- Volume 134, Issue 8, 1988

Incubation of human polymorphonuclear leucocytes (HPMN) with Chlamydia trachomatis elementary bodies (EB) or phorbol 12-myristate 13-acetate (PMA) resulted in the production of superoxide anions (O− 2) and hydrogen peroxide (H2O2). Exposure of HeLa cells to EB- or PMA- activated HPMN and to EB alone, for 2h, resulted in the formation of DNA strand scissions (nicks) in the HeLa cells. The nicks were visualized by incorporation of biotin 11-dUTP with its detection by streptavidin-peroxidase, and quantified by using [3H]dCTP in the in situ nuclear nick-translation reaction. Catalase, and to a lesser extent superoxide dismutase, reduced the amount of nicks induced by the EB- or PMA-activated HPMN. The possible relationship between the activity of PMN in chlamydial infections and the development of chronic diseases is discussed.

A central feature of the processes of aggregation and differentiation in the cellular slime mould Dictyostelium discoideum is the periodic excitatory cycle. Originally thought to involve primarily fluctuations in cyclic AMP levels, this excitatory cycle has since been shown to involve changes in several other second messengers including cyclic G MP, calcium and inositol trisphosphate. Previous work from this laboratory using specific inhibitors strongly suggested a role for eicosanoids in this stimulus-response process. Production of eicosanoids from fatty acid precursors is an oxygen-consuming process. In this paper, we report on oxygen consumption measurements in intact D. discoideum cells and in cell extracts. We demonstrate the existence of an azide-insensitive component of oxygen consumption which can be stimulated by the addition of arachidonate and other polyunsaturated fatty acids, and at least partially inhibited by meclofenamate and eicosatetraynoic acid, both of which block eicosanoid biosynthesis in higher organisms. These observations provide further evidence for the existence of an eicosanoid-metabolizing system in D. discoideum.

Candida albicans, grown aerobically in glucose-containing media, produced C14, C16 and C18 saturated long-chain alcohols only after the end of exponential growth. Contents of C14 alcohols were always lowest, and c16 and c18 alcohol contents about equal. Contents of all three classes of alcohol increased as the concentration of glucose in aerobic cultures harvested after 168 h incubation was raised from 1·0 to 30·0% (w/v). However, in 168 h anaerobic cultures, greatest long-chain alcohol contents in organisms were obtained using media containing 10% (w/v) glucose. Substituting glucose (10%, w/v) with the same concentration of galactose in aerobic cultures greatly decreased contents of long-chain alcohols, while inclusion of 10% (w/v) glycerol virtually abolished their synthesis. Supplementing anaerobic cultures with odd-chain fatty acids induced synthesis of odd-chain alcohols. Maximum conversion of fatty acid to the corresponding long-chain alcohol was observed with heptadecanoic acid. The effect of glucose on production of heptadecanol from exogenously provided heptadecanoic acid was similar to that observed on synthesis of the three major even-chain alcohols in media lacking a fatty-acid supplement. Cell-free extracts of organisms catalysed in vitro conversion of palmitoyl-CoA to 1-hexadecanol.

Caulobacter crescentus has an inducible pathway which is responsible for the degradation of histidine. Induction of this pathway occurs in the presence of both glucose and ammonia. Growth yield experiments indicate that only two of the three available nitrogens are used for growth suggesting that formamide may be produced as a waste product. However, formamide was not detected in the culture fluid and formate was formed instead. These results suggest that histidine may be degraded in a novel pathway which results in the production of 1 mol each of ammonia, glutamate and formate per mol of histidine. The third nitrogen from histidine appears to be sequestered in some kind of secondary metabolite.

A cytochemical staining method was developed for the demonstration of hydrogenase activity in hydrogenosomes of Trichomonas vaginalis. After fixation in low concentrations of glutaralde-hyde under strictly anaerobic conditions and incubation of these cells in the presence of H2 and the tetrazolium compound 2-(2′-benzothiazolyl)-5-styryl-3-(4′-phthalhydrazidyl)-tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. The method was used to demonstrate the hydrogenosomal nature of similar microbodies of the sapropelic ciliates Plagiopyla nasuta and Trimyema compressum.

Isogenic whi2 and WHI2+ strains of Saccharomyces cerevisiae were grown in a 2-litre bioreactor as batch cultures on a medium containing yeast extract and peptone with either glucose or ethanol as carbon and energy source. The concentration of dissolved oxygen within the medium was varied over the range of 0 to 100% saturation. Expression of the whi2 + phenotype only occurred above 40% oxygen saturation with either glucose or ethanol as carbon and energy source. Under these conditions the whi2 cells could be distinguished from whi2 + cells in that they were phase dark, highly budded and very small during the stationary growth phase, and reached final cell densities four to six times higher than WHI2+ cells. The results clearly show that the WHI2 gene of S. cerevisiae plays an important role in cell proliferation and that the availability of oxygen, or some product of oxidative metabolism, is involved in regulating the phenotypic expression of mutations within this gene.

