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.     

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.     

<Emphasis Type="Italic">Symbiobacterium</Emphasis> Lost Carbonic Anhydrase in the Course of Evolution

Recent genetic studies have elucidated that carbonic anhydrase (CA; EC 4.2.1.1), a ubiquitous enzyme catalyzing interconversion between CO₂ and bicarbonate, is essential for microbial growth under ambient air but not under high-CO₂ air. The irregular distribution of the phylogenetically distinct types of CA in the prokaryotic genome suggests its complex evolutionary history in prokaryotes. This paper deals with the genetic defect of CA in Symbiobacterium thermophilum, a syntrophic bacterium that effectively grows on CO₂ generated by other bacteria. Phylogenetic analysis based on 31 ribosomal protein sequences demonstrated the affiliation of Symbiobacterium with the class Clostridia with 100% bootstrap support. The phylogeny of β- and γ-type CA distributed among Clostridia supported the view that S. thermophilum and several related organisms lost this enzyme during the course of evolution. The loss of CA could be based on the availability of a high level of CO₂ in their living environments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Consumption of barley ameliorates the diabetic steatohepatitis and reduces the high transforming growth factor β expression in mice grown in α-minimum essential medium in vitro as embryos

Non-alcoholic fatty liver disease (NAFLD), which includes the subtype non-alcoholic steatohepatitis (NASH), is a major complication of type 2 diabetic mellitus (T2DM), even among non-obese patients. However, the exact cause of NAFLD/NASH in non-obese patients with T2DM is unclear. We studied a non-obese mouse model of T2DM created through the malnourishment of embryos by culture in vitro for 48 h in α-minimum essential medium (MEM) at the two-cell stage. We compared the development of steatohepatitis in these MEM mice with control mice that were similarly cultured in standard potassium simplex-optimized medium (KSOM). We also studied the effects of 10 weeks of consumption of barley, which contains large amounts of the soluble fiber β-glucan, on the steatohepatitis of the adult MEM mice. The size of lipid droplets, the area of fibrosis, and the mRNA expression of the transforming growth factor beta (Tgfb) gene in the liver were higher in adult MEM mice fed a rice-based diet than in KSOM mice fed the same diet. However, barley consumption reduced the area of fibrosis and TGFB expression in MEM mice. In conclusion, adult mice that are cultured in MEM at the two-cell embryo stage develop steatohepatitis and T2DM, accompanied by higher hepatic TGFB expression, than KSOM controls. Furthermore, the consumption of barley during adulthood ameliorates the steatohepatitis and reduces the TGFB expression.