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The immunogenicity of reassortants of the cold-adapted influenza A master strain A/Ann Arbor/6/60 is determined by both the genes for cold-adaptation and the haemagglutinin gene.

Two surface antigen segregants were prepared by co-infection of chicken embryo kidney cell cultures with reassortants of the cold-adapted influenza A master strain A/Ann Arbor/6/60-ca (H2N2) possessing the surface antigens of A/Queensland/6/72 (H3N2) and A/Hong Kong/123/77 (H1N1) and other genes that were common to the master strain. The segregants were shown by serological tests to possess H3N1 and H1N2 surface antigens but it was not possible to determine the presence of H1 or N1 genes by single-stranded RNA polyacrylamide gel electrophoresis. The immunogenic properties of A/Queensland/6/72-ca and H3N1 segregant CR6/35/2/9 were compared by immunising mice intranasally with graded doses of each virus twice at an interval of 3 weeks and then challenging with the wild-type A/Queensland/6/72 (H3N2). Clearance of the challenge virus occurred in mice immunised with the same vaccinating dose, indicating that the immunogenicity of both the ca H3N2 and H3N1 viruses was identical and similar findings were obtained for mice immunised with A/Hong Kong/123/77-ca and the H1N2 segregant CR6/35/1/19 and challenged with the wild-type A/Hong Kong/123/77. Therefore, there appears to be a good correlation between immunogenicity and the inheritance of the haemagglutinin gene.

Adaptation, Physiological↗

Assessment of myocardial perfusion during cold stress using thallium-201 scintigraphy in diabetic patients with abnormal changes in left ventricular function during cold stress.

Eleven type-I (insulin-dependent) diabetic patients with abnormal changes in left ventricular function in response to cold stress (CS) were investigated to try and determine the cause of these abnormal responses. Resting M-mode echocardiography demonstrated that all 11 patients had normal left ventricular dimensions and wall motion, thereby excluding overt cardiomyopathy. Thallium-201 scintigraphy was used to assess myocardial perfusion during CS and eight patients were found to have perfusion defects during stress which persisted in four. It is possible that CS unmasks evidence of myocardial ischemia but it is also possible that the abnormal responses to CS reflect altered vasomotor reactivity in the diabetic patient, producing coronary spasm. Left ventricular function may be influenced by many factors in diabetes and an abnormal CS test may not necessarily indicate structural disease. Such tests, however, may help in the further understanding of the pathophysiology of heart disease in diabetes.

Adult↗

Cold-adapted yeasts as producers of cold-active polygalacturonases.

Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2 degrees C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14 degrees C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40 degrees C and pH 5, and that from the Cryptococcus strains at 50 degrees C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40 degrees C, while that from the other strains had already lost activity at 30 degrees C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.

Carbon↗

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures.

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0 degrees C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24 degrees C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43 degrees C nor lambda phage propagation at an even lower temperature, 30 degrees C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15 degrees C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.

Adenosine Triphosphatases↗

A comparison of the effects of prior cold incubation on cerebral cortex function in a hibernator (Cricetus auratus) and a non-hibernator (Cavia porcellus)--II. High energy phosphate levels in cerebral cortex slices after in vitro cold incubation.

1. ATP and CP levels were measured in brain slices from golden hamster and guinea pig after varying periods of cold storage and subsequent incubation at 37 degrees C in the presence and absence of K+ salts. 2. ATP and CP levels were maintained at higher levels in hamster tissue. 3. The results are discussed in relation to the ability of a hibernator to transform and transport chemical energy at low temperatures.

Adenosine Triphosphate↗

The structure of a cold-adapted family 8 xylanase at 1.3 A resolution. Structural adaptations to cold and investgation of the active site.

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 A resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.

Binding Sites↗

Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodelling and ascites morbidity in broilers under normal and cold temperature.

