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At least 433 records · Page 24Linked to original sources

PubMed1996

Sequence and tissue-dependent RNA expression of mouse FAD-linked glycerol-3-phosphate dehydrogenase.

A 2432-bp cDNA for mouse FAD-linked glycerol-3-phosphate dehydrogenase, a nuclear-encoded enzyme associated with the inner mitochondrial membrane, was isolated from a Lambda ZAP phage library generated from brown adipocyte mRNA. The amino acid sequence was 95 and 93% homologous to the rat and human enzymes. PvuII and SacI polymorphisms between Mus spretus and C57BL/6J were used to map the mouse FAD-linked glycerol-3-phosphate dehydrogenase gene (Gdm1) to chromosome 2, 33 cM from the centromere. Northern blot analysis showed that brown adipose tissue predominantly expressed a 6.5-kb mRNA with lower expression of 4.5- and 2.4-kb forms, whereas brain and pancreatic islets almost exclusively expressed a 6.5-kb transcript, muscle expressed a 4.5-kb transcript, and testis expressed a 2.4-kb RNA form. Analysis of poly(A)+ RNA from brown adipose tissue suggested that all RNA forms are polyadenylated. Among the tissues examined, FAD-linked glycerol-3-phosphate dehydrogenase protein levels and enzyme activity were highest in brown adipose tissue, and a consistent correlation between protein levels and enzyme activity in all tissues was observed. RNA levels corresponded to protein levels in all tissues except testis, where high levels of 2.4-kb mRNA and relatively low protein were expressed. Exposing mice to cold temperatures induced the 6.5-kb mRNA and enzyme activity only in brown adipose tissue, suggesting a role for thermogenesis in this tissue. Although the molecular basis for the formation of the 6.5- and 4.5-kb mRNA's is not known, data suggest that the regulation of FAD-linked glycerol-3-phosphate dehydrogenase is complex and tissue-specific.

Adipose Tissue, Brown↗

PubMed2004

The wheat VRN2 gene is a flowering repressor down-regulated by vernalization.

Plants with a winter growth habit flower earlier when exposed for several weeks to cold temperatures, a process called vernalization. We report here the positional cloning of the wheat vernalization gene VRN2, a dominant repressor of flowering that is down-regulated by vernalization. Loss of function of VRN2, whether by natural mutations or deletions, resulted in spring lines, which do not require vernalization to flower. Reduction of the RNA level of VRN2 by RNA interference accelerated the flowering time of transgenic winter-wheat plants by more than a month.

Alleles↗

PubMed2000

Direct measurement of aluminum uptake and distribution in single cells of Chara corallina.

Quantitative information on the uptake and distribution of Al at the cellular level is required to understand mechanisms of Al toxicity, but direct measurement of uptake across the plasma membrane has remained elusive. We measured rates of Al transport across membranes in single cells of Chara corallina using the rare (26)Al isotope, an emerging technology (accelerator mass spectrometry), and a surgical technique for isolating subcellular compartments. Accumulation of Al in the cell wall dominated total uptake (71-318 microgram m(-2) min(-1)), although transport across the plasma membrane was detectable (71-540 ng m(-2) min(-1)) within 30 min of exposure. Transport across the tonoplast was initially negligible, but accelerated to rates approximating uptake across the plasma membrane. The avacuolate protoplasm showed signs of saturation after 60 min, but continued movement across the plasma membrane was supported by sequestration in the vacuole. Saturation of all compartments was observed after 12 to 24 h. Accumulation of Al in the cell wall reflected variation in [Al(3+)] induced by changes in Al supply or complexing ligands, but was unaffected by pH. In contrast, transport across the plasma membrane peaked at pH 4.3 and increased when [Al(3+)] was reduced by complexing ligands. Cold temperature (4 degrees C) reduced accumulation in the cell wall and protoplasm, whereas 2,4-dinitrophenol and m-chlorocarbonylcyanidephenyl hydrazone increased membrane transport by 12- to 13-fold. Our data suggest that the cell wall is the major site of Al accumulation. Nonetheless, membrane transport occurs within minutes of exposure and is supported by subsequent sequestration in the vacuole. The rapid delivery of Al to the protoplasm suggests that intracellular lesions may be possible.

