Effect of growth substrates on morphology of Nocardia corallina.

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of L-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8-8.5 hrs doubling times) and to undergo the shphre-rod-shpere growth cycle. Other amino acids, fatty acids or surgars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.     

Effects of diazepam on photosynthesis,respiration, rubidium uptake,and finestructure of Scenedesmus obliquus in synchronous cultures

Effects of diazepam (Valium) on photosynthesis, chlorophyll/photosynthesis ratios, respiration, uptake of rubidium ions, and ultrastructure of Scenedesmus obliquus synchronized by a light-dark regimen of \(14:\overline {10}\) hrs were determined. 80 and 160 μM diazepam, added to the nutrient medium at the start of the light-dark change (i.e., start of the cell cycle) gradually reduced rates of photosynthesis below the initial rates from the beginning of the experiment. Contents of chlorophyll, however, remained nearly unaffected. Consequently, the diazepam-treated cells had a higher chlorophyll/photosynthesis ratio—also with regard to respiration in order to calculate the gross photosynthesis. The occurrence of photorespiration cannot be assumed. The net influx or rubidium was slightly reduced by 100 μM diazepam 0.5 and 2.0 hrs after the start of the cell cycle and was strongly inhibited after 5 to 14 hrs. 80 and 160 μM diazepam caused separation of thylakoids, formation of giant mitochondria and enlargement of vacuoles. The results are discussed and it is finally suggested that diazepam acts on different membrane systems. Furthermore an ATP deficiency cannot be excluded.     

Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Regulation and characterization of two inducible amino-acid transport systems in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two amino-acid transport systems in Chlorella vulgaris: an arginine system (arginine and lysine) and a proline system (proline, glycine, alanine and serine). the same amino-acid transport systems are induced in the absence of glucose, when the cells are depleted of their nitrogen source as judged by a comparison of K_m values and the lack of additive induction by the two treatments Changes in the concentration of neither internal free amino acids nor of soluble carbohydrate pools correlate prefectly with the induction of amino-acid transport. Also exogenous cAMP had no effect on the induction of transport. Both aminoacid transport systems are able to accumulate free amino acids more than 1000-fold. The accumulation plateau is not due to a steady state of influx and efflux, but rather arises by a shut-off of influx. No significant effux is observed. The biological importance of this frequently observed behaviour in amino-acid transport is discussed.     

Agromonas oligotrophica gen. nov., sp. nov., a nitrogen-fixing oligotrophic bacterium

Phenotypic and chemotaxonomic characteristics of five isolates of acetylenereducing (nitrogen-fixing) oligotrophic bacteria from a paddy soil were investigated. They showed similar phenotypic characteristics: they were aerobic, asporogenous, gram-negative, motile by a polar flagellum, and irregular rods. On full strength nutrient broth (NB) growth was severely suppressed, but well supported on 10-to 10000-fold diluted NB. They consumed glucose but produced no acid, and also utilized phenolic acids such as ferulic acid or p-coumaric acid. The cellular fatty acid composition, quinone system and DNA base composition of the isolates were investigated. Cellular fatty acids mainly consisted of straightchain unsaturated C_{18 : 1} (62–81% of total fatty acids). Ubiquinone Q-10 and a high guanine-plus-cytosine content (65.1–66.0 mol%) were found. The taxonomic status of the isolates is discussed and a new genus, Agromonas, with a single species Agromonas oligotrophica sp. nov., is proposed for these isolates. The type strain of A. oligotrophica is JCM 1494.     

Growth Characteristics of Bartonella henselae in a Novel Liquid Medium: Primary Isolation, Growth-Phase-Dependent Phage Induction, and Metabolic Studies

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 × 10⁸ CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.     

Growth of Escherichia coli B/r/l in a semi-continuous system designed for the synchronization of cell division

Escherichia coli B/r/l was grown under conditions of periodic feeding. Glucose, the only carbon source, was supplied at intervals longer than the generation time of the organism. Thus, each period of glucose availability was followed by a period of depletion. This process gave rise to two synchronous populations, one dividing shortly after a new supply of fresh medium and the other dividing at a later stage within the feeding cycle. Thymine incorporation experiments suggested that the double population emerged as the result of a discriminatory blockage of DNA replication.     

