The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.     

Long non-coding RNA OIP5-AS1 promotes pancreatic cancer cell growth through sponging miR-342-3p via AKT/ERK signaling pathway

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a long non-coding RNA (lncRNA), has been reported to link with the progression of some cancers. However, its biological functions and underlying molecular mechanisms in pancreatic cancer are largely unknown. The aim of this study was to investigate the role of lncRNA OIP5-AS1 in pancreatic cancer. Quantitative real-time PCR analysis revealed that OIP5-AS1 is highly expressed in pancreatic cancer tissues versus adjacent non-tumor tissues. In vitro functional assays showed that downregulation of OIP5-AS1 or overexpression of miR-342-3p inhibited the proliferation, decreased Ki67 expression, and induced cell cycle arrest in pancreatic cancer cells. The expression of cyclinD1, CDK4, and CDK6 was decreased by knockdown of OIP5-AS1. Moreover, we found that OIP5-AS1 acted as a miR-342-3p sponge to suppress its expression and function. Dual-luciferase assay confirmed the interaction of OIP5-AS1 and miR-342-3p and verified anterior gradient 2 (AGR2) as a direct target of miR-342-3p. Results showed that depletion of miR-342-3p abolished the inhibitory effects of OIP5-AS1 knockdown on pancreatic cancer cell growth. The expression of Ki67, AGR2, cyclinD1, CDK4, CDK6, p-AKT, and p-ERK1/2 was reversed by silencing of miR-342-3p in pancreatic cancer cells with OIP5-AS1 knockdown. Further, knockdown of OIP5-AS1 suppressed tumor growth in a xenograft mouse model of pancreatic cancer. OIP5-AS1 induced pancreatic cancer progression via activation of AKT and ERK signaling pathways. Therefore, we demonstrate that OIP5-AS1 functions as oncogene in pancreatic cancer and its downregulation inhibits pancreatic cancer growth by sponging miR-342-3p via targeting AGR2 through inhibiting AKT/ERK signaling pathway.

     

Asr1p, a novel yeast ring/PHD finger protein, signals alcohol stress to the nucleus

During fermentation, yeast cells are exposed to increasing amounts of alcohol, which is stressful and affects both growth and viability. On the molecular level, numerous aspects of alcohol stress signaling remain unresolved. We have identified a novel yeast Ring/PHD finger protein that constitutively shuttles between nucleus and cytoplasm but accumulates in the nucleus upon exposure to ethanol, 2-propanol, or 1-butanol. Subcellular localization of this protein is not altered by osmotic, oxidative, or heat stress or during nitrogen or glucose starvation. Because of its exclusive sensitivity to environmental alcohol, the protein was called Asr1p for Alcohol Sensitive Ring/PHD finger 1 protein. Nuclear accumulation of Asr1p is rapid, reversible, and requires a functional Ran/Gsp1p gradient. Asr1p contains two N terminally located leucine-rich nuclear export sequences (NES) required for nuclear export. Consistently, it accumulates in the nucleus of xpo1-1 cells at restrictive temperature and forms a trimeric complex with the exportin Xpo1p and Ran-GTP. Deletion of ASR1 leads to sensitivity in growth on medium containing alcohol or detergent, consistent with a function of Asr1p in alcohol-related signaling. Asr1p is the first reported protein that changes its subcellular localization specifically upon exposure to alcohol and therefore represents a key element in the analysis of alcohol-responsive signaling.     

芦芽山阳坡不同海拔白杄径向生长对气候变暖的响应

使用树轮生态学方法研究了山西芦芽山建群种白杄(Picea meyeri)径向生长对气候变暖的响应状况, 发现随着气温升高, 不同海拔白杄生长与气候因子关系的变化存在差别。研究区气温可以分为1958-1983年的气温降低阶段和1984-2007年的气温升高阶段。由气温降低阶段进入气温升高阶段, 低海拔白杄树轮年表的序列间相关系数和第一主成分解释量均增大, 而高海拔白杄树轮年表的序列间相关系数和第一主成分解释量均减小, 表明气候条件对低海拔白杄生长的影响增强而对高海拔白杄生长的影响减弱。随着气温升高, 不同海拔白杄径向生长与气候因子的关系均出现了变化。1958-1983年, 低海拔(2 060 m)白杄生长与7月降水量显著正相关(p < 0.05), 而在1984-2007年, 这一关系表现为极显著正相关(p < 0.01), 同时与生长季中5-7月平均气温呈现显著负相关(p < 0.05)。海拔2 330 m, 白杄在1958-1983年与7月降水量极显著正相关(p < 0.01), 进入1984-2007年后与气候因子没有显著相关关系。海拔2 440 m, 白杄生长由1958-1983年的与气候因子没有显著相关关系转变为1984-2007年的与上一年10月平均气温显著负相关(p < 0.05)。高海拔(2 540 m)白杄生长在1958-1983年与上一年11月平均气温极显著负相关(p < 0.01), 在1984-2007年与上一年10月、当年1月平均气温和6月降水量均显著负相关(p < 0.05)。滑动相关分析结果表明, 随着气温升高, 低海拔主要气候因子对生长的影响增强, 而高海拔主要气候因子对生长的影响减弱, 这可能成为高海拔白杄生长对气温升高敏感性降低的原因。在气候变暖的驱动下, 海拔引起的白杄生长与气候因子关系的差异发生了变化。     

Neurite growth in 3D collagen gels with gradients of mechanical properties

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.     

