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Cold adaptation as a life style strategy.

Humans evolved in Africa, where climate would have required cold adaptation. Modern humans have housing which separates them from the environment. Metabolic disorders are prevalent in populations with excess Western diet and lack of exercise. A thrifty genotype has been proposed as an explanation for susceptibility. An alternative explanation is that cold adaptation is absent. But the human genome is complex, and there are many variations possible in the metabolic pathways. Behaviour which uses the circadian rhythms of metabolism may be helpful for fitness.

Acclimatization↗

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones.

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.

Adaptation, Physiological↗

Safety, efficacy, and effectiveness of cold-adapted influenza vaccine-trivalent against community-acquired, culture-confirmed influenza in young children attending day care.

OBJECTIVE: The goal was to evaluate the safety, tolerability, and efficacy of an investigational, refrigerator-stable formulation of live attenuated influenza vaccine (cold-adapted influenza vaccine-trivalent) against culture-confirmed influenza, acute otitis media, and effectiveness outcomes in young children in day care over 2 consecutive influenza seasons. METHODS: Children 6 to <36 months of age who were attending day care were assigned randomly in year 1 to receive 2 doses of vaccine or placebo intranasally, 35 +/- 7 days apart. In year 2, subjects received 1 dose of the same treatment as in year 1. RESULTS: A total of 1616 subjects (vaccine: 951 subjects; placebo: 665 subjects) in year 1 and 1090 subjects (vaccine: 640 subjects; placebo: 450 subjects) in year 2 were able to be evaluated for efficacy. The mean age at first vaccination was 23.4 +/- 7.9 months. In year 1, the overall efficacy of the vaccine against influenza subtypes similar to the vaccine was 85.4%; efficacy was 91.8% against A/H1N1 and 72.6% against B. In year 2, the overall efficacy was 88.7%; efficacy was 90.0% against H1N1, 90.3% against A/H3N2, and 81.7% against B. Efficacy against all episodes of acute otitis media associated with culture-confirmed influenza was 90.6% in year 1 and 97.0% in year 2. Runny nose or nasal discharge after dose 1 in year 1 was the only reactogenicity event that was significantly more frequent with cold-adapted influenza vaccine-trivalent (82.3%) than placebo (75.4%). CONCLUSIONS: Cold-adapted influenza vaccine-trivalent was well tolerated and effective in preventing culture-confirmed influenza illness in children as young as 6 months of age who attended day care.

Acute Disease↗

Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66, the master donor virus for live attenuated influenza vaccines (FluMist).

Cold adapted (ca) B/Ann Arbor/1/66 is the master donor virus for the influenza B (MDV-B) vaccine component of the live attenuated influenza vaccine (FluMist). The six internal genes contributed by MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the vaccine strains. Previously, it has been determined that the PA and NP segments of MDV-B control the ts phenotype while the att phenotype requires the M segment in addition to PA and NP. Here, we show that the PA, NP and PB2 segments are responsible for the ca phenotype of MDV-B when examined in chicken cell lines. Five loci in three RNA segments, R630 in PB2, M431 in PA and A114, H410 and T509 in NP, are sufficient to allow efficient virus growth at 25 degrees C. Substitution of these five amino acids with wt (wild type) residues completely reverted the MDV-B ca phenotype. Conversely, introduction of these five ca amino acids into B/Yamanashi/166/98 imparted the ca phenotype to this heterologous wt virus. In addition, we also found that the MDV-B M1 gene affected virus replication in chicken cells at 33 and 37 degrees C. Recombinant viruses containing the two MDV-B M1 residues (Q159, V183) replicated less efficiently than those containing wt M1 residues (H159, M183) at 33 and 37 degrees C, implicating the role of the MDV-B M segment to the att phenotype. The complexity of the multigenic signatures controlling the ca, ts and att phenotypes of MDV-B provides the molecular basis for the observed genetic stability of the FluMist vaccines.

Adaptation, Physiological↗

Increased thermoregulatory responsiveness in cold adapted but not in hyperthyroid hypermetabolic rats.