Intracellular concentrations of acetyl-CoA and malonyl-CoA in Escherichia coli K12 were determined by a malonyl-CoA:acetyl-CoA cycling technique. Under aerobic growth conditions with glucose the acetyl-CoA and malonyl-CoA concentrations varied over a range of 0.05-1.5 nmol (mg dry wt)-1 (20-600 μM) and 0·01-0·23 mol (mg dry wt)-1 (4·90 μm), respectively. The intracellular concentration of acetyl-CoA was highest in exponentially growing cells and it fell rapidly to less than 5% of the maximum level when the organism entered stationary phase after exhaustion of glucose. A linear relationship was observed between the intracellular concentration of total acyl-CoA and the logarithm of the concentration of glucose in the medium. Consequently, the acetyl-CoA/malonyl-CoA ratios also varied drastically, in a range of 0·6-41·7, under different conditions. Of several carbon sources tested, glucose was the most effective for promoting the synthesis of cellular acetyl-CoA. For cells grown on glycerol or acetate the maximum concentrations of total acyl-CoA were significantly lower. In cells incubated with citrate (not used as a carbon source by E. coli), the level was consistent with that in cells starved for exogenous carbon sources.

Growth Characteristics Of Polypaecilum Pisce And Basipetospora Halophila, Two Fungi Isolated From Dried Salt Fish, Were Examined At 20, 30, 34 And 37 °C Over A Range Of Water Activity (aW) Values (0·99–0·70) On Media Containing Various Concentrations Of Nacl Or Glucose/Fructose. At High Aw Growth Of P. Pisce Was Similar On Media Containing Either Solute, But Below 0·90 aW Growth Was More Rapid On Nacl Media, Indicating That P. Pisce Can Be Classified As A Halophilic Fungus. The Minimum aW For Growth On Nac1-Based Media Was 0·746; On Glucose/Fructose Media The Minimum Was 0·700 aW. Optimal Growth, I.E. Growth At 80% Or More Of The Maximum Growth, Occurred Between 0·96 And 0·91 aW at 30 °C. Over the wide range of temperatures studied, B. halophila exhibited more rapid growth on NaCl media than on glucose/fructose media over the entire range of aW at which growth occurred. The optimal growth rate on NaCl- based media occurred between 0·89 and 0·86 aW, an exceptionally low value. B. halophila can be classified as an extreme halophile, markedly intolerant of high aW, particularly on glucose/fructose-based media at temperatures above 30 °C.

Dunaliella tertiolecta accumulated glycerol at a rate of 200 fmol per cell h−1, when subjected to a hyperosmotic shock of 0·930 osmol 1−1 with NaCl or mannitol. In alkaline media, a slight inhibition of the osmotic response, to 130 fmol per cell h−1 at pH 9, was found. Acetate or ammonium ions markedly decreased osmoinduced glycerol synthesis under pH conditions where the uncharged form of these agents was present at concentrations exceeding 2 mm. The proton translocators dinitrophenol (DNP) (0·3–1 mm) and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) (0·01, 0·03 and 0·05 mm) disturbed the metabolic response at all pH values tested. Also, DNP disturbed glycerol retention in low pH media, whereas CCCP did not affect the glycerol level under iso-osmotic conditions. The osmoinduced glycerol synthesis was blocked almost completely in the presence of monovalent cation ionophores such as nigericin (0·01 mm) and monensin (10 µg ml−l) but not by the K+ ionophore valinomycin (0·01 mm). With all the ionophores investigated, a correlation was found between decreasing glycerol and ATP contents, indicating that glycerol synthesis requires ATP. The results are discussed in relation to the regulation of glycerol metabolism under different osmotic conditions.

Evidence is presented for a new pathway of acetone metabolism in Thiosphaera pantotropha. The initial step involves a carboxylation, probably resulting in the formation of acetoacetate. Cells grown on acetone and propan-2-ol fixed large quantities of 14CO2, in contrast to cells grown on acetate and acetoacetate. Growth on acetone and propan-2-ol, but not on other substrates, was dependent on the exogenous supply of CO2. NMR studies on the labelling pattern of the intracellular poly-β-hydroxybutyrate (PHB) confirmed that C2 units are produced from acetone after initial carboxylation. The metabolism of acetone and propan-2-ol was associated with drastic changes in the ultrastructure of the organisms. During growth on these substrates, proteinaceous, crystalline inclusions were observed. These were absent in acetate-grown cells. The nature of these crystalline inclusions remains to be elucidated.

The total soluble proteins were extracted from 38 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) and the similarity between them was investigated by polyacrylamide gel electrophoresis. Normalized densitometric recordings of the protein profiles were computer-compared by two fundamentally different modes: peak search and position analysis, and correlation coefficient analysis. Factors influencing the reproducibility of the system were investigated. The data indicated that the PPFMs were more heterogeneous than previously believed. The results from both analyses showed a high degree of congruence with each other and with data from previous studies.