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Mouse genetics meets molecular biology at Cold Spring Harbor. Mouse Molecular Genetics sponsored by the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, August 29-September 2, 1990.

As stated earlier, the digestion of roughly 100 short talks delivered within a few days and containing very condensed information is, even for experts, quite demanding. Sometimes one felt in danger of becoming "psyched out" by all the experiments described; perhaps smaller group discussions dealing with experiments that have not worked, as well as the elimination of redundancies in the presentations, would have added to the flavor of the meeting. But what is the "take home message" from the 1990 CSH Mouse Meeting? With all the described breakthroughs, has mouse molecular genetics and development arrived at a turning point? I am inclined to answer this question with a "yes" on the basis of the following considerations: The elegant studies of development in Drosophila and Caenorhabditis elegans were made possible by the power of genetics with the use of developmental mutations. These studies taught us that the sequential activation of a hierarchy of regulatory genes dictates the temporal and spatial patterns of expression of proteins that define cell phenotypes and the body plan, and thereby control development. With the identification of important regulatory genes responsible for many classical as well as experimentally induced mouse mutations, in conjunction with traditional transgenic studies and the power of deleting and altering genes via ES cell chimeras, the study of mouse development has now gained an important new dimension. It is feasible that the consequences of subtle but precise genetic changes, such as the modification of regulatory elements or DNA-binding domains, can be studied in the whole organism by use of ES cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Antegrade cold blood cardioplegia is not demonstrably advantageous over cold crystalloid cardioplegia in surgery for congenital heart disease.

OBJECTIVE: The superiority of blood cardioplegia in pediatric cardiac surgery has not previously been challenged in a controlled clinical trial. The purpose of this study was to compare antegrade cold blood versus cold crystalloid cardioplegia in pediatric cardiac surgery. METHODS: One hundred thirty-eight pediatric patients (mean age 32 months; 95% CL 24.2 to 39.8 months; range 1 day to 15 years) were prospectively randomized to receive either cold blood (4:1 dilution, blood/Plegisol, potassium chloride 15 mEq/L; n = 62) or cold crystalloid (Plegisol; n = 76) cardioplegic solution during a variety of operations for congenital heart disease. Multiple doses of cold (4 degrees C) cardioplegic solution was administered antegradely in addition to topical cooling during ischemic arrest. Myocardial recovery and outcome measures were assessed by five clinical end points: (1) inotropic support, (2) echocardiographic assessment of ventricular function, (3) overall complication rate, (4) length of stay in the intensive care unit, and (5) 30-day survival. Multiple logistic regression and multivariate analysis of variance were used to investigate which of the following clinical determinants were contributory: (1) cardioplegia, (2) urgency of operation, (3) aortic crossclamp time, (4) age, and (5) cyanosis. Population data did not differ between the two cardioplegia groups (p > 0.05). RESULTS: The most important clinical determinant of studied end points was the aortic crossclamp time (p < 0.05). The type of cardioplegic solution (blood vs crystalloid) was less important (p > 0.05). The only statistically significant difference between blood and crystalloid cardioplegia for the measured clinical end points was the level of intraoperative inotropic support (p < 0.05), although this did not correlate with any significant differences in measured ventricular function. CONCLUSION: Our results suggest no clear clinical advantage of antegrade cold blood cardioplegia over crystalloid cardioplegia during hypothermic cardioplegic arrest in pediatric cardiac surgery. The aortic crossclamp time was the strongest predictor of measured outcomes.

Bicarbonates↗

Cold chain monitoring during cold transportation of human corneas for transplantation.

BACKGROUND: As recommended by international standards the cornea should be maintained in a specific temperature range (2 degrees -8 degrees C) to guarantee its viability. However, there is no standard packaging method to maintain these conditions during transport. Our packaging system is similar to those used by the main eye banks in Spain and elsewhere in Europe. The objective is to monitor the cold chain in the current packaging method to validate the maintenance of temperature within the adequate range for a minimum 24-hour period. METHODS: The effects of the following variables were studied: number and freezing temperature of the cold packs; air volume in the packaging system; position of the cornea in the packaging system; and the wall section of the container. Exterior temperature was maintained constant at 20 degrees to 24 degrees C. The cold chain was monitored using a device that measures temperature continuously and for which a histogram of temperature variation can be downloaded to a computer for further analysis. RESULTS: When the cold packs were frozen to -40 degrees C or the number of cold packs increased to four, the temperature decreased quickly to 0 degrees C and the transport period was not prolonged. The main objective was to improve isolation by reducing inner air volume, and maintaining the position of the cornea in the container. CONCLUSIONS: The currently used cold packaging systems (not frozen, 4 degrees C) do not maintain the temperature within the accepted range for the required distribution period. The improved system maintains the cornea at between 2 degrees C and 6 degrees C for a minimum of 24 hours.

