Salmon calcitonin-induced stimulation of 1 alpha,25-dihydroxycholecalciferol synthesis in rats involving a mechanism independent of adenosine 3'':5''-cyclic monophosphate.

The effect of natural salmon calcitonin on accumulation in plasma of 1 alpha,25-dihydroxy-[3H]cholecalciferol from 25-hydroxy[3H]cholecalciferol in vivo was investigated in vitamin D-deficient thyroparathyroidectomized rats into which graded doses of the hormone were continuously infused by use of a balance study system. A dose-dependent increase in plasma concentrations of 1 alpha,25-dihydroxy[3H]cholecalciferol was observed with calcitonin infusion for 6--30h at a rate greater than 20 M.R.C. m-units/h. Infusion of parathyrin or cyclic AMP produced a similar stimulation [Horiuchi, Suda, Takahashi, Shimazawa & Ogata (1977) Endocrinoly 101, 969--974], but the maximal effect of calcitonin was additive to that of either parathyrin or cyclic AMP. Furthermore concurrent infusion of theophylline (0.5 mumol/h) did not potentiate the effect of submaximal doses (3 and 20 M.R.C. m-units/h) of calcitonin. Plasma concentrations of calcium showed a decrease with calcitonin infusion for 30h, but those of Pi remained unchanged. These results strongly suggest that the rat kidney is endowed with a calcitonin-sensitive 1 alpha-hydroxylase system that is separate from the parathyrin/cyclic AMP system and is independent of changes in plasma Pi.     

R plasmic Rtsl-mediated production of extracellular deoxyribonuclease in Escherichia coli.

Escherichia coli strains harboring Rtsl were found to excrete extracellular deoxyribonuclease. The DNase activity was greater in cells with pTW2, a mutant from Rtsl.     

Concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB)

Summary A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color -D-glucosyl and -D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975)     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Effects of x-ray irradiation on the subsequent gonadotropin secretion in normal and neonatally estrogenized female rats.

Female rats were irradiated with 190R of X-rays at 10 days of age and sacrificed 4, 7 or 12 months later. Their ovaries were histologically examined and serum levels and pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. Both serum levels and pituitary contents of LH and FSH rose significantly 4 and 7 months after irradiation, although the ovaries were markedly reduced in weight. On the contrary, 12 months after irradiation, the ovaries increased in weight and consisted mostly of polyhedral, hyperplastic interstitial cell masses, and both LH and FSH in the serum and pituitary were reduced to normal levels. These characteristic changes in the ovarian weight and histological appearance could not be observed in the similarly irradiated animals which were received daily injections of estrone for the first 30 days of postnatal life, i.e., daily injections of 50 mug for the first 10 days, 100 mug for the middle 10 days and 200 mug for the last 10 days. Serum LH levels of the estrogenized irradiated rats at 7 or 12 months of age did not elevate although those of FSH were significantly higher than the non-irradiated intact levels. From these results, a rise in the blood levels of LH and the FSH may be attributed to the increase in weight and the histological changes in the ovaries of the irradiated female rats, and the elevation of only FSh level may not result in the abnormal growth of the irradiated ovaries.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. II. Mode of growth of coxsackievirus A9 in HeLa cell cultures and the effect of sulfated polysaccharide on plaque formation.

For the purpose of clarifying the mechanism of plaque formation in HeLa cell cultures by coxsackievirus A9, which does not show definite CPE in fluid cultures, we investigated the growth pattern of the virus in HeLa cells, comparing plaque (HeLA)-forming and non-plaque (HeLa)-forming viruses. It was revealed that the yield of both viruses per cell was nearly the same, but non- plaque (HeLa)-forming virus was far less efficient in infecting HeLa cells. Dextran sulfate was effective in releasing more virus from cells, when HeLa cell cultures were infected with plaque (HeLa)-forming virus, but not in cultures infected with non-plaque (HeLa)-forming virus. From these experimental results, the mechanism by which plaques are formed in HeLa cell cultures by coxsackievirus A9 was discussed.     

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

Proliferative response of seminal vesicle cells to androgen and estrogen in neonatally castrated mice

Proliferation and death of androgen- and estrogen-responsive cells in seminal vesicles were compared between neonatally and adult (on Day 60 after birth) castrated mice. Daily injections of either testosterone propionate (TP) or estradiol-17 beta (E2) were started on Day 90 after birth; the incorporation of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) into the whole seminal vesicles was used as an index for proliferation. Although the peak of [125I]IdUrd uptake was observed 3 days after starting TP injections in both neonatally and adult castrated mice, the peak was lower and the period of proliferation was much longer in the former than in the latter. When TP injections were stopped, the fraction of surviving cells that synthesized DNA on Day 3 of TP injections was much larger in neonatally than adult castrated mice. The difference was attributed to the presence of TP-induced proliferation of fibromuscular cells in the neonatally castrated mice but not in the adult castrated mice; only the fibromuscular cells but not epithelial cells survived after stopping TP injections. Although injections of E2 increased the proliferation of epithelial cells but did not the weight of seminal vesicles in adult castrated mice, the same procedure increased the proliferation of both epithelial and fibromuscular cells and the weight in neonatally castrated mice. The E2-induced fibromuscular cells seemed to survive in the presence or absence of E2. The present results seem to indicate that androgen- and estrogen-induced proliferation of fibromuscular cells is irreversible in seminal vesicles of neonatally castrated mice and that the depletion of androgen in the seminal vesicle during neonatal and prepubertal periods is at least in part compensated by the administration of androgen, even after 90 days of age.