Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc.

Zymomonas mobilis is an unusual microorganism which utilizes both iron-containing alcohol dehydrogenase (ADHII) and zinc-containing alcohol dehydrogenase (ADHI) isoenzymes during fermentative growth. This organism is obligately ethanologenic, and alcohol dehydrogenase activity is essential. The activities of ADHI and ADHII were altered by supplementing growth medium with iron or zinc salts and by iron starvation. Growth under iron-limiting conditions (chelators, minimal medium) reduced ADHII activity but did not prevent the synthesis of the ADHII protein. The inactive form of this enzyme appeared quite stable, was not renatured by iron addition, and persisted in the cell. The iron-induced increase in ADHII activity required de novo synthesis which was blocked by antibiotic additions. The ability of Z. mobilis to synthesize ADHII and ADHI may be advantageous in nature.     

Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis.

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.     

The distribution of blood group antigens in rodent epithelia

Summary The pattern of distribution of antigens cross-reacting with antibodies to human blood group antigens A and B and two precursor molecules was examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice. Staining for blood group antigen A was negative. In all epithelia examined, blood group antigen B was present at the surface of basal and parabasal cells, and the H antigen at the surface of spinous cells. N-acetyllactosamine was present on the cell membranes in the upper spinous and granular cell layers of epidermis and forestomach epithelium and was not expressed in the oral epithelia except for a limited area in the dorsal tongue epithelium.Thus, the expression of antigen varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The observed sequence of expression of these antigens during maturation differs from that of human epithelia, but the present study provides a basis for further experimental studies of the role of cell surface antigens in epithelial homeostasis and maturation.     

Connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse

Summary Epithelial-mesenchymal interactions play an important role during embryogenesis but it is uncertain whether such interactions influence the maintenance of epithelial structure in the adult. To examine this problem, separated epithelial and connective tissue components of skin and mucosae from various regions of adult mice were homoor heterotypically recombined and transplanted to histocompatible hosts. The patterns of tissue architecture and keratinization of the resultant epithelia were examined for changes indicative of mesenchymal influences on the epithelial phenotype. Each type of epithelium, in some recombinations, fully conserved its normal pattern of phenotypic expression indicating that subepithelial connective tissue from all regions is permissive and that regionally-specific connective tissue influences are not necessary for conservation of epithelial specificity. In other recombinations, however, the epithelium acquired features of tissue architecture or keratinization typical of the epithelium normally associated with the connective tissue component, indicating directive influ ences from the connective tissue. The patterns of epithelial response observed suggest that there may be separate connective tissue influences on epithelial architecture and cytodifferentiation and that there is a regionally-related variation in the competence of epithelia to respond to these influences.Supported by NIH NIDR 2 R01 DE05190     

Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.     

Glycosylation of 5-Hydroxy-4-cyanoimidazole

Abstract

The sodium derivative of 5-hydroxy-4-cyanoimidazole with 2, 3, 5-tri-O-benzoylribofuranosyl bromide gave 5-(2, 3, 5–tri-0-benz-oyl-β-D-ribofuranosyl) oxy-4-cyanoimidazole whereas with 2, 3, 5-tri-0-benzyl-α/&β-D-arabinofuranosyl)-4–cyanoimidazole was produced. The compounds were characterised by ¹H-n.m.r. and mass spectrometry.