2,4-Dinitrophenol↗

PubMed2003

Effect of variable moderate chronic stress on ligature-induced periodontal disease in Wistar rats.

The aim of this investigation was to study the impact of stress on ligature-induced periodontal disease in rats by means of a variable moderate chronic stress model. Thirty male Wistar rats were randomly assigned to six groups. Control groups received only ligatures around the second maxillary molars, while experimental groups were exposed to stress in additional. Stress was imposed by means of flashing light, isolation, rat blood smelling, new environment exposure, immobilization in cold temperature and immobilization at room temperature. Stress was applied randomly, thereby diminishing adaptation of the animals to the model. The animals were killed after 29, 43, and 57 days. The distance between the cementum-enamel junction and the alveolar bone (CEJ-AB) was measured. Alveolar bone loss was statistically different between stressed and control animals, whereas differences were not observed between experimental periods. The mean CEJ-AB distance in animals exposed to stress was 154.50 microm smaller than the corresponding distance in the controls. It might be concluded that variable moderate chronic stress decreased alveolar bone loss in a ligature-induced periodontal disease model.

Alveolar Bone Loss↗

PubMed2002

Thermodilution cardiac output. Cold vs room temperature injectate and the importance of measuring the injectate temperature in the right atrium.

BACKGROUND: The feasibility of thermodilution cardiac output measurements with the more convenient room temperature thermal indicator instead of cold injectates has been repeatedly investigated. However, the issue has not been addressed with the appropriate statistical approach advocated by Altman and Bland. Furthermore, we wished to determine if the incorporation of a second thermistor in the thermodilution catheter, to measure the temperature of the thermal indicator where it is delivered into the right atrium/superior caval vein, would result in more precise cardiac output measurements. METHODS: Fifty patients were randomized to receive a single or dual thermistor pulmonary artery thermodilution catheter. Cardiac output was calculated as the average of four injections of 10 ml of isotonic saline. Precision (2 x SD of differences in replicate measurements) for the two catheters and injectate temperatures, and bias and limits of agreement between measurements, with cold and room temperature injectates, were determined. RESULTS: Precision was (0 degrees C) 0.42 l/min and (20 degrees C) 0.90 l/min, and bias and limits of agreement -0.83 l/min and -1.93-0.27 l/min for the single thermistor catheter. For the dual thermistor system precision was (0 degrees C) 0.34 l/min and (20 degrees C) 0.58 l/min. Bias and limits of agreement were -0.03 l/min and -0.61-0.55 l/min. CONCLUSION: The second thermistor is redundant if cold injectates are used. If one wishes to use room temperature injectates the single thermistor system is inadequate. A dual thermistor catheter is, on the other hand, acceptable.

Adult↗

PubMed1986

Dynamics of microtubule depolymerization in monocytes.

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.

Benzimidazoles↗

PubMed2001

Differential antinociceptive effect of transcutaneous electrical stimulation on pain behavior sensitive or insensitive to phentolamine in neuropathic rats.

The effects of transcutaneous electrical stimulation and systemic injection of phentolamine, a non-specific alpha-adrenergic antagonist, on the behavioral signs of mechanical allodynia and cold hyperalgesia in rats with nerve injury were investigated. Mechanical allodynia and cold hyperalgesia were evaluated by measuring the paw withdrawal frequency (PWF) resulting from repetitive application of a von Frey hair and the paw lift duration (PLD) at a cold temperature, respectively. After a unilateral nerve injury, both PWF and PLD increased in the injured hind paw. Application of low-frequency, high-intensity transcutaneous electrical stimulation (LFHI-TES) to the injured hind paw depressed the injury-induced increased PWF, whereas it had no effect on the injury-induced increased PLD. Naloxone reversed the LFHI-TES produced depression of PWF. Intraperitoneal administration of phentolamine depressed the injury-induced increased PLD without affecting the injury-induced increased PWF. Our results suggest that LFHI-TES, which activates the endogenous opioid systems, produces an antinociceptive effect that appears to be related to whether or not the pain is mediated by sympathetic activity.

