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PubMed1992

Incidence and coincidence of Listeria spp., motile aeromonads and Yersinia enterocolitica on ready-to-eat fleshfoods.

A survey for the presence of Listeria spp., Yersinia enterocolitica and motile aeromonads in 203 samples of ready-to-eat fleshfoods purchased from retail outlets was conducted. Overall, 39.4%, 3.4% and 23.2% of samples were positive for the presence of Listeria spp., Y. enterocolitica and motile aeromonads respectively. Two factors have been identified as contributing to contamination of fleshfoods by these cold-tolerant bacteria. These are (i) the method of sale; delicatessen-bought foods were notably more contaminated than similar products bought pre-packaged, and (ii) the method of preservation. For motile aeromonads fermented foods were the least contaminated, whereas smoked and cooked products had similar incidence rates. For L. monocytogenes, significantly more (41.9%) smoked products were contaminated than fleshfoods preserved by other methods. For Y. enterocolitica, only cooked products were contaminated. In the case of cooked fleshfoods it must be assumed that most contamination occurs post-cooking and that contamination rates are increased by poor food handling procedures. Of the three possible pairwise combinations of these organisms, the coincidence of Y. enterocolitica and motile aeromonads was the only one that differed significantly from a random distribution (P less than 0.001), indicating that fleshfoods contaminated with Y. enterocolitica are probably also contaminated by motile aeromonads.

Aeromonas↗
PubMed1993

Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica in pâté and a comparison with predictive models.

A reference or type strain and a food derived-strain of the cold-tolerant pathogens Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica were individually inoculated into samples of commercial pâté and incubated at 4 and 10 degrees C. The organisms were periodically estimated by presumptive counts, then values for the lag and generation times were calculated. Both strains of L. monocytogenes grew at both temperatures. The food strain of A. hydrophila grew only at 10 degrees C, and the type strain did not grow at either temperature. Similarly, the type strain of Y. enterocolitica did not grow at either temperature, whereas the food strain grew at both 4 and 10 degrees C. In some cases growth of non-test organisms may have inhibited the growth of these latter two species. The measured values of lag and generation times did not, in general, correlate well with those predicted by response surface models, taken from the literature and produced in this laboratory. It may be that the pâté contained an inhibitor that affected the growth of the organisms. The two strains of A. hydrophila and Y. enterocolitica showed significantly different growth characteristics, reinforcing the value of using a 'cocktail' of strains in growth experiments. Differences in predicted kinetic values from the models indicate that a model for any particular strain may not reflect the growth of naturally occurring contaminants of the same species.

Aeromonas hydrophila↗
PubMed2006

A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae.

The wilt fungus Verticillium dahliae Kleb. produces desiccation- and cold-tolerant resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. In an exploration of the molecular mechanisms involved in the development of these important structures, we have identified in V. dahliae a differentially expressed, class II hydrophobin gene (VDH1). vdh1 mutants generated through targeted gene disruption show a severe reduction in microsclerotia production, indicating that the gene is important for this type of development. Although vdh1 mutants do produce normal conidiophores and spores, desiccation-tolerance of the spores is reduced. The VDH1 gene is not, however, needed for normal disease development in tomato. VDH1's functions are multi-faceted, and seem generally relevant to long-term survival in V. dahliae.

Amino Acid Sequence↗
PubMed2026

Identifying the regulatory network of the key lipid metabolism transcription factor peroxisome proliferator-activated receptor in oysters.

Rising seawater temperatures driven by global warming have led to summer mass mortality events that pose significant challenges for the oyster industry. Peroxisome proliferator-activated receptor (PPAR) serves as a key transcriptional regulator of lipid metabolism and plays an essential role in thermal adaptation. However, the upstream regulatory mechanisms of PPAR remain poorly understood in marine organisms. In this study, we identified two PPAR subtypes (PPARα and PPARβ/δ) in oysters and compared transcriptomic data in different tissues and under various environmental stressors, with PPARα exhibiting higher expression levels and responsiveness to environmental stresses. We observed significantly higher PPARα gene expression levels and promoter activity in the relatively cold-tolerant Crassostrea gigas compared to C. angulata. The low expression of the inhibitory transcription factor CTNNB1 in C. gigas may contribute to higher gene expression of PPARα. Additionally, the expression genome-wide association study (eGWAS) identified 9 significant SNPs and 124 candidate regulatory genes associated with PPARα expression, including ubiquitination, phosphorylation, signaling pathways, lipid metabolism, and glucose metabolism. We provided the first experimental validation of the PPARα ubiquitination-degradation pathway in marine organisms via Co-IP, which was mediated by the E3 ligase RFWD3. The protein kinase SNF1 and signaling-related proteins PIKA and KCNK2 indirectly modulated PPARα downstream pathway activation to varying degrees. This study presents the first systematic investigation of PPARα expression regulation in marine organisms. It identifies key molecular regulators and provides novel insights into lipid metabolic regulation and molecular targets for genetic improvement of heat tolerance in oysters under global warming.