High performance of CO2–temperature gradient chamber newly built for studying the global warming effect on a plant population

To study the effect of global warming concomitant with rising CO₂ on plant growth under field-like conditions, a CO₂–temperature gradient chamber (CTGC) was created from a standard temperature gradient chamber (TGC). Despite great daily and seasonal changes in ambient air temperature, the gradient of air temperatures was simulated by 5°C warmed conditions at the air outlet, rising at a rate of 1°C at 5 m intervals. Also, CO₂ concentrations were linearly increased from the air inlet to the outlet; those at the air inlet and 25 m distance from the air inlet were 372 p.p.m and 756 p.p.m. (doubled), respectively. The CTGC proved successful in biological experiments conducted through a full growth season in 1998. This apparatus will be useful for understanding plant response to simultaneous changes in CO₂ and temperature. Being a combination of a TGC and a CO₂ gradient chamber, the improved CTGC allows parameters and validation data sets to both be obtained for models contributing to plant growth, species composition, and global carbon flux analysis.     

Theoretical analysis of gradient detection by growth cones.

Gradients of diffusible and substrate-bound molecules play an important role in guiding axons to appropriate targets in the developing nervous system. Although some of the molecules involved have recently been identified, little is known about the physical mechanisms by which growth cones sense gradients. This article applies the seminal Berg and Purcell (1977) model of gradient sensing to this problem. The model provides estimates for the statistical fluctuations in the measurement of concentration by a small sensing device. By assuming that gradient detection consists of the comparison of concentrations at two spatially or temporally separated points, the model therefore provides an estimate for the steepness of gradient that can be detected as a function of physiological parameters. The model makes the following specific predictions. (a) It is more likely that growth cones use a spatial rather than temporal sensing strategy. (b) Growth cone sensitivity increases with the concentration of ligand, the speed of ligand diffusion, the size of the growth cone, and the time over which it averages the gradient signal. (c) The minimum detectable gradient steepness for growth cones is roughly in the range 1-10%. (d) This value varies depending on whether a bound or freely diffusing ligand is being sensed, and on whether the sensing occurs in three or two dimensions. The model also makes predictions concerning the role of filopodia in gradient detection.     

Endogenous electrical currents and the resultant voltage gradients in the chick embryo

We have studied some of the electrophysiological properties of 2 1/2- to 4-day-old (stage 14-22) chick embryos. Using a recently developed two-dimensional vibrating probe, large currents were found to exit the posterior intestinal portal (p.i.p.) during the period of tail gut reduction. During this period, epithelial cells lining cloacal regions of the hindgut are dying, thus creating a low-resistance pathway for current flow out of the embryo. Currents entered the intact epithelium over other regions of the embryo. The outward currents at the p.i.p. were first detected at stage 15 and reached their average maximum current density of 112 +/- 10 microA/cm2 at stage 17. After stage 17, the magnitude of the currents decreased, dropping to 16 +/- 0.3 microA/cm2 by stage 22. The currents were reversibly reduced by about 50% when Na+ was replaced by choline in the bathing solution. The magnitude of the currents leaving the p.i.p. suggested the existence of a measurable intraembryonic voltage gradient. The transepithelial potential (TEP) of stage 14-21 embryos was measured lateral to the neural tube through the dorsal ectoderm. For all stages, the combined average TEP was 16 +/- 0.5 mV. Differences in the TEP between various regions of the embryo were used to calculate an intraembryonic voltage gradient. At stage 14, before outward current was found at the p.i.p., no significant intraembryonic voltage gradient was detected. At stage 17, when the outward current at the p.i.p. was maximum, a voltage gradient of 21 +/- 5 mV/mm (mean +/- SEM; N = 6) was measured in the caudal end of the embryo. This gradient in some cases was as steep as 33 mV/mm. This is well above the minimum level needed to affect the direction of embryonic cell migration in vitro. We hypothesize that this endogenous electrical field acts as a directional cue for neural crest cell movements in the developing chick embryo.     

Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.     

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.     

