinase inhibitors comprise a built-in defense mechanism of the seed against insects. Findings on the involvement of proteinase inhibitors, such as BBI, in prevention of tumorigenesis suggest a possible positive contribution of the inhibitors to the nutritional value of legume seeds. BBI is also an effective inhibitor of nephrotoxicity induced by the antibiotic gentamicin. BBI does not cause side effects and does affect the antimicrobial activity. The in vitro effects of proteinase inhibitors on animals should be interpreted with caution when related to humans.

Increased urinary trypsin-inhibitory activity in mercuric chloride induced nephrotoxicity in Wistar rats.

UNLABELLED: The relationship between trypsin-inhibitory activity (TIA) and the nephrotoxic effects of mercuric chloride (HgCl2)--as illustrated by proteinuria and by a drop in the glomerular filtration rate (GFR) measured by creatinine clearance test (CCT)--was investigated in Wistar rats. HgCl2, 150 or 250 micrograms/100 g BW per day was injected intraperitoneally three times a week for 2 weeks. Both groups showed a significant degree of proteinuria and urinary TIA. Group B (250 micrograms HgCl2/100 g BW) displayed a greater drop in GFR than group A (150 micrograms HgCl2/100 g BW). The urinary TIA was significantly correlated with proteinuria (group A: r = 0.87, group B: r = 0.84), but it was also significantly inversely correlated with the CCT (A: r = -0.96; B: r = -0.88). IN CONCLUSION: these results suggest that increased urinary TIA may be involved in and indicative of the pathogenesis of mercuric chloride induced nephrotoxicity.

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds.

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth--a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkaline pH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.

Antimicrobial gentamicin activity in the presence of exogenous protease inhibitor (Bowman-Birk inhibitor) in gentamicin-induced nephrotoxicity in rats.

In our previous studies, we found increased levels of urinary trypsin inhibitory activity in gentamicin-induced nephrotoxicity in rats. Following administration of the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI), no proteinuria was detected in gentamicin-treated rats, and a decrease in creatinine clearance was noted in only 50% of the injected rats. In the present study, we examined the antimicrobial activity of gentamicin against Escherichia coli in the presence of BBI in gentamicin-induced nephrotoxicity in rats. We found that 50% of rats with E. coli-positive blood cultures died of septicemia. All the rats injected with E. coli plus gentamicin or E. coli plus gentamicin plus BBI survived, the latter showing no proteinuria or deterioration in creatinine clearance. In conclusion, BBI, which is an effective inhibitor of gentamicin-induced nephrotoxicity, does not affect the antimicrobial activity of gentamicin sulfate.

Effect of interaction between gentamicin and pyridoxal-5-phosphate on functional and metabolic parameters in kidneys of female Sprague-Dawley rats.

Daily intraperitoneal (i.p.) injection of genatmicin at a dose of 70 mg/kg for 11 days produced nephrotoxicity in female Sprague-Dawley rats as evidenced by increased excretion of urinary protein and trypsin inhibitory activity as well as rise in renal individual class and total phospholipid. The observed proteinuria was associated with a significant twofold fall in creatinine clearance and histopathological changes, including the presence of hyaline casts and flattened epithelial cells within the lumen of proximal convoluted tubules. Although pyridoxal-5-phosphate (50 mg/kg) administered i.p. did not significantly alter creatinine clearance, histopathology, proteinuria, and urinary trypsin inhibitory activity, an increase in individual class and total phospholipid was noted in kidney. In rats simultaneously administered gentamicin and pyridoxal-5-phosphate, the observed fall in renal gentamicin content was associated with a return of individual class and total phospholipid to control values. However, the decline in creatinine clearance, enhanced proteinuria, and increase in urinary trypsin inhibitory activity in the simultaneous-treated group was similar or greater than that seen in the gentamicin-only injected rats. Morphological examination of simultaneous-treated rats revealed extensive alterations in proximal tubules including numerous mitotic figures, large vesicular nucleii, and prominent nucleoli in epithelial cells as well as hyaline casts within the lumen. Our data combined with results of previous studies suggest that sex and type of rat strain are important factors in aminoglycoside-induced nephrotoxicity. It is evident that a specific concentration of pyridoxal-5-phosphate may be necessary to provide protection against all manifestations of aminoglycoside-induced renal damage.

Purification and characterization of trypsins from the digestive tract of Locusta migratoria.

Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.

Growth, digestibility, and enzyme activities in the pancreas and intestines of guinea-pigs fed on raw and heated soya-bean flour.

The nutritional effects of giving raw (RSF) or heated (HSF) soya-bean flour to young guinea-pigs were investigated in trials 1 and 2, in which the levels of dietary protein were 120 and 190 g/kg diet respectively. The growth rate of animals fed on RSF was lower than that of those fed on HSF. Growth retardation of guinea-pigs fed on RSF was accompanied by a lower apparent digestibility of the protein (0.49-0.53) compared with HSF (0.67-0.76) and lower food conversion efficiency. In RSF-fed animals, increasing dietary protein affected growth and food conversion efficiency negatively. The pancreas of animals fed on RSF and HSF was similar in weight but secreted less trypsin, chymotrypsin and amylase, in RFS-fed animals. It was concluded that the mechanism by which raw soya-bean negatively affects the growth rate of guinea-pigs by reducing the activity of intestinal enzymes, differs from that suggested for rats and chicks, but is similar to that of pigs and calves.

Purification and characterization of Locusta migratoria chymotrypsin.

A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.

Enhanced urinary trypsin inhibitory activity in gentamicin-induced nephrotoxicity in rats.

