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PubMed2004

Low temperature blocks the stimulatory effect of human chorionic gonadotropin on steroidogenic acute regulatory protein mRNA and testosterone production but not cyclic adenosine monophosphate in mouse Leydig tumor cells.

Low temperatures slow down metabolism, partly because the kinetic energy of molecules is reduced and enzymes may be structurally impaired. We now report that relative to its maximal activity at 37 degrees C, adenylate cyclase (AC) still retained 25% functionality (determined as cyclic adenosine monophosphate [cAMP] production) at 4 degrees C in mouse Leydig tumor cells (MLTC-1) in response to 50 IU/L human chorionic gonadotropin (hCG), whereas steroidogenic acute regulatory (StAR) protein mRNA and testosterone production were completely impaired. The incubation of MLTC-1 with the phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine; IBMX) resulted in significantly increased intracellular cAMP concentration at all 3 temperatures, but this had no impact on testosterone production. AC, cAMP, and phosphodiesterase form an important intracellular second-messenger mechanism in many organisms, some that inhabit very low temperature niches. The cold-resistance of AC and phosphodiesterase may thus have evolved to cope with adverse conditions. Although hibernation may lead to decreased steroid hormone production, it is also likely that cold-mediated decreased steroid hormone production induces hibernation.

1-Methyl-3-isobutylxanthine↗
PubMed2003

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets.

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.

Adaptation, Physiological↗
PubMed2002

The effect of mild cold exposure on UCP3 mRNA expression and UCP3 protein content in humans.

OBJECTIVE: In rodents, adaptive thermogenesis in response to cold exposure and high-fat feeding is accomplished by the activation of the brown adipose tissue specific mitochondrial uncoupling protein, UCP1. The recently discovered human uncoupling protein 3 is a possible candidate for adaptive thermogenesis in humans. In the present study we examined the effect of mild cold exposure on the mRNA and protein expression of UCP3. SUBJECTS: Ten healthy male volunteers (age 24.4 +/- 1.6 y; height 1.83 +/- 0.02 m; weight 77.3 +/- 3.0 kg; percentage body fat 19 +/- 2). DESIGN: Subjects stayed twice in the respiration chamber for 60 h (20.00-8.00 h); once at 22 degrees C (72 degrees F), and once at 16 degrees C (61 degrees F). After leaving the respiration chamber, muscle biopsies were taken and RT-competitive-PCR and Western blotting was used to measure UCP3 mRNA and protein expression respectively. RESULTS: Twenty-four-hour energy expenditure was significantly increased at 16 degrees C compared to 22 degrees C (P<0.05). At 16 degrees C, UCP3T (4.6 +/- 1.0 vs 7.7 +/- 1.5 amol/microg RNA, P=0.07), UCP3L (2.0 +/- 0.5 vs 3.5 +/- 0.9 amol/microg RNA, P=0.1) and UCP3S (2.6 +/- 0.6 vs 4.2 +/- 0.7 amol/microg RNA, P=0.07) mRNA expression tended to be lower compared with at 22 degrees C, whereas UCP3 protein content was, on average, not different. However, the individual differences in UCP3 protein content (16-22 degrees C) correlated positively with the differences in 24 h energy expenditure (r=0.86, P<0.05). CONCLUSION: The present study suggests that UCP3 protein content is related to energy metabolism in humans and might help in the metabolic adaptation to cold exposure. However, the down-regulation of UCP3 mRNA with mild cold exposure suggests that prolonged cold exposure will lead to lower UCP3 protein content. What the function of such down-regulation of UCP3 could be is presently unknown.

Adult↗
PubMed1997

Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization.

The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5'-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37 degrees C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock Ipp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37 degrees C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37 degrees C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37 degrees C is prevented by destabilizing its mRNA.

Bacterial Proteins↗
PubMed2003

A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles.

BACKGROUND: This study outlines steps for isolating and culturing Helicobacter pylori from freshly voided fecal specimens and genotyping isolates for vacA alleles. MATERIALS AND METHODS: A family with four H. pylori-infected members participated in this pilot study. Criterion for participation was a positive test for H. pylori by the urea breath test. Fecal specimens from children were taken from a freshly soiled diaper, placed in cold buffer, and prepared for culture in less than 2 hours. Culturing of H. pylori utilized selective culture media and isolates were screened for negative Gram stain, positive catalase and oxidase tests, and positive H. pylori 16S ribosomal RNA polymerase chain reaction (PCR). Strain types were determined by vacA genotyping. RESULTS: The isolation procedure is relatively simple, although 5-7 days are required for H. pylori culturing. Isolation and purification of DNA eliminated PCR inhibitors and resulted in reliable analyses. All four family members were infected with the same H. pylori strain with a genotype of vacA s1a/m2. CONCLUSION: This research lays the foundation for developing a routine and direct noninvasive method to detect the presence of H. pylori in fecal specimens. It is especially convenient for diagnosing children and infants, as samples can be obtained from soiled diapers. Culturing H. pylori from fecal samples in certain cases is important for antibiotic resistant studies prior to treating infected patients and for strain genotyping in epidemiological studies to determine transmission.

