Enhanced bactericidal activity of platelet β-lysin forE. coli in the presence of membrane perturbation

Inhibition by sodium chloride of the growth of 19 strains ofLegionella pneumophila and of 10 strains of otherLegionella spp. was studied. Results from growth in buffered -ketoglutarate cysteine yeast extract (BAYE) broth containing 0 to 2.0% sodium chloride indicated that 15/19 laboratory strains ofL. pneumophila were capable of growing in 1.0% to 1.5% sodium chloride, whereas 4 strains ofL. pneumophila and 10 strains of 6 other species were not.L. micdadei andL. longebeachae were the most inhibited in BAYE broth, growing only in concentrations of 0.5% sodium chloride. These in vitro studies indicate thatL. micdadei andL. longbeachae might be differentiated from other species by their low tolerance to salt in BAYE broth, and thatL. pneumophila may be more tolerant to salt concentrations found in brackish water environments.     

Influence of media and temperature on bacteriocin production by<Emphasis Type="Italic"> Bacillus cereus</Emphasis> 8A during batch cultivation

Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil. The influence of temperature and media on the growth of B. cereus 8A and the production of this bacteriocin was studied during batch cultivation. Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml^–1. Bacteriocin was also produced in peptone, MRS, Mueller–Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth. Temperature had a strong influence on bacteriocin production, which was higher at 30 °C than at 25 °C. An important decrease in bacteriocin activity was observed at 37 °C. The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth. Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.     

Survival ofLegionella pneumophila in the aquatic environment

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [³H]thymidine-labeling. The [³H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 μm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: concentrations in Morris hepatomas of different growth rates

Cyclic AMP and cyclic GMP levels were examined in Morris hepatoma explants in vivo. All eight tumor lines examined had significantly elevated cyclic AMP and cyclic GMP levels when compared to normal liver from tumor-bearing rats. No apparent correlation was observed between the rates of tumor growth and cyclic nucleotide levels; however, two tumor lines (3924A and 7288ctc) had very high levels of cyclic GMP.     

Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3'',5''-monophosphate.

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

Temperature sensitivity of flocculation induction,conjugation and sporulation in fission yeast

Homothallic cultures of Schizosaccharomyces pombe, anaerobically grown to stationary phase in broth at 32°C, were induced by aeration to flocculate. Flocculation was followed by copulation, conjugation, zygote formation, meiosis and sporulation. Cultures grown to stationary phase at 32°C and then aerated at 37°C did not sporulate. Grown to stationary phase at 37°C, cultures were not immediately inducible when aerated at 32°C. To identify which events in the developmental sequence were thermosensitive, we grew and induced cultures at 32°C and then shifted them at various times to 37°C. We observed the following events to be thermosensitive: development of respiratory sufficiency, readiness (inducibility of a culture within 1 h), flocculation induction, copulation, conjugation and early sporulation (including meiosis). Respiration, flocculation and spore maturation were thermoresistant. Conjugation-induced lysis and post-developmental deflocculation were enhanced at 37°C.NRCC no. 17775     

CYCLIC GMP IN A NEUROBLASTOMA CLONE: POSSIBLE INVOLVEMENT IN MORPHOLOGICAL DIFFERENTIATION INDUCED BY DIBUTYRYL CYCLIC AMP

Abstract— DBcAMP induces morphological differentiation of mouse neuroblastoma cells grown in culture. DBcGMP or 8-Br cyclic GMP when added alone also induces a discrete morphological differentiation. When analogues of cyclic GMP were added together with dBcAMP, neurite outgrowth was strikingly enhanced as compared to the effect of dBcAMP alone. Intracellular concentrations of cyclic GMP were increased during dBcAMP treatment and cyclic AMP levels were increased during 8-Br cyclic GMP treatment. Both treatments produced an increased protein kinase activity, supporting the possibility that not only cyclic AMP but also cyclic GMP may be involved in the differentiation process.     

Influence of cyclic 3′,5′-adenosine monophosphate on uracil uptake by rifampicin treatedEscherichia coli cells

Summary Incubation of cells from a wild type strain ofE. coli with 0.3 mg/ml rifampicin for 15 minutes lead to a complete inhibition of RNA synthesis measured as the uracil incorporation into the trichloroacetic acid insoluble fraction. In these rifampicin-treated cells [¹⁴C]uracil incorporation tended to decrease during a further incubation at 37°. Addition of cyclic AMP increased the inactivation of the system responsible for [¹⁴C]uracil uptake. The cyclic nucleotide effect seems to be specific since ATP or 5AMP did not increase such inactivation.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Effect of Cold Temperatures on the Viability of Chromobacterium violaceum

