Smears from rat liver suspension as modified substrate for antinuclear antibody test.

Immunofluorescence still remains a standard method for documenting antinuclear antibodies (ANA). Cryostat cut sections of rodent liver or Hep2 cell nuclei have been used as substrate in the test but are often difficult to arrange in laboratories in developing countries. Hence, a modification was developed using smears from rat liver suspensions. The smears were compared with the cryostat cut sections over 338 sera samples of suspected cases of collagen diseases, rheumatoid arthritis, thyroid disorders, hepatitis B, enteric fever, tuberculosis and normal subjects. The sera from suspected collagen diseases cases were also compared with ANA test using Hep2 cells. The modified smear technique was well comparable and the clarity of the immunofluorescence was even better than for cryostat cut sections. Using the modified smear technique 272 sera out of 2,851 sera gave positive test for ANA. The homogenous, speckled and peripheral patterns were seen for 203, 66 and 3 samples respectively. To conclude: The smears prepared from homogenised rat liver suspension and fixed like bacterial smears offer a very convenient and reliable tissue substrate for ANA test.

Antibiotic-resistant endocarditis in a hemodialysis patient.

A chronic dialysis patient developed persistent bacteremia as a result of infection with Enterococcus faecium. During the patient's illness, resistance to ampicillin, gentamicin, vancomycin, and teicoplanin developed. Despite arteriovenous (AV) graft removal and an extensive but inconclusive search for the source of the infection, bacteremia persisted. On autopsy, the patient was found to have had aortic-valve endocarditis. Endocarditis is a well-known complication in dialysis patients. Multidrug-resistant organisms are becoming more prevalent in hospitalized patients as well. Risk factors for the development of endocarditis in dialysis patients include catheters, AV grafts, and calcific valvular disease, all in conjunction with frequent access to the circulation. Avoidance of temporary catheter use by prompt placement of AV fistulas or grafts and consideration of their early use, the meticulous care of catheters once in place, and treatment of the nasal carriage of Staphylococcus aureus may lower the incidence of bacteremia and therefore endocarditis in dialysis patients. The removal of infected catheters and/or AV grafts if prompt clearing of the blood with antibiotics does not occur is the next step, followed by valve replacement in selected cases. The routine use of vancomycin in the dialysis population should be reevaluated in light of the development of high-level antibiotic-resistant organisms.

Immunogenicity of low dose hepatitis B vaccine by the intradermal route & persistence of anti-HBs after three years.

Plasma derived hepatitis B vaccine given intradermally (0.2 ml; 4 micrograms HBsAg) at 0, 1, 6 months to 200 health care workers, produced seroconversion in 97.5 per cent. Antibody levels to hepatitis B surface antigen (anti-HBs) crossed 1000 mIU/ml in 62 per cent while 26.5 per cent had levels of 100 to 1000 mIU/ml. Anti-HBs levels persisted in the same range in 41.7 per cent but dropped by a log in 58.3 per cent subjects at the end of 3 yr. Protective antibodies above 10 mIU/ml were documented in 93.3 per cent vaccinees after 3 yr. The 0.2 ml vaccine by intradermal (id) route was also found to give a good booster effect in another group of 27 persons who had received full dose vaccine 5 yr earlier. Thus, 0.2 ml vaccine by id route was safe, gave high seroconversion and persistent antibody levels over 3 yr and could offer effective protection at an economic cost.

Studies on glutathione metabolism in ventral prostate and chemically induced prostatic carcinoma in rats.

Glutathione content and the activity of glutathione reductase were examined in ventral prostate and chemically induced 11095 squamous-cell prostatic carcinoma in rats. Castration produced a significant reduction in the levels of reduced (GSH) and oxidized (GSSG) glutathione and glutathione reductase activity in the prostate. Replacement of testosterone (50 mg/kg) daily for 7 days to castrated animals elevated the reduced glutathione level and the activity of glutathione reductase almost to normal limits. Squamous-cell carcinoma was implanted in castrated and intact animals. Tumor growth in normal rats produced a decrease of almost 30% in the weight of the ventral prostate at 21 days post-implantation, although the glutathione levels remained unaffected. Much greater activity of glutathione reductase was detected in the tumor in comparison to the values noted for the normal tissue. The tumor also showed significantly higher values for the GSH/GSSG ratio. No apparent difference could be found in the rate of the growth of tumors whether implanted in normal or castrated animals. The levels of reduced and oxidized glutathione and glutathione reductase activity also seemed identical in tumors obtained from both groups of animals. Administration of testosterone (50 mg/kg) or beta-estradiol (2 mg/kg) daily for 11 days to tumor-bearing castrated animals did not alter the levels of glutathione and glutathione reductase activity. A significantly higher level of blood reduced glutathione was found in tumor-bearing rats in comparison to that seen for the normal subjects. Our results demonstrate that androgen depletion and replacement therapy influence the metabolism of glutathione in rat ventral prostate. Squamous-cell carcinoma of the prostate appears to differ from the normal tissue with respect to the observed androgen effects. There is dissimilarity in the metabolism of glutathione in the two tissues since greater activity of glutathione reductase and lower values of reduced glutathione were seen in the tumor as compared to those of the ventral prostate. Treatment with beta-estradiol, an antiprostatic agent, does not seem to influence the growth or glutathione metabolism of squamous-cell carcinoma of the prostate. The observed changes in blood glutathione levels might prove to be useful as an index of rapid growth of the neoplastic tissue.

3',5'-cyclic-nucleotide-dependent protein kinase of squamous cell carcinoma of the prostate.

An adenosine 3'5'-cyclic-monophosphate (Cyclic AMP)-dependent protein kinase has been identified and partially purified from the rat prostate tumor induced by 20-methylcholanthrene. This enzyme is stimulated 2- to 3-fold by the nucleotide. Equilibrium studies at pH 5.0 suggest the presence of a major class of binding site for cyclic AMP with an association constant of approximately 10(8) M-1. The concentration of binding site is about 1 pmol/mg of protein of the enzyme preparation. The enzyme is stimulated by other cyclic nucleotides as well, but only by higher concentrations. In comparing the ability of different histone subfractions, casein and protamine, to serve as substrate for this particular protein kinase, maximal cyclic-AMP-dependent enzyme activity was observed with histones. The results suggest that factors contributing to the malignant growth of the prostatic tissue do not directly involve changes in the characteristics of a cyclic-AMP-dependent protein kinase.

Studies on the mechanism of action of 6-mercaptopurine. Interaction with copper and xanthine oxidase.

Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), as determined by Subhrama Rao & Raghav Rao's [(1955) J. Sci. Chem. Ind. Res. 143, 278], method is found to be 6.74. It is suggested that the formation of a stable complex between 6-mercaptopurine molecules and Cu2+ may be an additional mechanism of action of 6-mercaptopurine, particularly with reference to its anti-inflammatory properties.

Amino acids: modifiers of xanthine oxidase activity.

L-Glutamic acid has been found to be a positive and L-lysine a negative modifier of the xanthine oxidase activity at the optimum pH (7.4) of the enzyme. Increase in pH was observed to be associated with a progressive decrease in the inhibition produced by L-lysine.

Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3':5'-cyclic monophosphate in androgen action.

1. The ability of exogenously administered cyclic AMP (adenosine 3':5'-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as alpha-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2'-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16-24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized-castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a submaximal dose of testosterone (1.0mg/100g) on all those prostatic and seminal-vesicular enzymes that are increased by exogenous cyclic AMP. 11. The evidence indicates that cyclic AMP may be involved in triggering the known metabolic actions of androgens on secondary sexual tissues of the rat.