Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.     

The use of monoclonal antibodies for detecting the influenza virus

The possibilities of using influenza A (Leningrad) 385/80 (H3N2) virus matrix protein-specific FITC-labeled D8 monoclonal antibodies in immunofluorescence assays were investigated. The virus antigen accumulation was detected in chorioallantoic cells of chick embryos. Exhibiting the type-specific properties, the fluorescent antibodies stain the perinuclear space, cytoplasmic membrane, and granular structures in the cytoplasm of infected cells. The haemagglutination test tires in the corresponding specimens were at least 1:16.     

Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone

A procedure was developed for overexpression of Trypanosoma brucei pyruvate kinase in Escherichia coli. The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higher S(0.5) for phosphoenolpyruvate and a significantly smaller kcat than the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.     

The effect of an original analog of the C-terminal fragment of vasopressin on the behavior of white rats

Ethinylestradiol (EE) has evident paradoxical effects on cancer risk for human breast and hepatic cancer which parallel in some respects its effects on estrogen-induced neoplasms in the hamster kidney and liver. EE has been shown to be only weakly carcinogenic in the hamster kidney, but the most potent carcinogenic estrogen in the hamster liver following prolonged treatment. Unexpectedly, when EE and potent carcinogenic estrogens, such as diethylstilbestrol (DES), 17beta-estradiol (E2) and Moxestrol (MOX), are administered concomitantly, estrogen-induced carcinogenesis in the kidney is completely prevented. In studying this novel finding, we found that, compared with E2 exposure alone, EE at 0.05 and 1.0 nM significantly (P < 0.001) inhibited the rise in proliferation of cultured primary hamster proximal renal tubular (PRT) cells in the presence of E2 (1.0 nM). Consistent with these findings, combined EE + DES treatment for 5.0 months reduced hamster kidney c-myc, c-fos and c-jun RNA expression to 43, 37 and 52%, respectively, compared with levels observed after DES treatment alone. Interestingly, TAM + DES treatment for the same period also resulted in the same low level of RNA expression of these proto-oncogenes. c-MYC, c-FOS and c-JUN protein products were comparably reduced after either EE + DES or TAM + DES treatment. It appears that c-fos expression and c-FOS protein levels in the hamster kidney were more responsive to TAM inhibition. These data demonstrate that EE possesses unique anti-tumorigenic properties in vivo in the hamster kidney. Additionally, the observed anti-estrogen-like effect of EE on cell proliferation of cultured PRT cells suggests that EE may interfere critically with estrogen receptor (ER)-mediated mitogenic pathway(s) affected by potent carcinogenic estrogens, thus preventing subsequent gene dysregulation and, hence, tumor development. Based on competition studies, the differential binding of EE to hamster kidney ER relative to that of the other estrogens (E2, DES, MOX) appears not to contribute to the prevention of estrogen carcinogenesis at this organ site by EE.     

The clinical significance of MDR-1 gene expression in the hemopoietic cells of patients with acute leukemias in different phases of the disease

AIM: Analysis of cytostatic therapy effects on expression of gene MDR-1 in hemopoietic cells of patients with acute leukemia (AL) in complete clinicohematological remission (CCHR). MATERIALS AND METHODS: The study included 48 AL patients. 27 of them were untreated, 25 were resistant to or had recurrent AL. 4 patients were followed up. Bone marrow mononuclear fraction was investigated. Expression of MDR-1 gene in the cells was evaluated at hybridization. RESULTS: High expression of MDR-1 gene occurred in leukemic blast cells either upon achievement of CCHR or at least 6 months after its onset. When using schemes of chemotherapy containing two potential inductors of gene MDR-1, expression of this gene was registered significantly more frequently than in using schemes based on one inducing drug (p < 0.05). Frequency of occurrence of enhanced expression of gene MDR-1 in leukemic blasts significantly correlated with frequency of CCHR (p < 0.05). Correlation between occurrence of the gene's expression in normal hemopoietic cells in CCHR and occurrence of early AL recurrences was not found. CONCLUSION: The findings may facilitate design of new AL treatment programs.     

Polylysine decelerates kinetics of negatively charged gramicidin channels as shown by sensitized photoinactivation

Effect of a cationic polymer, poly(L-lysine), on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its negatively charged analogue O-pyromellitylgramicidin (OPg) in a bilayer lipid membrane is studied using a method of sensitized photoinactivation. This newly developed method is based on the analysis of transmembrane current transients induced by a flash in the presence of a photosensitizer. It has been shown previously that the time course of the flash-induced current decrease in most cases follows a single exponential decay with an exponential factor (tau, the characteristic time of photoinactivation) that correlates well with the single-channel lifetime. Addition of polylysine does not affect tau for gA channels, but causes a substantial increase in tau for OPg channels. This effect is reversed by addition of polyacrylic acid. The deceleration of the photoinactivation kinetics is ascribed to electrostatic interaction of polylysine with OPg probably resulting in OPg clustering. The latter can stabilize the channel state by reducing the rotational and lateral mobility of OPg monomers and dimers, and thus increase the single channel lifetime.     

Relaparotomy]

The study of intrarenal and upper urinary tracts urodynamics in the presence of the ureteropelvic obstruction and of the function of the lower urinary tract shows that congenital hydronephrosis was in 42.8% of cases associated with bladder dysfunction presenting with hyperreflex-type dysfunction. Maladapted hyperreflex bladder was diagnosed in 29.4%, adapted--in 13.5% of cases. The hyporeflex dysfunction occurred in 15.9% of cases. The urinary bladder dysfunction in children with congenital hydronephrosis accounts for 58.7%. These aggravate the same defects of the upper urinary tracts and deteriorate the course of chronic obstructive pyelonephritis.