Animals↗
PubMed2005

Phenotypic and genotypic characterisation of Bacillus cereus isolates from Bangladeshi rice.

We report the first phenotypic and genotypic characterisation of Bacillus cereus from Bangladeshi rice. Seven strains of B. cereus were isolated from two high-yielding rice varieties (BR5 and BRRI Dhan28) which are cultivated during different rice-growing seasons in Bangladesh. The strains were identified as B. cereus based on colonial and cellular (light microscopic) morphology and were confirmed using the API 50 CHB test system and 16S rDNA analysis. They could also be distinguished from each other based on their substrate-utilisation patterns using the API system. No psychrotrophic (cold-tolerant) isolates were obtained and all were mesophilic with a minimum growth temperature of 10 degrees C, but with an ability to grow at a higher rate than normal temperature (45 degrees C) for B. cereus. All strains had temperature optima between 35 and 45 degrees C, but two groups could be distinguished based on having either a narrow or a broad optimum range. The different strains could not be distinguished based on their phospholipid compositions including major fatty acyl chains which were typical for B. cereus. However, capillary-GC/MS analysis of trace fatty acyl components of the lipids has been used for the first time to group the isolates. Genotypic analysis of the rDNA region has also been used to distinguish the strains. Restriction fragment length polymorphism (RFLP) analysis of the spacer region between the 16S and 23S rRNA genes yielded two groups based on different base sequences, whereas sequence analysis of variable regions in the 16S rRNA gene gave four different groups by base differences at two positions. Despite their association with rice, none of the strains produced emetic toxin. The spore germination response of heat-activated spores of the Bangladeshi B. cereus strains was typical of the bacterium, but three different groups could be identified based on differences in germination rates. The use of this polyphasic approach to taxonomy has enabled all the B. cereus strains to be distinguished from each other.

Bacillus cereus↗
PubMed2006

Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster.

Antifreeze protein 1 (DAFP-1), from the beetle Dendroides canadensis, was expressed in Drosophila melanogaster. Mean thermal hysteresis values (the difference between freezing and melting points), indicative of antifreeze protein activity, in the hemolymph of transgenic flies were found to be as high as 6.23+/-0.10 degrees C (using the nanoliter osmometer). Direct comparisons of the capillary and nanoliter osmometer techniques for measuring THA were made, illustrating the much higher values obtained by the latter. Transgenic Drosophila had supercooling points, both in contact with ice and not, that were slightly, but significantly, lower than wild-type controls (1.5-2.0 degrees C and 2.0-4.0 degrees C, respectively). The results indicate functionality of DAFP-1 in Drosophila melanogaster (the ability of DAFP-1 to inhibit both inoculative freezing across the cuticle and freezing initiated by endogenous ice nucleators). The much larger effects of DAFPs in inhibiting inoculative freezing and ice nucleation in Dendroides canadensis relative to the transgenic Drosophila may partially result from the lower DAFP concentrations and activities in Drosophila, however the absence of multiple types of DAFPs and absence of tissue specific expression may also contribute. Transgenic Drosophila were also able to live significantly longer than controls at 0 degrees C and 4 degrees C, indicating that DAFP-1 is able to increase cold tolerance at above freezing temperatures.

Acclimatization↗
PubMedDate not supplied

Homozygous carnitine palmitoyltransferase 1a (liver isoform) deficiency is lethal in the mouse.