Two experiments were conducted to evaluate the effects of early feed restriction on lipid peroxidation, pulmonary vascular remodelling and ascites incidence in broilers under normal and low ambient temperature. In experiment 1, the restricted birds were fed 8h per day either from 7 to 14 d or from 7 to 21 d, while the controlled birds were fed ad libitum. In experiment 2, the restricted birds were fed 80 or 60% of the previous 24-h feed consumption of full-fed controls for 7 d from 7 to 14 d. On d 14, half of the birds in each treatment both in experiment 1 and experiment 2 were exposed to low ambient temperature to induce ascites. Body weight and feed conversion ratio were measured weekly. The incidences of ascites and other disease were recorded to determine ascites morbidity and total mortality. Blood samples were taken on d 14, 21, 28, 35 and 42 to measure the plasma malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). On d 42, samples were taken to determine the right/total ventricular weight ratio (RV/TV), vessel wall area/vessel total area ratio (WA/TA) and mean media thickness in pulmonary arterioles (mMTPA). Low-temperature treatment increased plasma MDA concentration. When broilers were exposed to a cool environment for 3 weeks, plasma SOD and GSH-Px activity were decreased compared with normal-temperature chicks. RV/TV, WA/TA and mMTPA on d 42 were increased in birds exposed to cold, consistent with the increased pulmonary hypertension and ascites morbidity. Early feed restriction markedly decreased plasma MDA concentration. The plasma SOD and GSH-Px activity of feed-restricted birds were markedly higher than those fed ad libitum on d 35 and d 42. All early feed restriction treatments reduced ascites morbidity and total mortality. On d 42, the RV/TV, WA/TA and mMTPA of feed-restricted broilers were lower than that of the ad libitum-fed broilers. The results suggested that early feed restriction alleviated the lipid peroxidation, promoted the activity of enzymatic antioxidant and inhibited pulmonary vascular remodelling. These changes might be associated with reduced ascites incidence.

Animals↗

Disruption analysis of the gene for a cold-regulated RNA-binding protein, rbpA1, in Anabaena: cold-induced initiation of the heterocyst differentiation pathway.

A cold-regulated operon, rbpA1-rpsU, encodes an RNA-binding protein and a ribosomal protein in Anabaena variabilis M3. The level of expression of this gene cluster was about ten times higher at temperatures below 30 degrees C than at 38 degrees C. To study the role of the RbpA1 protein in vivo, we constructed insertional disruptants of rbpA1. These strains were totally devoid of RbpA1 protein but contained a normal level of the ribosomal protein S21, a product of the rpsU gene. The disruptants were morphologically normal at 38 degrees C, but at 22 degrees C they produced unusual cells at a low frequency. These cells were probably at an initial stage of proheterocyst formation. Various molecular events that are related to heterocyst initiation, namely, excision of the 11-kbp DNA element in nifD and the accumulation of transcripts of xisA and hetR, also occurred in the disruptants at 22 degrees C in the presence of nitrate ions, but these events did not occur in the presence of ammonium ions or at 38 degrees C. The results suggest that RbpA1 is required for enhanced repression of heterocyst initiation at low temperatures in the presence of nitrate. Possible mechanisms of the action of RabA1 are discussed.

Anabaena↗

The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo--cytoplasmic partitioning.

Low temperature is one of the most important environmental stimuli that control gene transcription programs and development in plants. In Arabidopsis thaliana, the HOS1 locus is a key negative regulator of low temperature-responsive gene transcription. The recessive hos1 mutation causes enhanced induction of the CBF transcription factors by low temperature as well as of their downstream cold-responsive genes. The hos1 mutant plants flower early, and this correlates with a low level of Flowering Locus C gene expression. The HOS1 gene was isolated through positional cloning. HOS1 encodes a novel protein with a RING finger motif near the amino terminus. HOS1 is ubiquitously expressed in all plant tissues. HOS1--GFP translational fusion studies reveal that HOS1 protein resides in the cytoplasm at normal growth temperatures. However, in response to low temperature treatments, HOS1 accumulates in the nucleus. Ectopic expression of HOS1 in wild-type plants causes cosuppression of HOS1 expression and mimics the hos1 mutant phenotypes.