Isolation and description ofHaloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophile common to Great Salt Lake sediments

A new halophilic species is described that was isolated from the hypersaline (>20%) surface sediments of Great Salt Lake, Utah, via transfer from MPN end-dilution tubes that contained a complex organic medium. The organism was an obligate anaerobe that proliferated optimally at approximately 13% salt, but did not grow significantly at <2% or ≥30% salt. It stained Gram-negative, was nonmotile, nonsporing, and contained an outer-wall membranous layer. The complex lipids of the organism were fatty acid esters that did not change dramatically during growth at 5% or 25% NaCl. The DNA base composition was 27.0±1 mol% guanosine plus cytosine. The temperature range for growth was >5°C and <60°C, the pH range was between 6.0 and 9.0. The doubling time for growth in complex medium with 25% NaCl was 7 h. The organism utilized carbohydrates, peptides, and amino acids. Butyrate, acetate, propionate. H₂, and CO₂ were the major fermentation end products formed. Glucose, mannose, fructose,n-acetyl glucosamine, and pectin were used as energy sources for growth. Methylmercaptan was produced from methionine degradation. The nameHaloanaerobium praevalens gen. nov. sp. nov. is proposed for the type strain GSL which has been deposited as DSM 2228. The taxonomic relationships ofH. praevalens to other obligate halophiles and anaerobes, as well as its biological role in the Great Salt Lake microbial ecosystem, are discussed.     

Composition of the cell wall and other fractions of the autolyzed yeast form of Histoplasma capsulatum

Comparison of two strains ofHistoplasma capsulatum yielded data differing only in quantification, and the constituents observed and identified were galactose, glucose, mannose, glucosamine and amino acids. A comparison of hydrochloric acid and formic acid hydrolyses ofH. capsulatum fractions indicated hydrochloric acid to be of more value than 88 per cent formic acid hydrolysis for composition analyses. The removal of formyl esters from formic acid hydrolysates was found necessary and was accomplished byN HCl hydrolysis for 30 min. Two derivative artifacts were observed with formic acid hydrolysis; D-1, which was refractory to subsequent HCl hydrolysis, and D-2, which disappeared after HCl hydrolysis. Another artifact, D-3, was observed with 6N HCl hydrolysis of histoplasma cell wall fractions. The following conditions of hydrolysis were found to be useful: (1) glucose release was measured after hydrolysis inN HCl for 4 hr; (2) glucosamine release was measured after hydrolysis in 6N HCl for 9 hr; (3) amino acid release was accomplished by 6N HCl hydrolysis for 18 hr; and (4), hexoses released were determined by gas liquid chromatography (GLC) after hydrolysis in bothN HCl and in 88 per cent formic acid for 24 hr, followed byN HCl for 30 min. Several different types of carbohydrate polymers have been reported in the parasitic yeast form ofH. capsulatum. There is general agreement on the occurrence of amino acids as protein (8, 12, 13), chitin (7, 19) and several hexoses, including glucose and glucosamine, which are found in cell wall polymers (7, 8, 11–16, 19, 20, 24). The presence of uronic acid was also reported (14, 15), but not confirmed, by Domer, Hamilton & Harkin (8), and mannose was not found by all investigators (12). We undertook a study of graded acid hydrolyses and of composition analysis of the autolysis products of the yeast form by various procedures in order to add further to the above information.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

Phylogenetic significance of protein and nucleotide content in yeasts

A detailed summary of the content and composition of total proteins, RNA and DNA in 57 yeast-like microorganisms is presented. On the basis of the correlation between the content of amino acids in proteins the studied strains could be divided into 8 groups that differ not only in the content of proteins and amino acids but also in the RNA and DNA content. According to this characteristic the groups ofBasidiomycetes andAscomycetes could be discriminated. The present study should also serve as orientation for the screening of strains suitable for the production of fodder yeasts.     

Selection and characterization of acriflavine-induced mutants of Candida albicans

Acriflavine-treated cells of C. albicans plated on a medium containing glucose as the principal carbon source gave rise to numerous colonies, most of which grew when replica-plated onto a similar medium containing sodium acetate substituted for glucose. Of the small fraction of colonies from the glucose medium which failed to replicate, some were found to be true petites, deficient for cytochromes a and b; others possessed complete cytochrome spectra, like those of their wild-type parents, but, nevertheless, respired at reduced rates on both fermentable and non-fermentable substrates. The role of the conventional cytochrome system in a wild-type culture was indicated by the strong inhibition of its respiration by cyanide, azide and antimycin A.     

