Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin secretion induced by serotonin in rats

To study the possible involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in regulating the secretion of prolactin (PRL), the effect of anti-VIP rabbit serum on serotonin (5-HT)-induced PRL release was examined in urethane-anesthetized male rats. Anti-VIP serum (AVS) or normal rabbit serum (NRS) was infused into a single hypophysial portal vessel of the rat for 40 min at a rate of 2 microliters/min with the aid of a fine glass cannula and 5-HT was injected into a lateral ventricle 10 min after the start of the infusion. Intraventricular injection of 5-HT (10 micrograms/rat) caused an increase in plasma PRL levels in control animals infused with NRS and 5-HT-induced PRL release was blunted in animals infused with AVS (mean +/- SE peak plasma PRL: 118.9 +/- 19.8 ng/ml vs 54.7 +/- 16.2 ng/ml, p less than 0.05). These findings suggest that the secretion of PRL induced by 5-HT is mediated, at least in part, by hypothalamic VIP release into the hypophysial portal blood in the rat.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

Mitogenic responsiveness and monocyte-lymphocyte interaction of early and late rosette-forming cell populations of human peripheral blood lymphocytes.

Adherent cells in human peripheral blood mononuclear cells were removed by the attachment to the plastic surface of tissue culture dishes. After removal of adherent cells, early rosette-forming cells (early RFC), which were characterized by early (5 min) rosette formation with sheep blood cells (SRBC) at an SRBC to lymphocyte ratio of 8:1, were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradient. Total (60 min) rosette formation was carried out with the early RFC-depleted cell population on the gradient interface by the use of neuraminidase-treated SRBC at an SRBC to lymphocyte ratio of 20:1 and the resulting rosette-forming cells (late RFC) were sedimented by gradient centrifugation. These T cell subpopulations, early RFC-enriched and late RFC-enriched, were reasonably pure with respect to the ability to bind SRBC and contained less than 0.5% monocytes. Monocyte preparations, which were obtained after vigorous washing of the adherent cell layers on tissue culture dishes, responded to phytohemagglutinin P (PHA-P) or concanavalin A (Con A) with negligible incorporation of 3H-thymidine. There was no significance difference in the responsiveness to PHA-P between early RFC-enriched and late RFC-enriched populations either in the absence or in the presence of graded numbers of additional autologous monocytes. However, the response of early RFC-enriched population to Con A was significantly poor as compared with that of late RFC-enriched one unless additional monocytes were added. In the presence of 20% autologous monocytes in the culture, the Con A-induced response of early RFC-enriched population was markedly enhanced to reach close to that of late RFC-enriched population. These results suggest that early RFC and late RFC might be different from each other in their responsiveness and in their need for monocytes on the stimulation with Con A.     

Temporal pattern of changes in mitotic frequency in the epidermis and other larval tissues of Bombyx mori

Temporal changes in mitotic frequency were examined in various tissues through late larval life of Bombyx mori. From the second larval ecdysis to the third and from the third larval ecdysis to the fourth, there was a definite temporal change of mitotic pattern in each tissue. In the epidermis as well as in the tracheal epithelium, mitoses began to appear about 1 day after an ecdysis, and showed a maximum 1 to 2 days after an ecdysis. In the fat body, mitoses were observed continuously through the instars, and the mitotic frequency showed a maximum state just before an ecdysis. In the abdominal muscle the frequency was highest at about the middle of the period between two successive ecdyses. Furthermore, epidermal mitoses coincided with the time when the density of epidermal nuclei per unit area decreased to a half. This suggests that epidermal mitoses may be initiated by some process related to the increase in cell size.     

Effects of stereochemical structures of tetrahydrobiopterin on tyrosine hydroxylase.