Cold-adapted (CA) rats, unlike non-adapted (NA) ones, give exaggerated metabolic response to acute cold exposure, with paradoxical "overshoot" core temperature (Tc) rise in the cold, and they also give enhanced hyperthermia to central injection of prostaglandin E1 (PGE1). The adaptation-dependent differences might be explained either by the high thermogenic capacity of peripheral tissues in CA rats or by differences in the central processing of regulatory signals. If high tissue metabolism sufficiently explains the extreme responses of CA animals, other hypermetabolic states (with high resting metabolic rate, RMR), e.g. hyperthyroidism, should also be accompanied by enhanced reactions. In the present study thermoregulatory responses to acute cold exposure or to PGE1 were compared in hypermetabolic CA, similarly hypermetabolic thyroxine-treated (T4) and control non-hypermetabolic NA rats (mean RMR = 8.12, 8.47 and 6.03 W kg(-1), respectively). Cold exposure was followed by paradoxical core temperature (Tc) rise of 0.5 to 0.7 degrees C only in CA rats, but by Tc fall (0.8 to 2.1 degrees C) in NA and T4 animals. Identical central stimuli (PGE1) induced larger elevations of Tc and metabolic rate in CA rats than in similarly hypermetabolic T4 or in non-hypermetabolic NA animals (mean Tc rise of 1.9 degrees C in CA vs. 0.9 degrees C in T4 and 1.0 degrees C in NA rats). Vasodilatation thresholds were also similar in NA and T4, but lowered in CA animals. A hypermetabolic status, per se, does not seem to explain the enhanced thermoregulatory responsiveness of CA animals, adaptation-induced central regulatory changes may be more important for the "overshoot" phenomenon.

Acclimatization↗

Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris.

To investigate the mechanism of cold adaptation of isocitrate lyase (ICL; EC 4.1.3.1) from the psychrophilic bacterium Colwellia maris, Gln207 and Gln217 of this enzyme were substituted by His and Lys, respectively, by site-directed mutagenesis. His184 and Lys194 of ICL from Escherichia coli, corresponding to the two Gln residues of C. maris ICL, are highly conserved in the ICLs of many organisms and are known to be essential for catalytic function. The mutated ICLs (Cm-Q207H and Cm-Q217K, respectively) and wild-type enzymes of C. maris and E. coli (Cm-WT and Ec-WT) with His-tagged peptides were overexpressed in E. coli cells and purified to homogeneity. Thermolabile Cm-WT and mutated ICLs were susceptible to digestion with trypsin, while relatively thermostable Ec-WT was resistant to trypsin digestion, suggesting that the thermostability and resistance to tryptic digestion of the ICLs are related. Cm-Q207H and Cm-Q217K showed specific activities similar to Cm-WT at temperatures between 30 degrees C and 40 degrees C, but their activities between 10 degrees C and 25 degrees C were decreased, indicating that the two Gln residues of the C. maris ICL play important roles in its cold adaptation. Phylogenetic analysis of ICLs from various organisms revealed that the C. maris ICL can be categorized in a novel group, subfamily 3, together with several eubacterial ICLs.

Adaptation, Physiological↗

[The role of the paraventricular hypothalamic nuclei in the reaction of the hypophyseo-adrenal cortex system to cold adaptation].

The lesion of paraventricular hypothalamic nuclei (PVN) led to a delay in the development of cold adaptation in white rats, the latter starting with no activation of pituitary-adrenocortical system. After the PVN lesion, corticosteroid response to immobilization was inhibited. The delay in formation of the cold adaptation seems to be due to the changes in the pituitary-adrenocortical system response to the stress after the PVN lesion.

11-Hydroxycorticosteroids↗

Evaluation of cold-adapted and temperature-sensitive mutants of parainfluenza virus type 3 in weanling hamsters.

