Increased superoxide anion release from human endothelial cells in response to cytokines

To study the effects of macrophage and lymphocyte-derived factors on superoxide anion (O2-) generation and release from human umbilical vein endothelial cells (EC), cultured EC were stimulated by ultrapure interleukin 1 (IL 1) and recombinant interferon-gamma (IFN-gamma), and the O2- released into the supernatant was measured. Both of these cytokines enhanced O2- release in a dose and time-dependent manner. Addition of a combination of IL 1 and IFN-gamma, each in submaximal concentration, produced an additive effect on O2- release. It would appear from these findings that cytokines released by macrophages and lymphocytes during inflammatory reactions can promote O2- generation and release from human EC. O2- released from EC may alter the basement membrane of blood vessels and the surrounding connective tissue, and in this way promote the vascular injury and angiogenesis associated with local inflammation.     

Noninferior periodic operation of the biological reactor

The optimal periodic operation of the biological reactor was studied from the standpoint of the two-objective programming problem. The noninferior set with respect to the cell productivity and the conversion of the substrate into the biomass was determined by use of the optimization technique due to Miele. It was shown that the noninferior set was composed in general of the repeated batch branch and the repeated fed-batch branch, which occupy the high-productivity portion and the high-conversion portion of the noninferior set, respectively. However, the latter branch disappears in the case of growth kinetics with no substrate inhibition. In addition, the extreme points of the noninferior set yielding the maximal productivity and the maximal conversion represent such operations that are equivalent to the steady-state operation (chemostat culture) and the batch operation, respectively.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Responses of pituitary and adrenal medulla to insulin-induced hypoglycemia in patients with anorexia nervosa

The responses of pituitary and adrenomedullary hormones to insulin-induced hypoglycemia were studied in 10 patients with anorexia nervosa and 7 control females of comparable age. The increases in plasma GH and PRL were significantly smaller in the patients, while the responses of GH to arginine and of PRL to TRH were indistinguishable. Plasma cortisol attained similar peak levels in both groups with higher basal levels and smaller increments in the patients. The response of plasma epinephrine was markedly lower in the patients, although urinary epinephrine showed similar increase in both groups. These results suggest the possibility that the process by which hypoglycemic stimulus causes pituitary and adrenomedullary hormone secretion is deranged in patients with anorexia nervosa.     

Responses to epinephrine in patients with anorexia nervosa

To examine the sensitivity to epinephrine in patients with anorexia nervosa, 20-60 micrograms/kg body weight/min of epinephrine was infused for 30 min each in 5 patients and 5 controls. The increase in pulse rate and the decrease in diastolic blood pressure were significantly smaller in the patient group. Elevated plasma GH levels in the patients were markedly suppressed by epinephrine infusion. These results indicate the beta-adrenergic function is decreased at least in the cardiovascular system in patients with anorexia nervosa.     

Comparison of inhibitory effects of prolinal-containing peptide derivatives on prolyl endopeptidases from bovine brain and Flavobacterium

The inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in Ki values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with Ki values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with Ki values of nM order, while the Ki values of these compounds for the microbial enzyme were only of microM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.     

Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

Cloning and sequencing of Serratia protease gene.

The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease.     

Spirulina ferredoxin-NADP+ reductase. Further characterization with an improved preparation

The preparation procedure for Spirulina ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.2, FNR) was improved by adding protease inhibitors, phenylmethylsulfonylfluoride (PMSF) and EDTA, through the whole process of preparation and by introducing an affinity chromatography step on Blue Sepharose CL-6B. The addition of the inhibitors largely prevented the formation of the minor component (FNR I), and the affinity gel chromatography simplified the preparation process, shortening the exposure period of FNR to proteolysis. However, complete removal of the heterogeneity of FNR found at the amino (N)-terminal region was not achieved even by applying the new method. The affinity chromatography on the Blue Sepharose gel was also effective in purifying spinach FNR. The affinity of this gel for Spirulina FNR was compared with that for the enzyme derived from spinach leaves. The spinach enzyme had a higher affinity than the Spirulina one. Both enzymes showed the highest affinities to Blue Sepharose at 20--30 mM NaCl concentration. The N-terminal sequence analysis revealed that there was 4 forms, which were probably modifications produced by exopeptidase action during the preparation, or even in the living cells. The longest component gave the N-terminal sequence Ala-Lys-Thr-Asp-Ile-Pro-Val-Asn-Ile-Tyr-. The others lacked amino acids successively one by one from the N-terminus. In contrast, the carboxyl(C)-terminal residues of all 4 FNR forms were tyrosine. The probable C-terminal sequence was predicted to be -Trp-His-Val-Gln-Thr-Tyr based on a study of a cyanogen bromide peptide.