Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.     

Functional changes in vascular smooth muscle and endothelium of arteries during diabetes mellitus.

To investigate the influence of diabetes mellitus on the responsiveness of the vascular smooth muscle, the effects of various vasoactive agents on the reactivity of the vascular smooth muscle from diabetic animals have been undertaken, focusing on the functional changes in the endothelium, alpha-adrenoceptors, beta-adrenoceptors, voltage-dependent Ca(2+)-channels, receptor-operated Ca(2+)-channels, phosphatidylinositol turnover and potassium channels. Among the functional changes, it is a common phenomenon that decreases in acetylcholine-induced production of cyclic GMP are due to the attenuation of release of endothelium-derived relaxing factor through an impairment of endothelium; this observation was found in both rats and rabbits with diabetes mellitus. These functional changes in diabetes may be responsible for the vascular complications such as coronary heart disease, cerebrovascular disease, and an acceleration in atherosclerosis.     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: changes in nutrient concentrations,phytoplankton and zoobenthos

Large bag-type (75 m³) and tube-type (105 m³) enclosures were set up in the shallow eutrophic Lake Suwa and were each stocked with exotic planktivorous whitefish (Coregonus lavaretus maraena). The release of whitefish caused the increase in nutrient concentration in the tube-type enclosure whereas no such increase was observed in the bag-type enclosure. Bottom sediment seemed to be an important source of chironomid food for whitefish. The proportion of phytoplankton measuring<10μm and 20–40μm, which respectively corresponded toOchromonas spp. andCryptomonas sp., were lower in the fish enclosures than in the control, which might have been caused by high grazing pressure by rotifers. The predation by whitefish might have affected the species composition of phytoplankton through reducing copepod predation on rotifers, not through reducing the densities of cladocerans which directly feed on phytoplankton as many investigators have reported. The phytoplankton biomass was not affected much by the release of fish. Possible reasons are that the increase in density of rotifers reduced the biomass of available phytoplankton and also that inedible Cyanophyceae were in the decreasing phase of their seasonal succession and could not increase successfully in spite of elevated nutrient levels.     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: selective feeding of fish and predation effects on the zooplankton communities

Bag-type enclosures (75 m³) with bottom sheets and tube-type enclosures (105 m³) open to the bottom sediment were stocked with exotic whitefish (Coregonus lavaretus maraena) to study their predation effects on the plankton community. The fish fed mainly on adult chironomids during the period of their emergence (earlier part of the experimental period). Thereafter, the food preference was shifted to larvae of chironomids and crustacean zooplankters. The predation effects on the plankton community were not evident in the bag-type enclosures where zooplankton densities were consistently low. The fish reduced the crustacean populations composed ofBosmina fatalis, B. longirostris andCyclops vicinus in the tube-type enclosures where the prey density was high (above ca. 50 individuals 1⁻¹). The results suggested that the intensity of predation depended on the prey density. Rotifers increased in the fish enclosure, probably becauseCoregonus reduced the predation pressure byCyclops vicinus on rotifers and allowed the latter to increase. In the fish enclosures, no marked changes in species composition were observed. Zooplankton predated by the fish seemed to be distributed near the walls of the enclosures. Problems of enclosure experiments for examining the effects of fish predation on pelagic zooplankton communities are discussed.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [³⁵S]methionine into the α-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-α-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify α-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that α-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of α-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of α-amylase.     

Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.     

The preferential codon usages in variable and constant regions of immunoglobulin genes are quite distinct from each other.

The pattern of codon utilization in the variable and constant regions of immunoglobulin genes are compared. It is shown that, in these regions, codon utilizations are quite distinct from one another: For most degenerate codons, there is a selective bias that prefers C and/or G ending codons to U and/or A ending codons in the constant region compared with the bias in the variable region. This would strongly suggest that, in immunoglobulin genes, the bias in code word usage is determined by other factors than those concerning with the translational mechanism such as tRNA availability and codon-anticodon interaction. A possibility is also suggested that this differance of code word usage between them is due to the existence of secondary structure in the constant region but not in the variable region.