Cold-adapted (ca) and temperature-sensitive (ts) mutants of parainfluenza virus 3 were produced by serially passaging wild type (wt) parainfluenza virus 3 at 20 degrees C 45 times. Previously, plaque-purified viruses (clones) were selected from the wt parent and from cold passage levels 7, 12, 18, and 45 and characterized in vitro. In the present study, we evaluated at least one mutant from each cold passage level for attenuation in hamsters. Four of the five mutants tested were ts and ca (one each from cold passage levels 7, 12, 18, and 45) and one was ca but not ts (from cold passage 18). Groups of hamsters were inoculated intranasally with either parent wt or mutant virus. Four or five hamsters from each group were sacrificed prior to inoculation and daily on the first, second, third, fourth, and sixth or seventh day postinoculation and the amount of virus in nasal turbinates and lungs was quantitated. The quantity of virus recovered from the hamsters and the parainfluenza virus 3 antibody titers were inversely related to the cold passage level. Two of the mutants did not replicate in hamsters. Cold adaptation of parainfluenza virus 3 resulted in progressive attenuation of the virus in weanling hamsters. These highly attenuated mutants are suitable for evaluation in children as live virus vaccines.

Animals↗

Partial change of the ts-phenotype of cold-adapted influenza virus strain grown in canine kidney cells in the presence of trypsin.

The cold-adapted temperature-sensitive (ts) influenza virus strain A/Leningrad/134/17/57 (H2N2) multiplied well at 32 degrees C (optimal temperature); lower titres of infectious virus were obtained in developing chick embryos at 40 degrees C. In a canine kidney (MDCK) cell line and in primary calf kidney (CK) cells an increased reproduction of the virus was found at 40 degrees C especially in the presence of trypsin. The ratios of virus titres obtained at optimal versus higher temperatures (RCT40) were by 1,000 times lower than those found in chick embryos. Polyacrylamide gel electrophoresis revealed a comparable synthesis of the cold-adapted influenza virus strain polypeptides HA, NP, M and NS in MDCK cells, regardless whether they were incubated at optimal or non-permissive temperatures.

Adaptation, Physiological↗

Duration of immunity engendered by a single dose of a cold-adapted strain of Avian pneumovirus.

The duration of immunity after a single dose of a cold-adapted strain of Avian pneumovirus (APV) was studied. Turkeys were vaccinated at 1 wk of age and challenged with virulent virus 3, 7, 10, and 14 wk later. Nonvaccinated groups were also challenged at the same times. No clinical signs were observed in the vaccinated birds after vaccination or after any challenge. No viral RNA was shed by the vaccinated birds after any challenge. The nonvaccinated birds shed viral RNA after all challenges. Avian pneumovirus-specific humoral antibodies were detected in the vaccinated birds until 14 wk after vaccination. The results of this preliminary study indicate that inoculation with a single dose of a cold-adapted strain of APV at 1 wk of age provides protection until 15 wk of age.

Animals↗

Homotypic and heterotypic immunity of influenza A viruses induced by recombinants of the cold-adapted master strain A/Ann Arbor/6/60-ca.

The cold-adapted (ca) influenza A virus A/Ann Arbor/6/60-ca when administered intranasally to mice in two doses 3 weeks apart induces solid immunity to challenge 3 weeks later with heterotypic influenza A wild-type viruses (17). In the present study heterotypic immunity against viruses from different sub-types was shown to be relatively short-lived, having declined significantly 9 weeks after vaccination and being completely absent by 21 weeks. On the other hand, immunity against challenge viruses with surface antigens similar to the ca vaccinating virus or to other H 3 N 2 viruses remained high, even in the absence of detectable serum haemagglutination-inhibiting antibody. Both short- and long-term immunity induced by ca viruses was unaffected by earlier priming experiences with other wild-type or ca viruses. These results suggest that at least two mechanisms are involved in respiratory immunity to influenza viruses.

Animals↗

[The NS gene--a possible determinant of apathogenicity of a cold-adapted donor of attenuation A /Leningrad/134/47/57 and its reassortants].

Phenotypical properties of single-gene reassortants of attenuated cold-adapted strain A/Leningrad/135/47/57 (H2N2) and strain A/PR8/34 virulent for laboratory animals were studied. Only the group of reassortants inheriting NS gene from cold-adapted virus was fully attenuated for various animals species, similarly as reassortants with 6/2 genomic formula containing all the 6 internal protein genes from strain A/Leningrad/134/47/57. Reassortant 25A-1 single-gene for NS was temperature-sensitive (ts) on mammalian cells but formed plaques at 40 degrees C on chicken kidney cells. Reassortants with genomic formula 6/2 were temperature-sensitive in all types of cells used. Reassortant 25A-1 could synthesize normal amounts of polypeptides in MDCK cells at 39 degrees C, whereas protein synthesis of reassortants with 6/2 genomic formula was noticeably reduced at this temperature. Hence, a similar level of attenuation of both reassortant groups appears to be due to various molecular mechanisms. Possible role of NS2 gene mutation in attenuation of strain A/Leningrad/134/47/57 and its reassortants is discussed.