Association of three chicken proteins with the 34 kD target of rous sarcoma virus tyrosine kinase

Transformation of chicken embryo fibroblasts by avian retroviruses induces the tyrosine phosphorylation of a 34-39 kD cellular protein (p34). In vitro, p34 isolated from intestine interacts with F-actin in a Ca2+-dependent manner. We report here that, in the absence of Ca2+ chelators, three proteins co-purified with p34 extracted from a cytosolic or membrane fraction of chicken embryo fibroblasts; these two fractions account respectively for 10-20% and 50% of the total cellular p34. Isolated from the cytosoluble fraction of fibroblasts by sucrose gradient centrifugation and hydrophobic chromatography, p34 and the other proteins behaved as a homogeneous species upon non-denaturing gel electrophoresis, gel filtration, and CsCl density gradient centrifugation, thus indicating a strong association. Moreover, an analysis by electron microscopy following uranyl acetate staining revealed particles with a raspberry-like shape. This association was always disrupted by the calcium-chelating agent, EGTA.     

Tumor cell growth inhibition and extracellular signal-regulated kinase (ERK) phosphorylation by novel K vitamins.

2-(2-hydroxy-ethylsulfanyl)-3-methyl-1,4-naphthoquinone or CPD-5, a K vitamin analog, was previously indicated to be a potent growth inhibitor for Hep 3B hepatoma cells in vitro. Here, we show that CPD-5 and two newly synthesized analogs, 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5- nitro-1,4-naphthoquinone (PD-37) and 2-(2-hydroxy-ethylsulfanyl)-3- methyl-5-acetylamino-1,4-naphthoquinone (PD-42), are potent growth inhibitors of 13 different human cancer cell lines, with IC50 values in the range of 3-54 microM. Phospho-ERK was induced by each of three K vitamin analogs in every cell line in a dose-dependent manner, at growth inhibitory doses. ERK phosphorylation and growth inhibitory effects were strongly correlated, with p=0.0080 for CPD-5, p=0.0076 for PD-37 and p=0.0251 for PD-42. The induction of phospho-ERK and growth inhibition were antagonized by thiol-containing anti-oxidants, but not by catalase, consistent with a possible arylating mechanism. The data show a novel class of growth inhibitors with a wide spectrum of action that induces ERK hyper-phosphorylation, as a possible new growth inhibitory feature.     

BASAL LEVELS OF METALLOTHIONEIN I AND II EXPRESSION IN MOUSE EMBRYO FIBROBLASTS ENHANCE GROWTH IN LOW FOLATE THROUGH A CELL CYCLE MEDIATED PATHWAY

The impact of basal (non-induced) expression levels of metallothionein I and II on the growth of mouse embryo fibroblasts in standard DMEM/F-12 containing 8.8 microm folic acid, and in DMEM/F12 without hypoxanthine, thymidine or folic acid, containing 15 nm or 15 pm[6S]-folinic acid, was assessed by comparing wild-type MT (+/+) and homozygous null MT (-/-) cell lines. No difference in growth rate was observed between the two in DMEM/F12, although MT (-/-) cells displayed a 6-fold decrease in p27(Kip1), a two fold increase in p53 and a slight increase in p21(Waf1). After 6 days in culture, the growth rate for MT (-/-) cells in 15 nm or 15 pm[6S]-folinic acid was half that of MT (+/+). After an additional 6 days in 15 n m folate, both MT (+/+) and (-/-) cells maintained their respective growth rates, while those in 15 pm had ceased to grow. During the initial 6 days in 15 nm folate, neither cell population displayed an increase in apoptosis or a change in cell cycle distribution, even though MT (-/-) cells sustained an additional 4-fold increase in p21(Waf1)and a 6-fold decrease in cyclin E expression. At day 12, however, the MT (-/-) population, but not MT (+/+), underwent a 7-fold increase in apoptosis coupled with a 3 fold increase in S phase cells. Hence, the basal level of MT I and II constitutively expressed in MT (+/+) cells enhances growth in 15nM [6S]-folinic acid by preventing S phase arrest and apoptosis.     

Inhibition of mutant p53 phosphorylation at serine 15 or serine 315 partially restores the function of wild-type p53.

The tumor suppressor protein p53 is a phosphoprotein and has growth and transformation suppression functions. Phosphorylation of wild-type p53 is known to modulate its function. To investigate the role of phosphorylation in modulating the functions of mutant p53, we constructed a series of phosphorylation site mutants based on mutant p53 Ala143 (p53-143) and p53 His175 (p53-175). When transfected into p53-negative Saos-2 cells, parental mutant p53-143 and p53-175 abolished both growth suppression and induction of apoptosis. However, DNA-activated protein kinase (DNA-PK) or cyclin-dependent kinase (cdks) phosphorylation site double mutants partially restored the growth suppression and induction of apoptosis and recovered the p53-specific DNA binding activity. We also observed a difference in sensitivity to calpain from parental mutants p53-175 and p53-175/15 or p53-175/315. These results suggest that the lack of phosphorylation at either the DNA-PK or cdks site in p53 mutants partially restores the wild-type functions by altering their conformation.