Adult↗
PubMed2003

Altered expression of angiopoietins during blood-brain barrier breakdown and angiogenesis.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) belong to a novel family of endothelial growth factors that function as ligands for the endothelial-specific receptor tyrosine kinase, Tie-2. Ang-1 reduces endothelial permeability of noncerebral vessels and has a major role in vascular stabilization and maturation, whereas Ang-2 is thought to be an endogenous antagonist of the action of Ang-1 at Tie-2. Expression of these ligands at the mRNA and protein level were studied during both blood-brain barrier (BBB) breakdown and cerebral angiogenesis occurring in the rat cortical cold-injury model by RT-PCR analysis and immunohistochemistry respectively, during a time course of 6 hours to 6 days. In addition, immunohistochemical detection of fibronectin was used to detect BBB breakdown at the lesion site and dual labeling was used to determine whether the vessels demonstrating BBB breakdown expressed endothelial Ang-1 or Ang-2. Endothelial Ang-1 and Tie-2 proteins were present in all cerebral vessels of normal brain including those of the choroid plexuses, whereas both these proteins as well as Ang-2 were present in choroid plexus epithelium and in ependymal cells, suggesting that angiopoietins have an autocrine effect on these cell types as well. In contrast, in the early phase after injury during the known period of BBB breakdown, increased Ang-2 mRNA and protein and decreased endothelial Ang-1 and Tie-2 proteins were observed. Two to 6 days after injury, the progressive increase in Ang-1 mRNA and protein and the decrease in Ang-2 coincided with cerebrovascular angiogenesis. Confocal microscopy showed colocalization of both Ang-1 and Ang-2 in endothelium of lesion vessels, and our observation of colocalization of Ang-1 and Ang-2 in polymorphonuclear leukocytes and macrophages has not been reported previously. This study demonstrates that Ang-1 is an important factor in maintaining normal homeostasis in the brain. Thus Ang-1 therapy may have therapeutic potential in reducing BBB breakdown and the ensuing edema after massive brain injury.

Angiogenesis Inducing Agents↗
PubMed1997

Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R.

The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.

Amino Acid Sequence↗
PubMed2003

TRPM8 mRNA is expressed in a subset of cold-responsive trigeminal neurons from rat.

Recent electrophysiological studies of cultured dorsal root and trigeminal ganglion neurons have suggested that multiple ionic mechanisms underlie the peripheral detection of cold temperatures. Several candidate "cold receptors," all of them ion channel proteins, have been implicated in this process. One of the most promising candidates is TRPM8, a nonselective cationic channel expressed in a subpopulation of sensory neurons that is activated both by decreases in temperature and the cooling compound menthol. However, evidence for the expression of TRPM8 in functionally defined cold-sensitive neurons has been lacking. Here, we combine fluorometric calcium imaging of cultured rat trigeminal neurons with single-cell RT-PCR to demonstrate that there are distinct subpopulations of cold responsive neurons and that TRPM8 likely contributes to cold transduction in one of them. TRPM8 is preferentially expressed within a subset of rapidly responsive, low-threshold (approximately 30 degrees C), cold-sensitive neurons. A distinct class of slowly responsive cold-sensitive neurons that is activated at lower temperatures (approximately 20 degrees C) generally lacks detectable TRPM8 mRNA. Together with previous findings, our data support the notion that cold responsive neurons are functionally heterogeneous.

Animals↗
PubMed1992

Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization.

Brown adipocytes are thermogenic cells which play an important role in energy balance. Their thermogenic activity is due to the presence of a mitochondrial uncoupling protein (UCP). Until recently, it was admitted that in rodents brown adipocytes were mainly located in classical brown adipose tissue (BAT). In the present study, we have investigated the presence of UCP protein or mRNA in white adipose tissue (WAT) of rats. Using polymerase chain reaction or Northern blot hybridization, UCP mRNA was detected in mesenteric, epidydimal, retroperitoneal, inguinal and particularly in periovarian adipose depots. The uncoupling protein was detected by Western blotting in mitochondria from periovarian adipose tissue. When rats were submitted to cold or to treatment with a beta-adrenoceptor agonist, UCP expression was increased in this tissue as in typical brown fat. Moreover, the expression was decreased in obese fa/fa rats compared to lean controls. Morphological studies showed that periovarian adipose tissue of rats kept at 24 degrees C contained cells with numerous typical BAT mitochondria with or without multilocular lipid droplets. Immunocytochemistry confirmed that multilocular cells expressed mitochondrial UCP. Furthermore, the number of brown adipocytes and the density of mitochondrial cristae increased in parallel with exposure to cold. These results demonstrate that adipocytes expressing UCP are present in adipose deposits considered as white fat. They suggest the existence of a continuum in rodents between BAT and WAT, and a great plasticity between adipose tissue phenotypes. The physiological importance of brown adipocytes in WAT and the regulation of UCP expression remain open questions.