Adrenergic alpha-Antagonists↗

PubMed1984

Sliding of STOP proteins on microtubules.

Microtubules are stabilized against cold temperature disassembly by 145-kilodalton proteins [stable tubule only polypeptides (STOPs)] that block the end-wise dissociation of subunits from the polymers. We describe here several kinetic parameters of the interaction of STOPs with microtubules. STOPs will bind to microtubules either during assembly of the polymer or at steady state. The addition appears random on the polymers and does not require the mediation of tubulin subunits. Tubulin subunits compete with microtubules for STOP binding, but binding to the polymers is apparently irreversible. We demonstrate that STOPs do not exchange measurably between polymers at steady state. Nonetheless, a displacement of STOPs within a single polymer is readily demonstrable. We have determined that the displacement is apparently due to a surface translocation, or "sliding", of STOPs on microtubules.

Animals↗

PubMed2006

The cold and menthol receptor TRPM8 contains a functionally important double cysteine motif.

We have investigated the glycosylation, disulfide bonding, and subunit structure of mouse TRPM8. To do this, amino-terminal c-myc or hemagglutinin epitope-tagged proteins were incorporated and expressed in Chinese hamster ovary cells. These modifications had no obvious effects on channel function in intracellular calcium imaging assays upon application of agonists, icilin or menthol, and cold temperatures. Unmodified TRPM8 migrates with an apparent mass of 129 kDa and can be glycosylated in Chinese hamster ovary cells to give glycoproteins with apparent masses of 136 and 147 kDa. We identified two potential N-linked glycosylation sites in TRPM8 (Asn-821 and Asn-934) and mutated them to show that only the site in the putative pore region at position 934 is modified and that glycosylation of this site is not absolutely necessary for cell surface expression or responsiveness to icilin, menthol, and cool temperatures. Enzymatic cleavage of the carbohydrate chains indicated that they are complex carbohydrate. The glycosylation site is flanked in the pore by two cysteine residues that we mutated, to prove that they are involved in a conserved double cysteine motif, which is essential for channel function. Mutation of either of these cysteines abolishes function and forces the formation of a non-functional complex of the size of a homodimer. The double cysteine mutant is also non-functional. Finally, we showed in Perfluoro-octanoic acid-polyacrylamide gels that TRPM8 can form a tetramer (in addition to dimer and trimer forms), consistent with current thinking that functional TRP ion channels are tetrameric.

Amino Acid Motifs↗

PubMed2004

Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus.

More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.

Animals↗

PubMed2001

Effect of temperature on endplate potential rundown and recovery in rat diaphragm.

The amplitude of neuromuscular junction end-plate potentials (EPPs) decreases quickly within a train but recovers nearly completely from train to train during intermittent stimulation. Rundown has been shown to be dependent not only on the rate of transmitter release but also on the rate of replenishment of the depleted neurotransmitter at the site of release. Two groups of processes have been proposed for synaptic vesicle recycling, both of which involve multiple energy-requiring steps and enzymatic reactions and which therefore would be expected to be very temperature-sensitive. The present study tested the hypothesis that low temperature therefore increases the rate of EPP amplitude rundown. Studies were performed in vitro on rat diaphragm and used micro-conotoxin to allow normal-sized EPPs to be recorded from intact fibers. EPP amplitude rundown during intermittent stimulation at 20 and 50 Hz (duty cycle 333 ms) was greater at 20 degrees C than it was at 37 degrees C. Initially, temperature affected only intra-train rundown but, over longer periods of stimulation, both intra- and inter-train rundown were significantly accelerated by cold temperature. Cumulative EPP amplitudes were calculated by successively adding the amplitudes of each EPP during the stimulation period to provide an estimate of total neurotransmitter release in the neuromuscular junction. The cumulative EPP amplitude was significantly lower at 20 degrees C than it was at 37 degrees C during both 20 and 50 Hz stimulation. These data indicate that the mechanism involved in EPP amplitude rundown and recovery is temperature-sensitive, with a greater decrement in EPP amplitude at cold than at warm temperatures.