Polysaccharide production and the possible occurrence of GDP-d-mannose dehydrogenase inAzotobacter vinelandii

During the growth ofAzotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50mg EDTA per litre, water-insoluble capsular polysaccharide material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnose, guluronic acid and mannuronic acid. The activity of GDP-d-mannose dehydrogenase recorded in crude cell-free extracts fromAzotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.     

Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters

Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C₄ and C₅ acids, and branched and straight chain C₁₀, C₁₁, and C₁₂ acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [¹⁴C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C₄ and C₅ acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C₄ and C₅ branched acids were elongated by two carbon units to produce the branched C₁₀-C₁₂ groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters.     

An inducible riboflavin synthetase from a pseudomonad

Riboflavin synthetase activity is induced in a strain ofPseudomonas fluorescenspulida intermediate only under conditions permitting an accumulation of compound P, a 2,4-dioxopteridine, in the medium. The effect of different amino acids and sugars on the production of compound P and riboflavin synthetase was determined. The enzyme was partially destroyed by ammonium sulphate fractionation. It is inhibited by heavy metal ions and PCMB. PCMB inhibition can be almost completely reversed by GSH.     

Bile acid supplementation improves established liver steatosis in obese mice independently of glucagon-like peptide-1 secretion

Bile acids or its derivatives may influence non-alcoholic fatty liver disease development through multiple mechanisms. Intestinal L-cells secrete glucagon-like peptide-1 (GLP-1) and can be activated by bile acids (BA) influencing insulin resistance and hepatic steatosis development and progression. The aim of the present study was to assess the effects of cholic acid (CA) or ursodeoxycholic acid (UDCA) administration on portal and systemic levels of GLP-1 in genetically obese mice with established hepatic steatosis. Eight-week-old ob/ob mice were fed CA or UDCA during 4 weeks. Systemic and portal GLP-1 levels were measured as well as glucose tolerance test, serum and biliary parameters, hepatic triglyceride content, liver histology, and hepatic gene expression of relevant genes related to bile secretion. Eight-week-old ob/ob mice exhibited marked obesity, hyperinsulinemia, and fasting hyperglycemia. Administration of both CA and UDCA was associated to decreased hepatic triglyceride content and complete reversion of histological steatosis. BA-fed animals did not exhibit significant differences in glucose tolerance. In addition, neither CA nor UDCA administration significantly influenced portal or systemic GLP-1 levels. CA and UDCA strongly ameliorated established fatty liver in ob/ob mice independently of the GLP-1 incretin pathway. Thus, the anti-steatotic action of these bile acids is likely related to direct hepatic effects.     

P53 induction accompanying G2/M arrest upon knockdown of tumor suppressor HIC1 in U87MG glioma cells

Hypermethylated in cancer 1 (HIC1) is a novel tumor suppressor gene (tsg) frequently silenced by epigenetic modification, predominantly by methylation in different tumors. HIC1 functionally co-operates with p53 in cultured cells as well as in transgenic animals to suppress tumors and has binding site on its promoter. Its over expression often leads to cell cycle arrests. Although HIC1 proven to have role as tsg, its regulation to cell cycle and dependency upon p53 is grossly unknown. In this study, we investigated the role of HIC1 in cell cycle and proliferation of glioma cell line U87MG which has wild type p53, in both serum-containing and serum-deprived medium. Microscopic analysis and MTT assay showed reduced cell number and rate of proliferation upon HIC1 knock down compared to control siRNA (p = 0.025) and untreated cells (p = 0.03) in serum-containing medium and serum-free medium (p = 0.014 vs control siRNA; p = 0.018 vs untreated cells). Cell cycle analysis revealed an arrest at G2/M phase of cell cycle with no demonstrable increase in apoptosis with both medium. An increased expression of p53 concomitant with HIC1 knockdown was observed. Furthermore P21, a p53 responsive gene, along with p27 was significantly increased in comparison with controls. Our results demonstrated an important role of HIC1 for the normal progression of cell cycle, and at molecular level, it could affect the homeostasis of p53 as well as number of cell cycle-related genes, which may or may not be directly linked to p53.