Acclimatization↗

Hope against the cold: individual differences in trait hope and acute pain tolerance on the cold pressor task.

Hope theory (see Snyder, 1994) is presented as a useful framework for understanding reactions to pain. In Study 1, persons scoring higher on the trait Hope Scale (Snyder, Harris et al., 1991) kept their hands in the freezing water (of a cold pressor task) for significantly longer. In Study 2, the higher-hope males, and not females, as measured by both trait and state hope (Snyder, Sympson et al., 1996), recognized the onset of the pain threshold significantly later. Moreover, in Study 2, results showed that individual differences measures of optimism, self-efficacy, depression, and positive and negative affects did not relate to the pain onset and tolerance variables. The implications of hope as related to the pain process and related research are discussed.

Acute Disease↗

Evaluation of A/Alaska/6/77 (H3N2) cold-adapted recombinant viruses derived from A/Ann Arbor/6/60 cold-adapted donor virus in adult seronegative volunteers.

The influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) virus was evaluated as a donor of attenuating genes to new variants of influenza A virus. This ca donor virus was mated with the A/Alaska/6/77 (H3N2) wild-type virus, and three A/Alaska/6/77 (H3N2) ca recombinant viruses were produced. The parental origin of the genes in the three ca recombinants had been determined previously (2), and their virulence for adult seronegative volunteers was assessed in the present study to identify the genes present in the ca donor virus that confer attenuation. Each of the recombinants received the hemagglutinin and neuraminidase genes from the A/Alaska/6/77 (H3N2) wild-type parent. One ca recombinant (CR-29) received all six transferable genes from the ca parent and was found to be satisfactorily attenuated in the volunteers. The two other ca recombinants received five of the six transferable genes with a wild-type gene at the M or NS locus. The pattern of infection in humans with these latter two ca recombinants was similar to the CR-29 ca recombinant. These findings demonstrate that inheritance of a gene in ca recombinants at the M or NS locus segregates independently of attenuation and suggest that the M and NS genes present in the ca donor virus are not the major determinants of attenuation conferred by this virus.

Antibodies, Viral↗

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees.

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.

Adolescent↗

Comparative studies of wild-type and "cold-mutant" (temperature-sensitive) influenza viruses: polypeptide synthesis by an Asian (H2N2) strain and its cold-adapted variant.

The structure and replication of a cold-adapted, temperature-sensitive (TS) mutant of an Asian (H2N2) influenza virus was compared with that of its wild-type (WT) parent. Viruses were grown in a chicken kidney cell system, and at the nonpermissive temperature of 40 C, production of infectious TS virus was about 100,000-fold less than at 35 C, in contrast to WT virus. Major structural polypeptides of each virus grown at 35 C were similar, except that the hemagglutinin glycopolypeptide (HA) of the TS virions was slightly more heterogenous than that of WT virions. Synthesis of viral polypeptides was examined by sodium dodecyl sulfate acrylamide gel electrophoresis of pulse-labeled infected cells. This revealed a defect in the synthesis of TS viral hemagglutinin that was most pronounced at the nonpermissive temperature. Other TS viral polypeptides appeared to be synthesized normally at 40 C. A defect in the TS virus hemagglutinin was also indicated by serological studies that demonstrated that TS virus hemagglutinin had lost antigenic sites present on the WT virus. Thus, it is concluded that the virus mutant examined contains lesions in the hemagglutinin gene, although the possibility of additional unrecognized lesions is not excluded.

Adaptation, Biological↗

Cold-sensitive mutants of bacteriophage phi x174. II. Comparison of two cold-sensitive mutants.

Cold-sensitive bacteriophage phiX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants, cs70 and cs82, were examined. Mutant cs70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant cs70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant cs82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The cs82 block could not be bypassed by temperature shift. Since complementation analysis of cs70 and cs82 was not feasible due to the unique properties of these mutants, those phiX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant cs70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant cs70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant cs82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although cs70 was subject to less of (low-level) plating restriction by endogenous F(+) factors.

