Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Comparative cytological study of Phasianus colchicus,Meleagris gallopavo,and Gallus domesticus

Summary Since viable intergeneric hybrids between the chicken (Gallus domesticus) and the pheasant (Phasianus colchicus) have been reported, as well as interfamilial hybrids between the chicken and the turkey (Meleagris gallopavo), the chromosome complements of the pheasant and the turkey were compared with that of the chicken. In these three species belonging to the order Galli, the Z-chromosomes appeared to be identical, while the autosomal complements of the pheasant and the turkey differed radically from that of the chicken. It was noted with some surprise that the pheasant of the family Phasianidae and the turkey of the family Meleagridae have very similar chromosome complements, at least so far as gross morphology of somatic metaphase chromosomes is concerned.This work was supported in part by grant C-5138 from the National Cancer Institute, U.S. Public Health Service, and grant C-17601 from the National Science Foundation.The authors gratefully acknowledge the generosity of Rea's Game Birds, Paramount, California, who supplied the pheasant chicks, and the McPherin Hatcheries, Sunnymead, California, who furnished the turkey chicks. The authors also appreciate the editorial assistance of'Patricia A. Ray.     

Purification and Chemistry of Bacteriophage ?

From a stock of varkappa phage grown on Salmonella, a host-range mutant which attacks Escherichia coli was isolated. As in the case of Salmonella, only motile strains of E. coli are sensitive to varkappa. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 x 10(-9) ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage varkappa was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.     

The synthesis of polydeoxyribonucleotide by cell-free extracts of embryonic mouse tissue

The conditions under which soluble extracts prepared from mouse embryos incorporate [(3)H]thymidine 5'-triphosphate into polydeoxyribonucleotide have been studied. In common with similar preparations from other mammalian tissues, mouse-embryo DNA nucleotidyltransferase requires the four complementary deoxyribonucleoside 5'-triphosphates, primer DNA and a bivalent cation for activity. Unlike other mammalian DNA nucleotidyltransferases, the rate and extent of the incorporation of [(3)H]thymidine 5'-triphosphate is much greater with Mn(2+) than with Mg(2+) and, with either Mg(2+) or Mn(2+), maximum activity occurs at pH6.4. The difference between Mg(2+) and Mn(2+) varies markedly with pH, reaching a maximum of six- to eight-fold at pH6.4.