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Query: UMLS:C0085593 (chills)
 4,268 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Assessment of Fura-2 for measurements of cytosolic free calcium. 219 82

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 +/- 2.2 and 8.5 +/- 1.5 microM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 +/- 1.25 microM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, KCa' = 1.92 microM and a second, low-affinity site, KCa" = 1169.60 microM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of alpha-D-glucosyl-alpha-D-glucopyranoside and alpha-methyl-D-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 X 10(3) and 8 X 10(3) Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.
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PMID:Nematodirus battus: permeability changes, calcium binding, and phosphorylation of the eggshell during hatching. 620 83

Calmodulin (CaM) potentiates Ca(2+)-dependent signaling pathways in both the cytoplasm and nucleus. We have investigated the mechanism of CaM nuclear transport using tissue culture cell microinjection and a permeabilized cell import assay. The inhibition of CaM import by the translocation inhibitor wheat germ agglutinin (WGA) and by chilling, indicates that CaM import is facilitated, but because ATP depletion does not affect CaM import, the mechanism does not appear to be active. Chilling and WGA arrest persist in ATP-depleted cells, indicating that CaM is not retained in the cytoplasm by an ATP-dependent mechanism. In permeabilized cells, both Ca(2+)-CaM and Ca(2+)-free CaM are sensitive to extract-dependent WGA and chilling import inhibition. Titration experiments in microinjected and permeabilized cells indicate that a saturable cytosolic factor(s) mediates chilling and WGA arrest.
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PMID:Facilitated nuclear transport of calmodulin in tissue culture cells. 779 9

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.
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PMID:Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes. 1047 14

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.
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PMID:Osmotic and chill activation of glycine betaine porter II in Listeria monocytogenes membrane vesicles. 1076 57

The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in soybean (Glycine max (L.) Merr.) seeds during imbibition. Soybean seeds germinated readily over a large range of temperatures (10-35 degrees C), the thermal optimum being 25-30 degrees C. Low temperatures reduced the germination rate and no seed germinated at 1 degrees C. Pre-treatment of seeds at 1 degrees C reduced further germination at the optimal temperature (25 degrees C). This deleterious effect of chilling increased with duration of the treatment, and was maximal after 4 days. Osmoconditioning of seeds at 20 degrees C with a polyethylene glycol-8000 solution at -1.5 MPa for at least 24 h followed by drying back the seeds to their initial moisture content reduced their chilling sensitivity and even allowed germination at 1 degrees C. Chilling of control seeds resulted in a sharp decline in in vivo ACC-dependent ethylene production and in an increase in electrolyte leakage in the medium, which indicated deterioration of membrane properties. Osmoconditioned seeds placed at 1 degrees C did not show any reduction in their ability to convert ACC to ethylene nor any strong increase in electrolyte leakage. Imbibition of both control and osmoconditioned seeds at 1 degrees C resulted in a marked increase in ATP level (more than 50% of the total nucleotides) and energy charge; however, the latter cannot be considered as an indicator of chilling since it remained high (0.74-0.88) throughout the cold treatment. Chilling treatment longer than 6 days induced accumulation of malondialdehyde in the embryonic axis, which was more marked in control seeds than in osmoconditioned seeds, suggesting that chilling sensitivity was associated with lipid peroxidation. Imbibition of seeds at 1 degrees C resulted in an increase in superoxide dismutase, catalase and glutathione reductase activity, which was generally higher in osmoconditioned seeds than in control ones. This stimulation of the antioxidant defence systems occurred during the 4 first days of chilling and decreased then in control seeds while it remained high in osmoconditioned ones. Re-warming seeds at 25 degrees C resulted in an increase in all enzyme activity involved in antioxidant defence. However this effect of re-warming decreased in control seeds after 4 days of chilling, whereas it was maintained in osmoconditioned seeds.
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PMID:Osmoconditioning reduces physiological and biochemical damage induced by chilling in soybean seeds. 1129 12

