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Biomedical subjects

N Tsukagoshi

Publications and source records attributed to N Tsukagoshi.

108 records · Page 6Linked to original sources

Structure and synthesis of a lipid-containing bacteriophage. Effects of lipids containing cis or trans fatty acids on the reconstitution of bacteriophage PM2.

Infectious PM2 virus paticles could be reconstituted in vitro from a mixture of nucleocapsid, phospholipids containing cis fatty acids, and proteins I and II. The presence or absence of acyl phosphatidylglycerol, a minor lipid component of thevirion, did not affect the reconstitution of infectious particles, even though it was incorporated into the particles when present. When phosphatidylglycerol was completely replaced by acyl phosphatidylglycerol in the reconstitution mixture, no infections particles were formed. Lipids containing either cis or trans fatty acids were also used for reconstitution in vitro of the lipid-containing bacteriophage PM2. Regardless of the ratio of phosphatidlyglycerol to phospatidylethanolamine in the reconstitution mixture, infectious particles were formed and had almost the same phospholipid composition when lipids containing cis-palmitoleic acid were used; no infectious particles were obtained when lipids containing trans-palmitoleic acid were used. In the latter case, virus-like particles were, however, formed. Reconstituted particles containing cis fatty acids were infectious when tested on wild type Pseudomonas BAL-31 as well as on the unsaturated fatty acid auxotroph grown in the presence of either cis or trans-palmitoleic acid. Reconstituted particles containing trans fatty acids were not infectious on any of these cells. When trans fatty acids as well as cis fatty acids were present in the reconstitution mixture, then there was a lower yield of infectious particles. Particles with either cis or trans fatty acids had all four viral proteins and adsorbed to BAL-31 host cells in a specific manner.

Bacteriophages↗

Structure and assembly of lipid-containing viruses, with special reference to bacteriophage PM2 as one type of model system.

The simpler lipid-containing viruses (influenza, Semliki Forest, PM2) may have a phospholipid bilayer sandwiched between an outer shell of protein and an internal nucleocapsid possessing helical or icosahedral symmetry. Extensive physical and chemical studies have enabled us to form a more detailed picture of the structure of bacteriophage PM2 and controlled stepwise degradation of the virion has helped us to localize the four viral proteins. The surface protein (II) of PM2 is basic and interacts with the acidic phosphatidylglycerol of the bilayer to stabilize the membrane. The nucleocapsid protein (III) has proteolipid characteristics and may interact with the phospholipids in a hydrophobic fashion. The spikes are formed from protein I and the fourth protein (IV) is closely associated with the DNA. It is possible to reassemble the virus by reversing the degradation steps. Assembly has been especially useful in revealing the processes whereby the proteins and lipids interact to form the bilayer. Furthermore, results of in vivo studies of phospholipid synthesis and both in vivo and in vitro studies of viral protein synthesis have enabled us to form a reasonably complete picture of the biosynthesis of PM2.

Bacteriophages↗

Identification of acyl phosphatidylglycerol as a minor phospholipid of Pseudomonas BAL-31.

Compound X, a minor phospholipid of Pseudomonas BAL-31 and bacteriophage PM2, has been identified as X-3-phosphatidyl-1'-(3'-acyl)-glycerol, or acyl phosphatidylglycerol. The water-soluble product obtained by mild alkaline hydrolysis showed the same RF value as that of glycerophosphoryl-glycerol. The chemical analysis gave the ratio 1 : 3 : 2 for phosphate-acyl ester-glycerol. The position of the third acyl group was determined by nuclear magnetic resonance techniques.

Bacteriophages↗

Phase transitions in the membrane of a marine bacterium, Pseudomonas BAL-31.

An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL-31, host cell of the lipid-containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cis16:1 or trans16:1 showed one transition at 9.4 degrees C and 12.4 degrees C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL-31 had one transition at approximately 12 degrees C, on the other hand there was no clear cut phase transition using extracted lipids. Replication of bacteriophage PM2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in tran16:1. Other cases were not studied.

Bacteriophages↗

A density label for membranes.

Bromostearic acid satisfies the essential fatty acid requirement of an unsaturated fatty acid auxotroph of Escherichia coli K12. Over 55% of the fatty acids derived from lipids isolated from membranes of cells grown in bromostearate-supplemented medium contained bromine, as established by gas chromatography and mass spectrometry of the methyl esters. During equilibrium centrifugation in sucrose gradients, the cytoplasmic membranes isolated from bromostearate-grown cells banded at a density approximately 0.06 g/cm(3) greater than membranes isolated from oleate- or elaidate-grown cells, affording a complete separation between membranes which contain brominated fatty acids and those which do not.

Bacteriological Techniques↗

HAP-Like CCAAT-binding complexes in filamentous fungi: implications for biotechnology.

Regulatory CCAAT boxes are found frequently in eukaryotic promoter regions. They are bound by different CCAAT-binding factors. Until now, a single CCAAT-binding complex has been reported in fungi. It is also found in higher eukaryotes and is highly conserved among eukaryotic organisms. This multimeric protein complex is designated HAP, AnCF, CBF, or NF-Y. The complex consists of at least three subunits. In fungi, only the HAP complex of Saccharomyces cerevisiae had been known for a long time. The recent cloning of genes encoding the components of the corresponding complex (AnCF/PENR1) of Aspergillus nidulans and characterization of CCAAT-regulated genes in A. nidulans, as well as other filamentous fungi, led to a deeper insight into the role of this transcription complex, in particular in aerobically growing fungi. An overview of the function of HAP-like complexes in gene regulation in filamentous fungi is presented. Some of the genes that have been found to be regulated by HAP-like complexes encode enzymes of biotechnological interest, like taka-amylase, xylanases, cellobiohydrolase, and penicillin biosynthesis enzymes. The importance of HAP-like complexes in controlling the expression of biotechnologically important genes is discussed.

Biotechnology↗