The effect of low, nonfreezing temperatures on the viability of five strains of Chromobacterium violaceum was studied. The viability of cultures grown at 30 C was determined after exposure to various diluents held at 0 to 2 C. A culture diluted at its growth temperature served as the control. Cells of strain N were most sensitive in the early part of the exponential phase of growth. Cells of strains 252 and 341 were most sensitive in the late exponential, early stationary phase of growth. Cells of strain 9 showed greatest loss of viability during the maximal stationary phase. Strain 69 was completely resistant throughout its growth cycle to cold injury. Cell viability was much greater in buffered salts solution than in distilled water, broth, or physiological saline, whether cultures were diluted at room temperature or in the cold. The proportion of cells surviving after exposure to cold, however, was the same regardless of the composition of the diluent. Loss of viability was progressive at 0 to 2 C and reached a maximum after 2 hr. There was no loss of viability of cells exposed to 20 C, but there was some loss at 12 C. Strain 341 cultivated at 15 C was much less sensitive to 0 to 2 C than when it was cultivated at 30 C. The composition of the growth medium seemed to have no effect on the survival of cells exposed to cold. The polyamines, spermine and trimethylenediamine, as well as erythritol and sucrose, exerted some protective action against the effects of cold but not uniformly for all strains studied.     

Induction of competence in nonencapsulated and encapsulated strains ofHaemophilus influenzae

A simple procedure for induction of competence in nonencapsulated and encapsulated strains ofHaemophilus influenzae is described, which consists of growing cells without shaking in brain-heart infusion broth under aerobic conditions. Competence emerged at the end of the exponential phase and reached a peak at the stationary phase. InH. influenzae Rd competence was maintained for at least 6 h at 37°C, whereas in two encapsulated clinical isolates ofH. influenzae type b a decrease in competence was observed after 4 h. Competence was maintained for 24 h at 22°C and 4°C as well as by freezing the cells in 15% glycerol and storing them at –70°C. Transformation frequencies of three chromosomal markers—streptomycin, nalidixic acid, and erythromycin resistance—were 0.5% to 1% inH. influenzae Rd and about tenfold lower in the two encapsulated clinical isolates ofH. influenzae type b. The advantage of this procedure is that it is simpler than the previously described procedures and yields stable, highly transformable cells. Unlike the standard M IV method, the static aerobic procedure does not interfere with the capsule synthesis and can be used for testing transforming activity of encapsulated virulent isolates ofH. influenzae.     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

Comparative lipid content ofCandida lipolytica grown on glucose and onn-hexadecane

Candida lipolytica, grown onn-hexadecane as the sole source of carbon and energy, contained 17.1% lipids in the logarithmic phase of growth, and 7.3% lipids in the stationary phase of growth. When the yeast was grown on glucose, it contained 6.2% lipids in the logarithmic phase of growth, and 3.6% lipids in the stationary phase of growth. Fatty acids, that could be extracted by petroleum ether after saponification, constituted the major part of the fatty acids ofC. lipolytica in its logarithmic phase of growth on glucose. They constituted only a minor amount of the fatty acids in the stationary phase of growth on glucose. The reverse was true when the yeast was grown onn-hexadecane. The broth contained more free, petroleum ether-soluble fatty acids when the cellular lipid content was high than when it was low. Overnight starvation ofC. lipolytica grown onn-hexadecane in a carbon-free nutrient medium, removed the residual cell-bound hydrocarbon, increased the cell population by one half and decreased the cellular lipid content (as % of dry yeast) by one third. Various methods for the determination of lipids, described as appropriate for yeasts were compared. The highest yields were obtained by extraction of the freeze-dried paste, at room temperature, with a 1:1 chloroform-methanol mixture.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

cGMP、cAMP及两种cAMP衍生物对大鼠肝细胞核体外转录系统的影响

 本文介绍了以α-鹅膏蕈碱和低浓度KCl为手段建立了RNA聚合酶Ⅰ、RNA聚合酶Ⅱ活性的细胞核转录系统进而研究了cGMP、cAMP、cAMP丁酯及cAMP硫代环磷酰二乙胺对大鼠肝细胞核中RNA聚合酶Ⅰ与Ⅱ活性的影响。结果显示cGMP可以提高RNA聚合酶Ⅰ的活性;cAMP主要提高RNA聚合酶Ⅱ的活极,而cAMP分子结构变化产生的丁酯及硫代环磷酰二乙胺衍生物可增强cAMP的这种作用,为深入研究cAMP的构效关系提供了实验依据。     

Growth phase-dependent surface properties of Legionella pneumophila and their role in adhesion to stainless steel coated QCM-D sensors

Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.