Isolation and identification of prostaglandin I 2 stimulating factor from human plasma

To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as acid by its ultraviolet and infrared absorption spectroscopy, and ¹H nmr and ¹³C nmr spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma (about 50 μg/ml) was half of the deproteinized human plasma, and was about 30 fold stronger than that of L-tryptophan, a cofactor of prostaglandin hyperoxidase.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

The parasitoidOoencyrtus nezarae (Hymenoptera: Encyrtidae) prefers hosts parasitized by conspecifics over unparasitized hosts

Summary Two laboratory experiments were conducted to examine the ovipositional preferences of the egg parasitoidOoencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) for parasitized and unparasitizedMegacopta punctatissimum Montandon (Hemiptera: Plataspidae). Females that had never oviposited or that had not oviposite for 3 days preferred recently parasitized hosts more than unparasitized hosts. The preference for recently parasitized hosts appeared to be mediated by the punctures in already parasitized hosts made by the ovipositor of the first female. Survival of the parasitoid progeny was lower in recently parasitized hosts than in unparasitized hosts. However, handling time of parasitized hosts was extremely short relative to that of unparasitized hosts, because the superparasitizing female could use the punctures made by the previous females. It is concluded that the females preferred the parasitized hosts over unparasitized hosts because the benefit of saving time and energy for drilling was more than the cost of progeny survival.     

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Effect of diabetes mellitus induced by streptozotocin on renal Superoxide dismutases in the rat

Two forms of superoxide dismutase, CuZn-SOD and MnSOD, have been investigated in the kidneys of streptozotocin-induced diabetic rats using both radio-immunoassay and immunoenzyme staining. The rats were killed 2, 8 and 12 weeks after the induction of diabetes mellitus and the kidneys excised. Two weeks after the induction of diabetes, the kidneys were hypertrophied because of the proliferation of renal tubular epithelium. However, the total CuZnSOD content of the kidneys did not increase and, because of the epithelial proliferation, the CuZnSOD concentration in each proximal tubular cell was decreased. Armanni-Ebstein lesions were found in the distal tubules 8 and 12 weeks after the induction of diabetes. The cells in these lesions were intensely stained for CuZnSOD, suggesting an adaptive response to the enhanced oxidative stress. The MnSOD staining in the thick ascending limbs of Henle's loops was enhanced in the diabetic kidneys, while that in the cortical tubules was unaltered. MnSOD was assumed to increase in response to hypermetabolism associated with the proliferation of renal tubules. This was most marked in the cells which were rich in mitochondria, again suggesting an adaptive response to enhanced oxidative stress induced by diabetes mellitus. The glomeruli of both the diabetic and control groups were not stained for SODs, and no significant microscopic change was found even 12 weeks after the induction of diabetes mellitus.     

Cloning and expression of cDNAs from enterically-transmitted non-A, non-B hepatitis virus.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.     

Changes in conformation of spin-labeled calmodulin by phospholipids

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.