We delineated in rats, the relationship between trypsin inhibitory activity in the urine and the nephrotoxic effects of gentamicin, eg, proteinuria and deterioration of glomerular filtration rate (GFR), measured by creatinine clearance. Gentamicin, 70 mg/kg per day, was injected intraperitoneally for 6-10 successive days. Serum and urine gentamicin levels were determined by a microbiological test. Trypsin inhibitory activity was assayed by the casein digestion method. The results showed a steady increase in urinary trypsin inhibitory activity starting from the fourth injection day. The increased levels of urinary trypsin inhibitory activity were associated with increased levels of urinary gentamicin excretion (r = 0.36, p less than 0.02, n = 50 after the fourth injection day), and were significantly higher than in control groups (p less than 0.001). The urinary trypsin inhibitory activity was inversely correlated with the GFR (r = -0.45, p less than 0.01, after the second injection day). The serum trypsin inhibitory activity remained unchanged throughout the study period in all groups. These data suggest that increased urinary trypsin inhibitory activity may be involved in the pathogenesis of gentamicin-induced nephrotoxicity.

Photoreactive, active derivatives of trypsin and chymotrypsin inhibitors from soybeans and chickpeas.

The photoreactive arylsufenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (STI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-STI indicated that only one of the two tryptophans, 93 or 117, present in STI was modified. Amino acid analysis of the two separated CNBr-cleavage products of 2,4-NAPS-STI showed that only tryptophan 93 underwent modification. 2,4-NAPS-STI fully retained its inhibitory activity against trypsin. The covalent attachment of 2,4-NAPS-STI to tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride. Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas were prepared by selective modification of the epsilon-amino groups of 2,4(5)-NAPS-Cl. The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues.

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

Nanomolar concentrations of Bowman-Birk soybean protease inhibitor suppress x-ray-induced transformation in vitro.

Experiments reported here indicate a crude soybean extract, if defatted with acetone, effectively blocks cell transformation in vitro. An active component of this crude extract is the Bowman-Birk trypsin and chymotrypsin inhibitor. The chymotrypsin-inhibitory region of the Bowman-Birk inhibitor is responsible for suppressing in vitro transformation. Another low molecular weight soybean trypsin inhibitor does not significantly suppress transformation. The Bowman-Birk inhibitor (i) has an irreversible effect on the transformation process, (ii) can suppress radiation-induced transformation even when added to cultures many days after the carcinogen exposure, and (iii) is effective in its ability to suppress transformation when present in the medium at a concentration as low as 0.125 nM.

Protease inhibitors in sera as possible indicators of familial hypothyroidism.

Both synthesis and degradation of proteins are reduced in the hypothyroid state. The possible involvement of serum trypsin and chymotrypsin inhibitors has been studied in a family that, for four successive generations, had clinical or subclinical hypothyroidism. Increased trypsin- and chymotrypsin-inhibiting activity was demonstrated in the sera of the four clinically hypothyroid women. Cellulose acetate electrophoresis of the sera of these patients and of two other subclinical hypothyroid family members disclosed the distinct appearance of an additional fraction in the alpha 2-globulin zone. The serum protein electrophoretic pattern changes observed in familial hypothyroidism might be genetically determined. Such changes could precede the clinical onset of the disease, thus serving as a possible indicator of the hypothyroid state.

The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans.

Four decades of studies on the isolation, characterization, properties, structure, function and possible uses of the Bowman-Birk trypsin- and chymotrypsin-inhibitor from soybeans are reviewed. Starting from Bowman's Acetone Insoluble factor, designated Ai, AA and SBTIAA, the Bowman-Birk inhibitor (BBI) was found to be a protein molecule consisting of a chain of 71 amino acids cross linked by 7 disulfide bonds, with a tendency to self-associate. BBI possesses two independent sites of inhibition, one at Lys 16-Ser 17 against trypsin and the other at Leu 43-Ser 44 against chymotrypsin. It forms a 1:1 complex with either trypsin or chymotrypsin and a ternary complex with both enzymes. Ingestion of BBI by rats, chicks or quails affects the size and protein biosynthesis of the pancreas. Establishment of the full covalent structure of BBI revealed a high homology in the sequences around the two inhibitory sites, suggesting evolutionary gene duplication from a single-headed ancestral inhibitor. Scission of BBI by CNBr followed by pepsin results in two active fragments, one that inhibits trypsin and the other, chymotrypsin. Replacements and substitutions in the reactive sites result in changes in inhibitory activity and in specificity of inhibition. Conformation studies, labeling of BBI with a photoreactive reagent, chemical synthesis of cyclic peptides that include inhibitory sites, in vitro synthesis of BBI, and species specificity regarding the inhibited enzymes are described. The significance of BBI as a prototype of a family of inhibitors present in all legume seeds is discussed.

Selective broadcast interconnection: a novel scheme for fiber-optic local-area networks.

We introduce a passive, unswitched scheme for directly interconnecting N stations, each of which has C transmitters and receivers. Implementations using fiber optics with spatial multiplexing and optionally wavelength multiplexing are discussed. This scheme utilizes the same resources as standard topologies with C parallel buses but outperforms them in two respects: (1) the aggregate throughput is proportional to C(2) rather than to C and (2) the power of each transmitter need reach only N/C, instead of N, receivers.

Photoreactive derivative of Kunitz's soybean trypsin inhibitor. Preparation by selective modification of a tryptophan residue and formation of a covalent complex of the modified inhibitor with trypsin.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.