Three acclimation groups [i.e. non-diapause (LD), diapause (SD) and diapause, cold-acclimated (SDA)] of the adult bugs Pyrrhocoris apterus differed markedly in their levels of chill tolerance. Survival time at a sub-zero, but non-freezing, temperature of -5 degrees C (Lt50) extended from 7.6 days, through 35.6 days, to >60 days in the LD, SD and SDA insects, respectively. The time necessary for recovery after chill-coma increased linearly with the increasing time of exposure to -5 degrees C, and the steepness of the slope of linear regression decreased in the order LD>SD>SDA. The capacity to prevent/counteract leakage of Na(+) down the electrochemical gradient (from haemolymph to tissues) during the exposure to -5 degrees C increased in the order LD<SD<SDA. As a result, the rates of counteractive outward movement of K(+), and of the E(K) dissipation, decreased in the same order. The least chill-tolerant insects (LD) showed the highest rate of body-water loss. Most of the water was lost from the haemolymph compartment. The ability to regulate a certain fraction of ion pools into the hindgut fluid was the highest in the SDA group, medium in the SD group and missing in the LD group. The adenylate energy charge in the fat body cells was constant in all three groups. The total pools of ATP, ADP and AMP, however, decreased in the SD and SDA groups but remained constant in the LD group. The inability of insects to maintain ion gradients at sub-zero temperature is discussed as an important cause of pre-freeze mortality.
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PMID:On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus. 1503 45

We have examined the role of alcohol dehydrogenase (ADH, E.C.1.1.1.1) in chilling tolerance using maize (Zea mays L.) Adh1(-)Adh2(-) doubly null mutant. Adh1(-)Adh2(-) doubly null seedlings were found to have lowered survival rates compared to non-doubly null maize seedlings (Silverado F(1)) when held at 2 degrees C for varying periods. Exposure to ethanol did not increase the chilling tolerance in either Silverado F(1) or Adh1(-)Adh2(-) doubly null. ADH activity in Silverado F(1) remained steady when held at 2 degrees C for up to 3 d. ADH1 protein accumulation in chilled Silverado F(1) seedlings remained unchanged throughout the period of cold exposure. Chilling led to a significant inhibition of the P-H(+)-ATPase (E.C. 3.6.3.6) activity in Adh1(-)Adh2(-)doubly null, but minimal inhibition was seen in Silverado F(1). Though P-H(+)-ATPase activity in Adh1(-)Adh2(-) decreased, P-H(+)-ATPase protein levels remained constant during the chilling period. Levels of ATP slightly fluctuated in both types of seedlings during the duration of chilling. Lipid peroxidation levels in Adh1(-)Adh2(-) doubly null increased with chilling exposure, but not in the Silverado F(1). We suggest that ADH activity may play a role in chilling tolerance that is not related to maintenance of glycolysis and ATP production as has been observed during oxygen depravation.
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PMID:Relationship between alcohol dehydrogenase activity and low-temperature in two maize genotypes, Silverado F1 and Adh1-Adh2- doubly null. 1559 4

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O(2) and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.
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PMID:Preparation and functional characterization of thylakoids from Arabidopsis thaliana. 1622 40

The soil bacterium Bacillus subtilis frequently encounters a reduction in temperature in its natural habitats. Here, a combined transcriptomic and proteomic approach has been used to analyse the adaptational responses of B. subtilis to low temperature. Propagation of B. subtilis in minimal medium at 15 degrees C triggered the induction of 279 genes and the repression of 301 genes in comparison to cells grown at 37 degrees C. The analysis thus revealed profound adjustments in the overall gene expression profile in chill-adapted cells. Important transcriptional changes in low-temperature-grown cells comprise the induction of the SigB-controlled general stress regulon, the induction of parts of the early sporulation regulons (SigF, SigE and SigG) and the induction of a regulatory circuit (RapA/PhrA and Opp) that is involved in the fine-tuning of the phosphorylation status of the Spo0A response regulator. The analysis of chill-stress-repressed genes revealed reductions in major catabolic (glycolysis, oxidative phosphorylation, ATP synthesis) and anabolic routes (biosynthesis of purines, pyrimidines, haem and fatty acids) that likely reflect the slower growth rates at low temperature. Low-temperature repression of part of the SigW regulon and of many genes with predicted functions in chemotaxis and motility was also noted. The proteome analysis of chill-adapted cells indicates a major contribution of post-transcriptional regulation phenomena in adaptation to low temperature. Comparative analysis of the previously reported transcriptional responses of cold-shocked B. subtilis cells with this data revealed that cold shock and growth in the cold constitute physiologically distinct phases of the adaptation of B. subtilis to low temperature.
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PMID:Adaptation of Bacillus subtilis to growth at low temperature: a combined transcriptomic and proteomic appraisal. 1651 63


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