Action Potentials↗

PubMed2001

Mutagenesis of beta-tubulin cysteine residues in Saccharomyces cerevisiae: mutation of cysteine 354 results in cold-stable microtubules.

Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in beta-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae beta-tubulin individually to alanine and serine residues. Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4 degrees C, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the alpha- and beta-subunits. The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation. When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains. The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin.

Alanine↗

PubMed1993

The effect of anesthesia on the photoresponses of four sympatric species of Drosophila.

Five isofemale strains from each of four sympatric species, Drosophila immigrans, D. repleta, D. melanogaster, and D. affinis, were lightly anesthetized with ether, CO2, Flynap, or cold temperature. The photoresponse of each treatment group was measured in an open field and a Y-tube apparatus. Relative light values were compared with those of the unanesthetized control groups of each species, as well as among the different treatments themselves and among species. Significant differences between species were apparent for all treatments in the Y-tube and for all treatments except ether and CO2 in the open field. Response to anesthesia may differ between species due, in part, to their differing genotypes. Comparing treatment groups within species in the Y-tube design, all species except D. melanogaster showed significant differences between treatments. In the open field, only D. affinis was significantly affected. Particularly in the Y-tube, anesthetized flies generally differed significantly from the controls, indicating that the use of anesthesia during a behavioral study could cause deviations in behavior from that of normal unanesthetized flies.

Anesthesia↗

PubMed2001

The effect of dietary chloride and bicarbonate on blood pH, haematological variables, pulmonary hypertension and ascites in broiler chickens.

1. The effect of supplementing grower diets with bicarbonate or chloride on haematological variables, pulmonary hypertension syndrome and ascites in broilers exposed to cold temperature was investigated. 2. High concentrations of dietary chloride had no effect on the pH of the venous blood but a low chloride/high bicarbonate diet significantly increased blood pH. There was no consistent effect of dietary chloride or bicarbonate concentrations on growth performance, although in 1 experiment birds given a low chloride/high bicarbonate diet consumed less food and gained less weight than controls. 3. Birds fed on high-chloride diets tended to have a higher incidence of ascites and pulmonary hypertension than controls. Birds fed on low-chloride and high-bicarbonate diets had significantly lower pulmonary hypertension and lower heart weights, which may have indicated a decrease in pulmonary and systemic blood pressure. 4. We conclude that increasing dietary bicarbonate and reducing dietary chloride has potential as a low cost and effective method to reduce the pulmonary hypertension which leads to ascites in broiler chickens.

Animals↗

PubMed1978

Contribution of an electrogenic sodium pump to the membrane potential in the intestinal epithelial cell.

The transmural potential difference of isolated rat duodenum and the membrane potential of its epithelial cell were examined during changes of the ionic medium at both warm (31--35 degrees C) and cold temperatures (2--5 degrees C). In the range of low external K+ concentrations, cooling produced an immediate and reversible depolarization of the membrane potential (around -10 mV) greater than that estimated from a simple thermodynamic temperature effect (RT/F). Such a temperature sensitive component of the membrane potential at 31 degrees C was completely abolished by anoxia and serosal ouabain. Readmission of K+ to the bathing fluid of duodenum previously deprived of K+ and loaded by Na+ caused a remarkable transient hyperpolarization of the epithelial membrane, which was abolished by serosal ouabain. Decreases in the value of transmural potential difference were also produced by cooling, anoxia and serosal ouabain, but these were so small that concurrent changes in the membrane potential could be regarded as chiefly reflecting changes in the effective emf of the cell. Thus, an electrogenic Na+-K+ exchange pump located on the serosal membrane of the duodenal epithelial cells was shown to contribute significantly to the membrane potential under normal conditions (up to around -7 mV). The coupling ratio of Na+-efflux and K+-influx associated with this pump was estimated by the Mullins-Noda equation (1963) to be approximately 4:3 under the physiological steady-state conditions.

