Phosphatidylinositol is an essential phospholipid of mycobacteria

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.     

Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria

We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.     

Effects of environmental temperature on life tables of Rhodnius neivai Lent, 1953 (Hemiptera: Reduviidae) under experimental conditions.

Changes in life tables of Rhodnius neivai due to variations of environmental temperature were studied, based on nine cohorts. Three cohorts were kept at 22 degrees C, three at 27 degrees C and three at 32 degrees C. Cohorts were censused daily during nymphal instars and weekly in adults. Nine complete horizontal life tables were built. A high negative correlation between temperature and age at first laying was registered (r=-0,84). Age at maximum reproduction was significantly lower at 32 degrees C. Average number of eggs/female/week and total eggs/female on its life time were significantly lower at 22 degrees C. Total number of egg by cohort and total number of reproductive weeks were significantly higher at 27 degrees C. At 32 degrees C, generational time was significantly lower. At 27 degrees C net reproductive rate and total reproductive value were significantly higher. At 22 degrees C, intrinsic growth, finite growth and finite birth rates were significantly lower. At 22 degrees C, death instantaneous rate was significantly higher.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Hemolymph patterns of free amino acids in the brine shrimp Artemia franciscana after three days starvation at different salinities

The hemolymph pattern of free amino acids was examined in the brine shrimp, Artemia franciscana (Great Salt Lake origin). After one-month acclimation to 35 or 60 ppt salinity at 27 degrees C, the animals were transferred to 10, 35 or 60 ppt salinities to continue acclimation for 3 days without feeding at 27 degrees C. The osmolarity of one of the new media was raised by glycerol addition. In the hemolymph, 8 amino acids such as taurine, alanine, threonine, serine, lysine, glycine, arginine and leucine, comprised approximately 70% of the total content of free amino acids. This pattern suggested internal proteolysis due to starvation at high temperature. The total content of free amino acids significantly increased at 10 and 60 ppt salinities in comparison to 35 ppt. The hemolymph patterns from the 10 ppt and glycerol-added media showed a singularly high peak of taurine or alanine.     

Stimulus-aggregation coupling in platelets activated with PAF-acether

Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.     

Production and characterization of functional substances from a by-product of rice bran oil and protein production by a compressed hot water treatment

A by-product of rice bran oil and protein production was treated with water and compressed hot water at 20 degrees C to 260 degrees C for 5 min, and at 200 degrees C and 260 degrees C for 5 to 120 min. Each extract was evaluated for its yield, radical scavenging activity, carbohydrate, protein, total phenolic and furfural contents, molecular-mass distribution and antioxidative activity. The maximum yield was obtained at 200 degrees C. The radical scavenging activity and the protein, total phenolic and furfural contents of the extract increased with increasing temperature. However, the carbohydrate content abruptly decreased when treated at above 200 degrees C. The extract treated at 260 degrees C for 5 min exhibited suppressive activity toward the autoxidation of linoleic acid. Each extract obtained at temperatures lower than or equal to 200 degrees C exhibited emulsifying ability.     

Effects of zinc on lipids of erythrocytes from carp (Cyprinus carpio L.) acclimated to different temperatures

We compared the effect of zinc (0.01, 0.1, 0.5 and 1 mM) at two temperatures (5 and 20 degrees C) on erythrocytes from summer and winter acclimatised carp. An increase in temperature from 5 to 20 degrees C increased the unsaturation index (UI) and relative proportion (UI/SFA) of unsaturated to saturated fatty acids in total lipids of the red cells. At 5 degrees C, the unsaturation index of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) decreased (30-40%) in the presence of 1 mM zinc. The change in unsaturation of phospholipids in the presence of zinc at 5 degrees C is probably responsible for the alteration in structural integrity of erythrocyte membrane as observed by hemolysis and the decreased thiol group content in the erythrocytes. In light of this result, zinc may be considered an environmental hazard for these fish at low temperatures.     

Variation in lipid A structure in the pathogenic yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37 degrees C, Y. pestis lipopolysaccharide (LPS) contained the tetra-acylated lipid IV(A) and smaller amounts of lipid IV(A) modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV(A) modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra-acylated lipid A. When grown at 21 degrees C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-alpha, whereas the lipid A synthesized by the three species at 37 degrees C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature-dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.     

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.     

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.     

Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts

The trehalose content of exponentially growing Saccharomyces cerevisiae cells rapidly increased in response to a temperature shift from 27 to 40 degrees C and decreased again when the temperature was shifted back from 40 to 27 degrees C. These changes were closely correlated with increases and decreases in the thermotolerance and desiccation tolerance of the cells. Our results support the hypothesis that trehalose functions as a protectant against heat and desiccation.     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

工厂化黄瓜穴盘育苗昼温适应性

在人工气候室内以黄瓜穴盘苗为材料,测定不同昼温处理下(昼温分别为30℃、27℃、24℃、21℃、18℃、15℃,夜温均为15℃)黄瓜幼苗下胚轴长、下胚轴粗、第一叶片和第二叶片的长和宽、地上部和地下部干物质积累量、叶片含水率及叶片的叶绿素荧光特性,并用主成分分析法和聚类分析法对不同昼温处理下的黄瓜穴盘苗质量进行分析.结果表明:不同昼温处理下黄瓜穴盘苗各生长指标存在显著性差异,幼苗质量的昼温反应表现为24℃＞21℃＞27℃＞30℃＞18℃＞15℃;通过主成分分析和系统聚类可以把各温度处理分为:最适温度处理(24℃/15℃)、适宜温度处理(21℃/15℃)和不适宜温度处理3类;不适宜温度处理又可分为高温抑制类(27C/15℃,30℃/15℃)和低温抑制类(15℃/15℃,18℃/15℃)2类.     

DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.     

Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from Chinese hamster cells

Four heat-resistant variants were isolated after treatment of Chinese hamster lung cells with the mutagen ethyl methane sulfonate, followed by a single-step selection procedure consisting in a severe hyperthermic treatment of 4 h at 44 degrees C. The isolated clones had a stable resistant phenotype for at least 150 generations during which they showed a 5,000-fold increased survival to a 4-h treatment at 44 degrees C when compared to wild-type cells. Comparative two-dimensional electrophoretic analyses of proteins revealed that, like induced thermotolerant wild-type cells (i.e., cells induced to a transient physiological state of thermotolerance by a sublethal heat conditioning treatment administered 18 h before), the heat-resistant variants had, at normal temperature, an increased content of a heat-shock protein with Mr of 27,000 (HSP27). In three of the four heat-resistant variants, the increased content of HSP27 was correlated with a two-fold increase in the constitutive level of the mRNA encoding HSP27. Chinese hamster HSP27 is composed of three species that differ in their relative isoelectric point, among which the two most acidic forms are phosphoproteins. In both the heat-resistant variant and wild-type cells, heat shock induces a rapid enhancement of the phosphorylation of HSP27: maximal phosphorylation occurs within 10 min upon changing the incubation temperature from 35 degrees to 44 degrees C. A concomitant shift in silver-staining intensity is rapidly detectable between the three isoforms, which seems to indicate that the two phosphorylated species represent post-translational modifications of the more basic species. It is concluded that most likely the enhanced expression of HSP27 is linked to the resistant phenotype of the variants. The study provides supporting evidence that both the content and phosphorylation status of HSP27 are determining factors in the ability of cells to survive hyperthermic treatments.