Adipose Tissue↗
PubMed1994

Vibration exposure and prevention in Japan.

Working conditions of vibration exposure have generally improved, but many difficult problems must be solved such as (1) hygienic improvements in a variety of vibrating tools; (2) improving working conditions, for example, by limiting the time of operation in spite of economic difficulties such as those faced by those who work on a piece rate basis; (3) gathering more complete information about the risk population because of the large number of self-employed in informal employment sectors; and (4) finding work places after rehabilitation for patients, particularly in mountainous rural areas or in small scale industries. Historical observation of vibration and preventive measures in Japanese national forests was presented on the basis of the results of a retrospective cohort study in Kyushu, Japan. Prevalence rate of VWF remarkably changed from 58.4% in the groups that began to operate chain saws in 1960 to only a few cases in the groups who started the operation after 1971. When we compare the relationships between the results of long term cohort study and the consequences of preventive measures of vibration syndrome, the most important factor is the decrease of vibration exposure (improvement in chain saws plus the time restriction system). The comprehensive prevention system used in Japanese national forests consists of the following: (1) Health care system; (2) Work regulation system; (3) System for improving mechanized tools; (4) Warming system to protect against cold conditions; and (5) Education and training system.

Humans↗
PubMed1993

Properties of elastase from Atlantic cod, a cold-adapted proteinase.

An intestinal elastase was purified from Atlantic cod (Gadus morhua) of apparent molecular mass 24.8 kDa as determined by SDS-PAGE and with isoelectric point above pI 9.3. Heat stability and stability towards acidic pH was reduced in the cod enzyme as compared with porcine intestinal elastase. N-terminal amino-acid sequence analysis of cod elastase showed considerable similarity with porcine elastase. The cod enzyme was less sensitive to phenylmethylsulfonyl fluoride inhibition than porcine elastase, but sensitivity towards other inhibitors was similar. Kinetic properties were examined using the substrate Suc-Ala-Ala-Ala-p-nitroanilide and the cod enzyme was found to have more than 2-times turnover rate (kcat) as compared with the porcine enzyme, and slightly higher Km values. Thus, the catalytic efficiency (kcat/Km) of Atlantic cod elastase was about 2-times higher than observed with porcine elastase, which indicates an adaptive response towards the low temperature environmental in which the cod lives. Substrate specificity was studied by digestion of oxidized B-chain of insulin and by using synthetic substrates. Digestion was most rapid at the carbonyl side of alanine residues, but also occurred at valine and leucine residues.

Amino Acid Sequence↗
PubMed2000

Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans.

Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.

Aspergillus nidulans↗
PubMed2006

Integration of transcriptomic and metabolic analyses for understanding the global responses of low-temperature winemaking fermentations.

Wine produced at low temperature is often considered to have improved sensory qualities. To investigate the effects of temperature on winemaking, the expression patterns during the industrial fermentation process carried out at 13 degrees C and 25 degrees C were compared, and correlated with physiological and biochemical data, including viability, fermentation byproducts and lipid content of the cells. From a total of 535 ORFs that were significantly differentially expressed between the 13 degrees C and 25 degrees C fermentations, two significant transcription programmes were identified. A cold-stress response was expressed at the initial stage of the fermentation, and this was followed by a transcription pattern of upregulated genes concerned with the cell cycle, growth control and maintenance in the middle and late stages of the process at 13 degrees C with respect to 25 degrees C. These expression patterns were correlated with higher cell viability at low temperature. The other relevant transcriptomic difference was that several genes implicated in cytosolic fatty acid synthesis were downregulated, while those involved in mitochondrial short-chain fatty acid synthesis were upregulated in the fermentation process conducted at 13 degrees C with respect to that at 25 degrees C. These transcriptional changes were qualitatively correlated with improved resistance to ethanol and increased production of short-chain (C(4)-C(8)) fatty acids and their corresponding esters at 13 degrees C as compared to 25 degrees C. While this increase of ethyl esters may account in part for the improved sensory quality of wine fermented at 13 degrees C, it is still unclear how the esterification of the short-chain fatty acids takes place. On the basis of its strong upregulation at 13 degrees C, we propose a possible role of IAH1 encoding an esterase/ester synthase in this process.

Cold Temperature↗
PubMed2004

Impairment of mitochondrial oxidative phosphorylation in rat fatty liver exposed to preservation-reperfusion injury.

