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.

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.

A prospective study of some diarrhoeagenic Escherichia coli in infants with diarrhoea in Mosul, Iraq.

The incidence of diarrhoeagenic Escherichia coli was investigated in 304 infants with diarrhoea in Mosul, Iraq by using standard biological assays and reversed passive latex agglutination (RPLA) procedures. Enterotoxigenic E. coli (ETEC) were found in 12.8% of the cases--27 (8.8%) strains produced heat-labile toxin (LT) only, 8 (2.6%) heat-stable toxin (ST) only and 4 (1.3%) produced both toxins (LT-ST)--whereas enteropathogenic E. coli (EPEC) were responsible for about 13.8% of the incidence of diarrhoea in the community. Detailed analysis revealed that 12 serotypes were involved. A greater number of cases of acute enteritis were seen in infants aged 0-18 months than in those aged between 19 and 36 months. The data indicate a highly significant level of resistance to most common antimicrobial drugs among diarrhoeagenic E. coli isolated, but most were highly sensitive to nalidixic acid, cephalothin and gentamicin.

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.

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.

Purification and properties of protease F, a bacterial enzyme with chymotrypsin and elastase specificities.

It has been previously demonstrated that commercial bacterial fibrinolysin (EC 3.4.21.7) selectively cleaves the bond between Met-53 and Ala-54 in ovine prolactin (199 amino acids). A one-step purification procedure on DEAE-cellulose for Protease F, which is the active component of bacterial fibrinolysin, and properties of the purified enzyme are reported. The enzyme is homogeneous as judged by acrylamide gel electrophoresis. Its molecular weight, calculated from gel filtration experiments on Sephadex G-100, is around 13,800. Amino acid analyses do not reveal the presence of any half-cystines. The presence of one tryptophan residue per enzyme molecule was resolved from the fluorescence spectrum. Amino terminal analysis showed that leucine was at the amino terminal position. Protease F hydrolyzes casein and synthetic specific substrates for chymotrypsin and elastase esterases but not for trypsin esterases. It is fully inhibited by phenylmethylsulfonyl fluoride, by chicken ovoinhibitor, and by Chymotrypsin Inhibitor I from potatoes but not by the trypsin-chymotrypsin inhibitors from soybeans and chick peas or by tosyl-L-phenylalanine chloromethyl ketone. The enzyme is stable at room temperature and in the cold, it is not affected by dialysis or by freezing and thawing, but it is inactivated during freeze-drying. The circular dichroism spectra of Protease F indicate an approximate 20% alpha-helix content of the enzyme with a considerable similarity to those of subtilisin, elastase, and beta-trypsin. The relatively low molecular weight of Protease F, the absence of intrachain disulfide bridges, and the fact that it is inhibited by several, but not all, chymotrypsin inhibitors suggest that it may differ phylogenetically from the known serine proteases.

Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors.

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.