To better understand carnitine palmitoyltransferase 1a (liver isoform, gene=Cpt-1a, protein=CPT-1a) deficiency in human disease, we developed a gene knockout mouse model. We used a replacement gene targeting strategy in ES cells that resulted in the deletion of exons 11-18, thus producing a null allele. Homozygous deficient mice (CPT-1a -/-) were not viable. There were no CPT-1a -/- pups, embryos or fetuses detected from day 10 of gestation to term. FISH analysis demonstrated targeting vector recombination at the expected single locus on chromosome 19. The inheritance pattern from heterozygous matings was skewed in both C57BL/6NTac, 129S6/SvEvTac (B6;129 mixed) and 129S6/SvEvTac (129 coisogenic) genetic backgrounds biased toward CPT-1a +/- mice (>80%). There was no sex preference with regard to germ-line transmission of the mutant allele. CPT-1a +/- mice had decreased Cpt-1a mRNA expression in liver, heart, brain, testis, kidney, and white fat. This resulted in 54.7% CPT-1 activity in liver from CPT-1a +/- males but no significant difference in females as compared to CPT-1a +/+ controls. CPT-1a +/- mice showed no fatty change in liver and were cold tolerant. Fasting free fatty acid concentrations were significantly elevated, while blood glucose concentrations were significantly lower in 6-week-old CPT-1a +/- mice compared to controls. Although the homozygous mutants were not viable, we did find some aspects of haploinsufficiency in the CPT-1a +/- mutants, which will make them an important mouse model for studying the role of CPT-1a in human disease.

Animals↗
PubMed1991

Hypothermic protection--a fundamental property of "antifreeze" proteins.

For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4 degrees C for 24 h in the presence of type I alanine rich alpha helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that cold-sensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins.

Animals↗
PubMed2003

Lipid diffusion in the thylakoid membranes of the cyanobacterium Synechococcus sp.: effect of fatty acid desaturation.

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.

Boron Compounds↗
PubMed2000

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET).

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.

Catalysis↗
PubMed2001

Gallotannin biosynthesis: beta-glucogallin: hexagalloyl 3-O-galloyltransferase from Rhus typhina leaves.

Leaves of staghorn sumac (Rhus typhina) contain several galloyltransferases that catalyze the beta-glucogallin dependent transformation of 1,2,3,4,6-pentagalloylglucose to gallotannins. Among these, an enzyme has been isolated that preferentially acylates the 3-position of the hexagalloylglucose, 3-O-digalloyl-1,2,3,4,6-tetra-O-galloylglucose, to afford the corresponding heptagalloylglucose being characterized by a 3-O-meta-trigalloyl side-chain. The enzyme, for which a M(r) of ca. 260,000 was determined, was purified to apparent homogeneity. SDS-PAGE suggested an alpha4beta4-conformation of the native enzyme. It had a pH-optimum and an isolelectric point at pH 5.6, was most stable at pH 4.0-4.3, and displayed excellent heat-stability and in particular an extreme cold-tolerance. We propose the systematic name "beta-glucogallin: hexagalloylglucose 3-O-galloyltransferase" for this new enzyme.

Acylation↗
PubMed2003

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris.

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.

Chlorella↗
PubMed2000

The occurrence and ecology of Trichinella in marine mammals.

Trichinella in marine mammals has a circumpolar arctic distribution and a narrow range of host species. It is commonly found in polar bears (Ursus maritimus), and increasingly in walruses (Odobenus rosmarus) where it presents a significant zoonotic hazard. This has resulted in the implementation of food safety programs in some arctic communities to test harvested walrus meat for Trichinella larvae prior to consumption. Trichinella has been reported infrequently in bearded seals (Erignathus barbatus) and ringed seals (Phoca hispida), and was once observed in a Beluga whale (Delphinapterus leucas). Cannibalism is probably the most important factor in maintaining a Trichinella cycle in polar bears. Arctic carnivores such as polar bears, arctic foxes (Alopex lagopus) and domestic dogs (Canis familiaris) have a high prevalence of Trichinella infection and the carcasses of at least some of these animals are deposited in the ocean. Scavenging of these carcasses by walruses probably occurs, but may not account for the high prevalence the parasite seen in this host species. Predation, carrion feeding and cannibalism have been documented for walruses and a sylvatic cycle similar to that of bears may exist in walrus populations. Seals and whales are likely infected through infrequent exposure to infected carcasses, either directly by scavenging or indirectly by consuming amphipods or fish that have fed on infected carcasses. The inefficiency of this mechanism may account for the low prevalence of Trichinella in seals and whales. It is known that isolates from marine mammals are cold tolerant, and infectious for man, and have been identified as Trichinella nativa (T2). Molecular and other phylogenetic studies would be useful to facilitate studies on the inter-relationship of Trichinella cycles involving marine and terrestrial mammals in the arctic and subarctic, and in the investigation of human outbreaks of trichinellosis in these areas.