Adaptation, Physiological↗

Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase.

In an effort to explore the role of glycine clusters on the cold adaptation of enzymes, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Gly261 and Gly262. The replacement of Gly262 by Ala resulted in an inactive enzyme. Substitution of Gly261 by Ala resulted to an enzyme with lower stability and increased energy of activation. The double mutant G261A/Y269A designed on the basis of side-chain packing criteria from a modelled structure of the enzyme resulted in restoration of the energy of activation to the levels of the native enzyme and in an increased stability compared to the mutant G261A. It seems therefore, that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme.

Adaptation, Physiological↗

PCR restriction analysis of genome composition and stability of cold-adapted reassortant live influenza vaccines.

Using cold-adapted master donor strains of influenza virus as a model, an approach was developed that exploits unique nucleotide differences between the donor strains and wild-type influenza viruses for rapidly and simply determining the genome composition and genetic stability of live attenuated vaccine reassortants. The approach is based on PCR amplification of approximately 150-300-nucleotide-long regions of individual RNA segments that include the unique nucleotide positions, followed by restriction nuclease treatment of the DNAs obtained with specific restriction endonucleases. Restriction sites recognized by chosen nucleases either existed or were created during PCR in the genome of one (but not the other) parent strain. The technique requires a minimal amount of infectious virus (approx. 100 microliters of allantoic or tissue culture fluid with a haemagglutination titre 1:4-1:8 or less) and allows rapid (within about 10 h) determination of the origin of the RNA segment or the presence of a mutation. The method is beneficial for genome composition analysis of reassortant vaccine strains as well as for investigation of the genetic stability of live attenuated vaccines during replication in vaccinees.

Acclimatization↗

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions.

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.

Adaptation, Physiological↗

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution.

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Adaptation, Physiological↗

[Effect cold adaptation to the ouabain-sensitive component of kidney respiration in rats].

In the course of intermittent cold adaptation (0 degrees C, for 16 weeks) the rats demonstrated an increase in the rate of oxygen consumption by a suspension of kidney pieces. The increase of oxygen consumption by 60% is ouabain-sensitive. Together with the data on Na, K-ATPase activation this indicates the main role of the Na-pump in the increased respiration of the kidneys on cold adaptation.

Acclimatization↗

Genetics of cold-adapted B/Ann Arbor/1/66 influenza virus reassortants: the acidic polymerase (PA) protein gene confers temperature sensitivity and attenuated virulence.

The cold-adapted B/Ann Arbor/1/66 influenza virus (ca B/AA/1/66) expresses temperature-sensitive (ts), cold-adapted (ca) and attenuation phenotypes. Reassortants which inherit one or more genes from ca B/AA/1/66 and all other genes from a virulent, wild-type influenza virus, B/Houston/1732/76, were produced and evaluated in order to identify the gene(s) responsible for the ts, ca and attenuation phenotypes. Only reassortants which inherited the PA gene from ca B/AA/1/66 expressed the ts phenotype in MDCK cells at 39 degrees C. None of the reassortants tested expressed the ca phenotype in embryonated eggs at 25 degrees C. The virulence of several reassortants was evaluated in ferrets. Inheritance of the PA gene from ca B/AA/1/66 was correlated with significant febrile attenuation and the apparent restriction of viral replication in the lower respiratory tract. Isolation of a virulent, non-ts revertant virus inheriting only the PA gene from ca B/AA/1/66 established a direct relationship between expression of the ts phenotype and attenuated virulence. Evidence for the contribution of at least one other gene from ca B/AA/1/66 to attenuation was observed. Thus, based on the methods used to determine reassortant gene compositions, these results indicate that the PA gene is primarily responsible for attenuation of ca B/AA/1/66 and its reassortants.

Cold Temperature↗