1. Four stereochemical isomers of tetrahydrobiopterin, i.e., 6-L-erythro-, 6-D-erythro-, 6-L-threo-, or 6-D-threo-1,2-dihydroxypropyltetrahydropterin, have been synthesized and used as cofactors for tyrosine hydroxylase (EC 1.14.18.-) purified from the soluble fraction of bovine adrenal medulla. The L-erythro- (the putative natural cofactor) and D-threo isomers showed a striking similarity in their cofactor activities for tyrosine hydroxylase; the remaining two isomeric tetrahydrobiopterins, D-erythro and L-threo isomers, also had very similar cofactor characteristics. 2. The Km values of the L-erythro and D-threo isomers as cofactor were found to be dependent on their concentrations. When their concentrations were below 100 muM, the Km values of the L-erythro and D-threo isomers were fairly low (about 20 muM). However, the Km values were markedly higher (about 150 muM) at concentrations above 100 muM. The same kinetic behavior was also observed with the tetrahydrobiopterin prepared from a natural source (bullfrog). In contrast, the Km value of the L-threo or D-erythro isomer was found to be independent of the concentration and remained constant throughout the concentration examined. 3. The Km values of tyrosine did not show much difference (from 20 muM to 30 muM) with respect to the structure of the four isomeric cofactors. At high concentrations tyrosine inhibited the enzymatic reaction with any one of the four tetrahydrobiopterin cofactors. 4. Oxygen at high concentrations was also inhibitory with any one of the four stereochemical isomers as cofactor. Approximate Km values for oxygen with the tetrahydrobiopterins as cofactor were 1-5%. 5. In contrast to the four isomers of tetrahydrobiopterin, when 6-methyltetrahydropterin or 6,7-dimethyltetrahydropterin was used as cofactor tyrosine or oxygen did no inhibit the enzymatic reaction at high concentrations, and the Km values toward the pterin cofactor, tyrosine, and oxygen were significantly higher than the Km values with the tetrahydrobiopterins as cofactor.     

Xyloglucan and r{beta}-D-Glucan in Cell Walls of Rice Seedlings

Cell walls of 4-day old rice seedlings were extracted successivelywith ammonium oxalate-oxalic acid, 4% KOH and 24% KOH. A rß-D-glucanpreparation and a xyloglucan preparation were isolated fromthe 4% KOH extract and 24% KOH extract, respectively. Methylationanalysis and enzymic degradation studies of the polysaccharidesshowed that the former was built up predominantly of repeating-oligosaccharideunits of 3-O-rß-cellobiosyl-D-glucose and 3-O-rß-cellotriosyl-D-glucosein a molar ratio of 2.6 : 1.0, and the latter was of repeating-oligosaccharideunits of -D-xylosyl-(16)-rß-D-glucosyl-(14)-[-D-xylosyl-(16)]-rß-D-glucosyl-(14)-D-glucose,-D-xylosyl-(16)-rß-D-glucosyl-(14)-D-glucose and cellobiose. ¹ Present address: Department of Botany, Iowa State University,Ames, Iowa 50011, U.S.A. (Received August 29, 1981; Accepted January 12, 1982)     

Regulatory relation between insulin receptor and its functional responses in primary cultured hepatocytes of adult rats

The relation between changes of insulin receptor and various metabolic responses were studied in adult rat hepatocytes in primary culture. In cells cultured for 3 h without insulin, the number of high affinity sites and the dissociation constant (Kd) of insulin receptor, determined from a Scatchard plot, were 1.05 x 10(5) sites/cell and 1.5 x 10(-9) M, respectively. The receptor number increased 2-fold, but the Kd value remained constant during 2-days culture in insulin-free medium (up-regulation). Addition of dexamethasone (Dex), growth hormone, glucagon or triiodothyronine did not change the number of insulin receptors or the Kd value. In contrast, 1-day culture in insulin (1 x 10(-7) M) medium decreased the receptor number by half (down-regulation) without change of the Kd value. Short-term responses of glycogenesis, amino acid transport and lipogenesis by insulin increased as the receptor number increased. In these cases, the sensitivity to insulin (Ka: half dose for the maximum response) did not change in cells with different receptor numbers, but the maximum response changed. These results show that hepatocytes, unlike adipocytes, do not have spare receptors of insulin. During down-regulation, the receptor number decreased by only half, but the insulin responses were lost almost completely. The receptor number returned to the normal level after culture in insulin-free medium for 12 h, but recovery of the responses took longer, suggesting that for the insulin response not only change of receptor number, but also other regulatory mechanisms for post-receptor processes, such as desensitization, are involved.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from $\text{E. coli}$ was determined by the method of Maxam-Gilbert. The sequence covers the genes for the β and ε subunits of F₁ along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the β and ε subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. A possible nucleotide binding site in the β subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of F₁F₀ were different in different genes, suggesting that the amount of each subunit in the F₁F₀ is determined to some extent on a translational level.