Animals↗
PubMed2003

Diversity in and adaptation to breath-hold diving in humans.

Several features of potential adaptation to breath-hold diving in diving populations and extreme divers are reviewed. Thermal adaptation consists of an improvement in cold tolerance, as witnessed by a decrease in critical water temperature, and implies an elevation of the shivering threshold associated with a greater body insulation. This is indicative of either a strong peripheral vasoconstriction or a more effective countercurrent heat exchange. Respiratory adaptation consists of a blunted ventilatory response to carbon dioxide and an enlargement of lung volumes. Finally, the occurrence of a diving response has been demonstrated. An extreme peripheral vasoconstriction is associated with a dramatic increase in arterial blood pressure. The consequent stimulation of arterial baroreceptors causes an extreme drop of heart rate. Bradycardia is not compensated by a higher stroke volume, with consequent decrease in cardiac output. This decrease, however, is not such as to undermine perfusion to vital organs. Redistribution of blood flow occurs, and some organs such as skeletal muscle may become unperfused, as indicated by the high blood lactate concentrations at low metabolic rate. It is not possible to state, however, whether these changes reflect genetic adaptations or an adaptive response to a prolonged environmental stress.

Adaptation, Physiological↗
PubMed2001

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos.

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%-64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.

Amino Acid Sequence↗
PubMed2001

Thermal hysteresis proteins.

Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

Adaptation, Biological↗
PubMed1999

Characterization of Ribes rubrum callus cultured under low temperature.

Ribes rubrum callus was cultured at the temperature of 15 degrees C for 28 d. The contents of proteins and lipids in the callus cultured at 15 degrees C in a 28 d culture was significantly increased, being 1.1- and 1.2-fold, respectively, of those in callus cultured at 25 degrees C. Comparisons of the contents of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidyldiacylglycerol (PG) revealed that in the callus cultured at 25 degrees C, the content of MGDG was significantly decreased and those of the others significantly increased compared to those in callus cultured at 15 degrees C, in a 7 d culture. But, in a 28 d culture, the contents of these lipids in callus at 15 degrees C became the same as those in callus cultured at 25 degrees C. In regard to the protein composition, the contents of proteins 30 KDa or less in callus cultured at 15 degrees C in a 28 d culture were significantly higher than those in callus cultured at 25 degrees C. These consequences reflect that not only increase in the polyunsaturated fatty acid (PUFA) ratio of total fatty acid (TFA) with cold treatment reported by us previously [Hirano, M., J. Ferment. Bioeng., 83, 608-611 (1997)], but also an increase in low-molecular-weight protein content might be responsible for the cold-tolerance of R. rubrum callus.

Journal Article↗
PubMed2002

Ventilatory pattern and chemosensitivity in unanesthetized, hypothermic ground squirrels (Spermophilus lateralis).

The present study examined the effects of severe hypothermia in the absence of anesthesia on breathing pattern, ventilatory control and chemosensitivity in a cold tolerant species capable of seasonal hibernation. Hypothermia was induced in ground squirrels and ventilation and heart rate were recorded in animals breathing air at a body temperature (Tb) of 5 and 10 degrees C. The animals were then exposed to hypercapnic (2, 4 and 6% CO(2)) and hypoxic (12, 10, 8 and 4% O(2)) gas mixtures. We found that severe hypothermia in ground squirrels caused the breathing pattern to change from a continuous pattern to patterns that are commonly observed during hibernation. This suggests that temperature and metabolism alone are important factors in producing these patterns. The relative ventilatory sensitivity to hypercapnia was retained in the ground squirrel during hypothermia while ventilatory sensitivity to hypoxia was totally abolished. This is in contrast to hibernation where a small but significant hypoxic ventilatory response is present along with an enhanced relative response to hypercapnia. This suggests that changes in Tb alone can not account for the changes seen in ventilatory sensitivity during hibernation.

Animals↗