Samuel M. Rosen

Lipopolysaccharide of the Gram-Negative Cell Wall Biosynthesis of a complex heteropolysaccharide occurs by successive addition of specific sugar residues

The use of mutants of Salmonella typhimurium in which biosynthesis of specific lipopolysaccharide precursors is blocked has made possible both biosynthetic studies and structural analyses which provide the basis for the structure of the core polysaccharide shown in Fig. 6. The simplest mutant, which is unable to synthesize UDP-glucose, forms only the backbone structure, containing heptose, phosphate, and keto-deoxyoctonate. To this backbone are attached side chains containing glucose, galactose, and N-acetylglucosamine. The resulting core structure is found in the lipopolysaccharide of the rough strain, as well as in that of the GDP-mannose- deficient mutant. In the wild type organism, long O-antigenic chains composed of repeating units containing galactose, mannose, rhamnose, and abequose are linked to the core, perhaps to the N-acetylglucosamine residue, as indicated in Fig. 6. The rough phenotype could presumably arise from mutation either at the level of nucleotide sugar synthesis or at some stage in assembly or attachment of the O-antigenic side chains. The pathways of nucleotide sugar synthesis appear to be normal in most rough strains of S. typhimurium (42), a finding which suggests loss of a lipopolysaccharide transferase reaction in these mutants. The site of the enzymatic defect has not yet been established in these cases, but two distinct genetic types of rough mutants have been detected (18). It is interesting to speculate about the function of the lipopolysaccharide. The lipopolysaccharide can account for as much as 5 percent of the dry weight of the cell, and its synthesis clearly involves major expenditure both of energy and of material. Yet loss of the antigenic side chains, or even of a major part of the core structure, appears to have little or no effect on the ability of the organism to survive under laboratory conditions, since the rough and mutant strains grow as well as the wild type does. However, only the wild types, possessing the complete antigenic side chains, are pathogenic. It is possible that the lipopolysaccharide is an important factor in aiding the bacterium to evade host defense mechanisms, such as phagocytosis. Such a role is well established for the capsular polysaccharides of the pneumococci. No mutants have thus far been detected which lack the backbone or lipid portions of the lipopolysaccharide. It may be that these parts of the lipopolysaccharide play an essential role in the physiology of the organism

Properties of an adenyl cyclase partially purified from frog erythrocytes

Abstract A particulate adenyl cyclase has been purified 150–200 fold from the erythrocytes of adult Rana Pipiens . The enzyme catalyzed the synthesis of cyclic 3′,5′-AM 32 P from α-labeled AT 32 P in the presence of NaF, Mg 2+ or Mn 2+ and a sulfhydryl compound. Catecholamines stimulated enzymic activity in the absence of fluoride to the extent of 50–75% of that demonstrable in the presence of fluoride. The ability of different catecholamines to stimulate enzymic activity paralleled their potency as β-adrenergic compounds. Sensitivity to stimulation by catecholamines was a labile function preserved only by storage of the enzyme in liquid N 2 . The purified adenyl cyclase fraction did not contain cyclic 3′,5′-nucleotide phosphodiesterase activity, exhibited optimal activity at pH 8.0, and was inhibited by Zn 2+ or Ca 2+ . dATP was the only nucleoside triphosphate other than ATP which was able to serve as a substrate for the formation of a cyclic 3′,5′-nucleotide. Phase microscopy, density gradient centrifugation, enrichment for ATPase activity, and the content of sialic acid and lipid phosphorus all suggested that the particulate enzyme was a fragment of the cell membrane.

Purification and properties of a specific fructose 1,6-diphosphatase from Candida utilis☆

Abstract A procedure is described for the isolation of crystalline fructose 1,6-diphosphatase from Candida utilis. The enzyme specifically hydrolyzes the C-1 phosphate group of fructose 1,6-diphosphate with the production of stoichiometric amounts of fructose 6-phosphate and Pi. It has a molecular weight of approximately 100,000, an alkaline pH optimum (9.0–9.5), a requirement for Mg++ or Mn++, and a low Km for fructose 1,6-diphosphate. This enzyme shows no activity at pH 7.5–8.0 unless EDTA is added. With 0.1 mM EDTA the activity becomes nearly equal to that observed at pH 9.5 in the absence of EDTA. This effect of EDTA was also observed in crude extracts. Other compounds, including histidine, diethyldithiocarbamic acid, cysteine, KCN, o-phenanthroline, glutathione, and α,α-dipyridyl, also induced enzymic activity at pH 7.5 but much higher concentrations were required than with EDTA. The induced activity at pH 7.5 was strongly inhibited by low concentrations of AMP. In the presence of 1.0 mM fructose 1,6-diphosphate there was no inhibition of enzymic activity by equivalent concentrations of sedoheptulose 1,7-diphosphate, fructose 6-phosphate, or fructose 1-phosphate. The enzyme was relatively insensitive to inhibition by Pi, and diisopropylfluorophosphate; significant inhibition occurred with Pb++, Ca++, and fluoride.

A bacterial activator of frog erythrocyte adenyl cyclase

Abstract Several preparations of clostridial neuraminidase were found to contain a substance which was an activator of the adenyl cyclase present in the membrane of the frog erythrocyte. Activation was most marked in the presence of catecholamines although it was also detectable in the presence of NaF or in the absence of either fluoride or catecholamines. The activator was neutralized by a commercial polyvalent anticlostridial antitoxin. It was characterized as nondialyzable, relatively heat stable, insensitive to treatment with DNAse or RNAse, and separable, by electrophoresis, from neuraminidase activity. The activator did not stimulate adenyl cyclase activity in tadpole erythrocytes or in rabbit cerebral cortex, preparations which are not stimulated by β-adrenergic amines. When added to frog erythrocyte adenyl cyclase, it potentiated the activity of only those catecholamines which fulfilled the structural requirements for stimulating this enzyme.

Assessing the Treatment of the Alcoholic Patient in an Urban Hospital

During the six month period June — December, 1976, 365 of 4,500 adult medical patients were discharged with either a primary or secondary diagnosis of alcoholism at a large New York City medical center. These patients were treated on either a general medical service or a special alcoholism unit within the center complex. Generally, those patients with alcoholism secondary to another problem (e.g., pneumonia) were admitted to general medicine, while those who voluntarily sought treatment for alcoholism or whose medical problems were minor or easily controllable were admitted to the alcoholism unit.