Synthesis and deposition of spore coat proteins during sporulation of Bacillus megaterium

Rabbit (anti-spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fraction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000-dalton protein, which is a major protein, did not cross-react with any other major (36,000, 22,000, 19,500, and 17,500-dalton) proteins. Specific antibody to the 22,000-dalton protein did not cross-react with the 48,000, 36,000, 19,500, 17,500, and 16,000-dalton proteins, but did cross-react with the 44,000, 25,000, and 12,000-dalton proteins.     

<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.     

Changes in histories during the vernalization of wheat embryos

The effect of cold-treatment on chromatin-histone in winterwheat embryos was studied. As the embryos were vernalized, theamount of whole histone increased about two-fold, but the histonecontent in excised embryos incubated in a medium containingsugar was changed little by prolonged chilling. These resultssuggest that the increase in histone content is not necessarilyconnected with the vernalizing response. Disc electrophoretic patterns of whole histone from vernalizedembryos gave a relatively distinct band of the faster movingcomponent of Fraction F-1 histone which was very scanty in non-vernalizedembryos but distinct in spring wheat embryos. Although therewas no increase in whole histone content, the faster-movingband of F-l was more conspicuous and bands of the other fractionswere fainter in the vernalized excised embryos. These observationssuggest that this particular component of Fraction F-l histoneis connected with the vernalizing response. As vernalization proceeded, the amino acid composition of wholehistone changed and the lysine-arginine ratio became higher,in agreement with the observed increase in the lysine-rich fraction(F-l) of histones. Nuclear size increased about two-fold during the period of vernalization,supporting the view that in embryos exposed to low temperature,cell division hardly proceeds. (Received September 27, 1972; )     

An improved method for large-scale purification of recombinant human glucagon

Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem. 11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.     

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.