BACKGROUND/AIMS: As the impairment of the cellular energy metabolism contributes to the failure of fatty liver grafts after transplantation, we aimed to determine whether steatosis affects the oxidative phosphorylation activity during preservation. METHODS: Rat normal and fatty livers were preserved for 18 h and then reperfused with warm oxygenated solution. The oxidative phosphorylation, the F(0)F(1)-ATPase and the Complex I activities were assessed in isolated mitochondria before and after preservation, and during reperfusion. The ALT release and portal pressure were monitored during reperfusion. RESULTS: The baseline phosphorylation activity was similar in normal and steatotic mitochondria. After cold preservation, the respiratory control index and state 3 respiration decreased significantly only in steatotic livers. Reperfusion induced a further deterioration in either group. Contrary to normal liver, uncoupling of fatty liver mitochondria allowed the recovery of the maximal respiration rate only using succinate (Complex II-dependent substrate), but not glutamate-malate (Complex I-dependent). Complex I dysfunction was confirmed spectrophotometrically. The ATPase activity was also significantly lower in fatty livers. Finally, ALT release and portal pressure were greater in steatotic livers. CONCLUSIONS: The alteration of the oxidative phosphorylation activity during preservation is greatly exacerbated by fatty infiltration likely resulting from damage of the respiratory chain Complex I and of the F(0)F(1)-ATP synthase.

Adenosine Triphosphate↗
PubMed1992

Mechanisms of intrinsic tone in ferret vascular smooth muscle.

1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 degrees C there appeared to be a relatively large population of attached, non-force-generating cross-bridges, indicating that separate mechanisms are involved in regulating cross-bridge attachment and the force per cross-bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2+ or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]i) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2-dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Ca2+]i suggests the presence of a constitutively active Ca(2+)-independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.

Animals↗
PubMed1997

[Detection of ras gene mutation in various stages of gastric cancer by PCR/RFLP SSCP and DNA sequencing].

Investigate mutation of ras gene family in various stage of gastric cancer in China. PCR-RFLP, PCR-SSCP and PCR-DNA sequencing were used to detect mutation rates of H-ras, K-ras and N-ras gene. Mutation rates of H-ras at 12 codon in metaplasia, atypical hyperplasia, and progressive gastric cancer is 16.7% (6/36), 31.2% (15/48), 34.7% (25/72), respectively. In groups of superficial gastritis and normal control, no mutation were found. Mutations of H-ras 61 codon and N-ras 12 codon in various groups were the same as normal. Only 2 cases of K-ras 12 codon mutation were detected in gastric cancer by PCR-SSCP, but it was not identified by DNA sequencing. It may be of polymorphism. All H-ras 12 codon mutation were G-->T mutation. There are significant difference between groups of metaplasia, dysplasia, and gastric carcinoma comparing with group of normal control (P < 0.05, P < 0.01, P < 0.01). H-ras 12 codon mutation maybe an early event and maybe play important role in gastric carcinogenesis. Although K-ras mutation rate is high in colon cancer and leukemia it seemed to be relationship with gastric cancer. High frequency of H-ras 12 codon mutation maybe the characteristic of gastric cancer and associate with high incidence of gastric cancer in China. Three methods used in this experiment were compared that SSCP method is more sensitive than RFLP and cold SSCP is simpler and likely to be used in clinic.

Gastric Mucosa↗
PubMed1995

Expression of the antifreeze protein gene in transgenic goldfish (Carassius auratus) and its implication in cold adaptation.

Ocean pout (Macrozoarces americanus) antifreeze protein (AFP) genes (approximately 10(6) copies) were microinjected into the oocytes of goldfish (Carassius auratus). Out of a total of 303 oocytes injected, 235 (77.6%) were normally fertilized after in vitro maturation and insemination, and 136 (57.9%) of the fertilized eggs were hatched. Dot blot analysis of genomic DNA from 2-month-old fish indicated that 30 of the fish (22%) were positive for the AFP transgene. The numbers of integrated AFP genes ranged from a single copy to multiple copies per cell. Two P1 male founders, Y45 and GV16, were crossed separately with a control female, and the inheritance of the AFP transgene was analyzed. Results from Southern blotting and polymerase chain reaction were found to be 7% (5 out of 82) and 58.8% (20 out of 34), respectively, indicating that these two founders are mosaic. The percentage of positive F2 generated from Y45F1 crossed with a control female was 56%, consistent with a Mendelian inheritance. Mature AFP detected by immunoblotting was expressed in both F1 and F2 offspring. These studies indicate that the antifreeze protein gene is successfully transferred and expressed in goldfish. Transgenic goldfish are significantly more cold tolerant than controls when challenged with low temperatures. These studies suggest that the AFP gene may have application in providing cold tolerance in addition to freeze resistance for a variety of fish species.

Acclimatization↗