Microbial Blood-coagulating Protease Screening Test and Some Properties

A potent fungus for blood-coagulating enzyme was isolated from soil during screening tests of about 1500 strains of microorganisms. The fungus was identified as a strain belonging to Cephalosporium. This enzyme could be produced effectively by solid culture on wheat bran and partially purified to about 800 fold. This enzyme was one of the alkaline proteases. It was stable over a pH range of 7~10 and was activated by Ca²⁺ The enzyme could not coagulate 1% fibrinogen solution. The enzyme mixed with diluted blood plasma coagulated the fibrinogen solution. The enzyme also coagulated the citrated plasma from humans, bovines, mice, goats and hens.     

On open access and optimal landings tax

This paper develops two types of simple models on the dynamic interaction between the stock of fish and the effort expended by fishers: continuous-time/discrete-time models in which a landings tax is incorporated as a control variable available to the management authority. The continuous-time model can describe several ideal options of the optimal tax program; however, unfortunately, it is incapable of choosing the best option. Hence, using the alternative tractable discrete-time model and a computational method, the remaining task of determining a unique optimal tax program is accomplished. The fishery thus managed exhibits a regulated open access.     

Trypanocidal activity of the stearylamine-bearing liposome in vitro

Liposome made of stearylamine and phosphatidylcholine showed the trypanocidal activity in vitro. Cytotoxicity of the liposome against Trypanosoma cruzi appeared to be the strongest in trypomastigotes followed by amastigotes and epimastigotes. Lysis of the human erythrocyte was undetectably low under the conditions that the liposome kills more than 95% of trypomastigotes. The liposome seems to damage the plasma membrane.     

An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

Summary We have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum. This work was supported by the Japanese Ministry of Education, Science and Culture and in part by grants from the National Institutes of Health. Editor's Statement This article reports a creative technical application of the author's previous work on lipid metabolism in lymphoid cells allowing an efficient, alternative selection procedure for isolation of hybridomas.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid β-oxidation. The impairment was reversed to the wild-type levels by treatment with 17β-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid β-oxidation and in maintaining lipid homeostasis.     

A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1^F/F ESCs, we isolated highly pure Ddx1^F/F and Ddx1^−/− ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress–p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.     

Solid-state NMR characterization of triacylglycerol and polysaccharides in coffee beans

It is important to understand the structural characteristics of triacylglycerol (TAG), polysaccharides and trace elements in coffee beans, so that residues can be reutilized in applications including biodiesel oils. Here, we performed ¹H and ¹³C solid-state NMR measurements on Indonesian green beans, roasted beans, and spent coffee grounds (SCGs). In the NMR spectra, there were liquid-like TAG containing linoleic acids based on observed signals of -CH=CH-CH₂-CH=CH- group in an acyl chain, which play a role in decreasing TAG’s melting point. We found TAG was still abundant in the SCGs from NMR spectra. After lipids were removed from SCGs, the intensity of the TAG signal decreased considerably, with approximately 64% of the TAG was successfully extracted. We described the chemical structure of TAG in coffee beans and demonstrated that it is possible quantify the amount of extracted TAG using solid-state NMR.     

The cloning and expression of chymosin (rennin) genes in microorganisms

Chymosin, also known as rennin, a milk-clotting enzyme obtained from the stomach of calves, is used in the manufacture of cheese. The production of this enzyme by recombinant DNA technology is now becoming possible. A new source of this enzyme to replace or supplement the animal product or similar, naturally occurring fungal enzymes will be of great economic value.     

Simultaneous Gas Chromatographic Microdetermination of Indole,Skatole and p-Cresol in Gastrointestinal Contents of Domestic Animals

Primary rat hepatocytes form discontinuous monolayers even at their maximum density. Here, we show that staurosporine promotes attachment and spreading of hepatocytes onto culture substrates, so that hepatocytes form a close, continuous monolayer. This treatment did not attenuate major hepatic functions. Therefore, this technique is promising for making seamless cell-sheet structures, which will be applicable for cell-polarity experiments or artificial liver construction.