Animals↗

PubMed1983

The role of choline phospholipids in hypertonic cryohemolysis.

Hemolysis resulting from a warm-to-cold temperature shift in a hypertonic environment (hypertonic cryohemolysis) is studied with the use of phospholipases as membrane probes of the phospholipids of the outer leaflet of the bilayer. Bee venom phospholipase A2 which attacks only phosphatidylcholine (PC) in the intact erythrocyte results in inhibition of cryohemolysis produced by both hypertonic sodium chloride and sucrose. In both cases, about 25% of the loss of PC occurs before any such inhibition, suggesting the possibility of functionally separate domains of PC in the outer leaflet of the bilayer. Sphingomyelinase also attacks only sphingomyelin in the intact erythrocyte and results in inhibition of cryohemolysis due to hypertonic sodium chloride but not of that due to sucrose. In each case, inhibition of the enzymatic hydrolysis by EDTA abolished the effect on cryohemolysis. It is postulated that cryohemolysis is inhibited when phospholipid interaction with membrane (cytoskeletal) proteins are abolished, but present knowledge of membrane structure does not permit a detailed mechanism to be proposed.

Cold Temperature↗

PubMed1991

Zones of differential sensory block during extradural anaesthesia.

We have measured spinal segmental levels of anaesthesia to light touch (LT), pinprick (PP) and cold temperature discrimination (TE) during 2% lignocaine extradural anaesthesia in 22 patients, to determine if zones of differential sensory block develop during extradural anaesthesia and, if so, the extent to which TE extends beyond PP or LT levels and how age affects differential block. The median thoracic dermatomal levels were 4.5 for LT, 2.0 for PP and 2.0 for TE. Zones of differential sensory block developed within 5 min of extradural injection of local anaesthetic, and persisted for the next 55 min. In all instances, PP extended more cephalad than LT, and TE extended above PP levels. There were no differences in the extent of zones between the two groups of patients with mean ages of 28 and 48 yr. Thus, during extradural anaesthesia, sympathetic denervation extended one to two spinal segments above the sensory levels of LT and PP anaesthesia, age (28 vs 48 yr) affected neither the cephalad extent nor the width of zones of differential block, and PP levels of anaesthesia were closer to presumed levels of sympathetic block than were LT levels.

Adult↗

PubMed2005

Identification and characterization of mutants capable of rapid seed germination at 10 degrees C from activation-tagged lines of Arabidopsis thaliana.

Five cold temperature germinating (ctg) mutants, completing germination at 10 degrees C faster than wild type, have been recovered from activation-tagged populations of Arabidopsis thaliana. Three (ctg10-D, 41-D, and 144-D) were tagged and segregated 3:1 for BASTA resistance in the F2 when crossed with wild type. None of the tagged ctg mutants was disturbed in sensitivity to abscisic acid or glucose but all were less sensitive to GA4+7 and osmoticum. The other two mutants (ctg156 and ctg225) were recessive, BASTA sensitive, and exhibited a transparent testa (tt) phenotype. They were more sensitive to abscisic acid, paclobutrazol, and GA4+7 than wild type but had similar sensitivity to osmoticum. Dimethylaminocinnamaldehyde staining of seeds from the two tt mutants, compared with stained seeds from the publicly available tt lines 1-10, suggested that ctg156 was a new allele of tt1, while ctg225 was similar to tt7-1. However, reciprocal crosses determined that ctg156 was not allelic to tt1 while ctg225 was a new allele of tt7. When the gene was sequenced from ctg225 it was missing 10 bp in the second exon, resulting in the incorporation of two spurious amino acids (G282E and D283A) followed by a stop. The screen successfully recovered mutants completing germination faster than wild type at 10 degrees C.

Abscisic Acid↗