Adsorption↗

Chronic glomerular nephritis in a man with an IgM cold autoantibody of anti-I specificity and a cold lymphocytotoxic antibody.

This communication describes a young man with chronic glomerular nephritis whose serum contained cold antibodies directed against both red cells and lymphocytes. A renal biopsy revealed unusual histological features including extensive deposits of IgM and complement. Although abnormal immune responses are not uncommon in chronic renal failure, it is suggested that there may be a causal relationship between the underlying renal lesion and the abnormal antibodies.

Adult↗

Dissociation of temperature-gradient and evaporative heat loss during cold gas hyperventilation in cold-induced asthma.

We examined temperature-gradient and evaporative energy losses during cold gas inhalation challenges in patients with exercise-induced asthma by using gases with similar water-carrying capacities but significantly different volume heat capacities. Seven subjects were asked to hyperventilate mixtures of 80% helium/20% oxygen (HeO2) or 80% sulfur hexafluoride/20% oxygen (SF6O2) for 5 min at a fixed target minute ventilation of 20 x FEV1 and an inspired gas temperature of 0 degrees C. Each subject equilibrated his or her lungs with the appropriate gas mixture prior to testing: PETCO2 and FIO2 were monitored and maintained at constant values (CO2 = 0.05; O2 = 0.20) by CO2 scrubbing and addition of compressed gas to the system. Gas composition, inspired and expired flow rates, and gas temperatures at the airway opening were recorded in real time using a computer-based data collection system that calculated respiratory heat loss on a per breath basis. Bronchoconstriction was quantitated using specific airway conductance measured before and serially after each challenge. The degree of bronchoconstriction correlated closely with evaporative respiratory heat loss (r = 0.658 p less than 0.05), but poorly with both temperature-gradient (r = 0.114, p greater than 0.20) and total (r = 0.268, p greater than 0.15) heat loss. These findings suggest that total respiratory heat loss is not the primary stimulus in exercise-induced asthma, and further suggest that total water loss, or focal heat/water loss, may be important in inducing bronchospasm in this subset of asthmatics.

Adult↗

Common cold and influenza symptom management: the use of pharmacokinetic considerations to predict the efficacy of a twice-daily treatment for colds and flu.

OBJECTIVE: The objective of the two pharmacokinetic studies reported here was to compare the relative bioavailability of an ibuprofen/pseudoephedrine modified-release capsule with each of the active ingredients given alone as standard formulations. STUDY DESIGN: Evaluation of two open, randomised, cross-over studies, one single dose and one multiple dose, in healthy male volunteers. METHODS: Healthy volunteers were randomised in a cross-over design to single or multiple doses of a combination of ibuprofen (600 mg) plus pseudoephedrine (90 mg) in a slow-release formulation and the individual active products alone as standard formulations; ibuprofen 400 mg, pseudoephedrine 60 mg. RESULTS: The single-dose study demonstrated that the bioavailabilities of ibuprofen and pseudoephedrine achieved with the slow-release formulation were not significantly different from those with standard tablets of each ingredient alone. In addition, mean plasma levels of ibuprofen predictive of clinical efficacy were achieved within 0.5-1 h and lasted for 10-12 h thereafter. The time required to reach clinically effective blood levels of pseudoephedrine was longer, starting at approximately 2 h. However, the plasma levels predicted that the clinical effect would then last for at least a further 12 h. Trough levels from the multiple-dose study showed that clinically relevant analgesic and decongestant plasma levels were maintained for 24 h during twice-daily dosing. The slow-release formulation was well tolerated with only mild adverse events. CONCLUSIONS: Blood levels would predict that the present slow-release formulation of ibuprofen plus pseudoephedrine should offer reliable day and night control of cold and flu and sinus symptoms and be associated with a favourable safety profile.

Adolescent↗