Cornea↗

Levels of noradrenaline in plasma during thermogenesis induced by cold-exposure or by noradrenaline infusion in warm- and in cold-acclimated rats.

Oxygen consumption and concentration of noradrenaline (NA) in arterial plasma have been measured in warm-acclimated (WA) and in cold-acclimated (CA) rats during exposure to cold and in barbital-sedated WA or CA rats receiving intravenous infusions of NA at various doses at room temperature. In WA and in CA rats in the cold, NA in plasma increased rapidly from a resting level of 0.1 to 0.2 ng/ml at thermoneutrality to values 5 to 10 times greater at -19 degrees C. The increases were directly related to the degree of cold exposure and were maintained throughout exposure. In barbital-sedated WA or CA rats infused with NA the plasma levels of NA which stimulated calorigenesis were approximately 10 times greater than those resulting from overflow of NA into the blood during intense cold exposure and were in the range found by others to be effective in stimulating O2 consumption in isolated brown adipocytes.

Acclimatization↗

Effects of repeated short-term cold exposures on cold induced thermogenesis of women.

The effects of repeated exposures to resting cold air (10 degrees C) on the shivering and thermogenic responses of women to standard cold stress were investigated. Ten women, aged 18 to 34 years, were divided into two groups of five women each. One group, the acclimated (A) was exposed ten times within 2 weeks, the first and the last exposures being the pre- and post-tests, respectively. The second group, the control (C) was exposed twice within 18 days. Measurements of rectal and skin temperatures, oxygen uptake, time to onset of shivering (TOS), and perceived cold were performed during all exposures. Shivering responses were evaluated by electromyography and visually. A significant (P less than 0.05) increase was seen in TOS (from 26.2 min to 55.6 min), and a significant decrease was seen in thermoregulatory heat production (from 14.78 kcal/h to -2.64 kcal/h) in group A; these changes were evident after about five exposures. It is concluded that the women became cold acclimated as a result of the repeated short-term resting cold air exposures.

Acclimatization↗

Cold-receptor cells supply both cold- and warm-responsive projection neurons in the antennal lobe of the cockroach.

The temperature receptor cells on the cockroach antennae are all excited by rapid cooling. In the antennal lobe, however, cold- as well as warm-responsive neurons occur. They are excited either by rapid step-like cooling or rapid step-like warming. Responses to such temperature transients do not show, however, whether antennal lobe neurons convey information on slowly changing temperatures typical of temperature gradients used for orientation. In contrast slow temperature changes permit an analysis of the effects of both instantaneous temperature and its rate of change. We compared the effect of slow temperature oscillations on the responses of antennal cold-receptors cells and cold- and warm-responsive projection neurons. In all cases the discharge rates were modulated by the temperature oscillations. They displayed a double dependence on instantaneous temperature and its rate of change. Information about cooling and warming, first contained in the output of a single cold-receptor cell diverges to form the parallel pathways of cold- and warm-responsive projection neurons, thereby in particular improving the detection of fluctuations in temperature.

Animals↗

Laboratory properties of cold-adapted influenza B live vaccine strains developed in the US and USSR, and their B/Ann Arbor/1/86 cold-adapted reassortant vaccine candidates.

The adaptation of two influenza B strains (B/Leningrad/14/55 and B/Ann Arbor/1/66) to replication at 25 degrees C is described. Comparison of the two viruses indicates that both also exhibit temperature sensitive phenotypes, although that of the virus B/Leningrad/14/55 is less pronounced. When inoculated into ferrets both viruses replicate well in the trachea, but only the B/Leningrad/14/55 cold-adapted virus replicates in the lungs. This virus exhibited a moderate level of attenuation in the animals, in contrast to the B/Ann Arbor/1/66 cold-adapted virus, which was fully attenuated. Reassortant viruses deriving the surface antigens of the contemporary wild type virus B/Ann Arbor/1/86 and most or all of their other genes, from one or other cold-adapted parent, were virtually indistinguishable from their respective cold-adapted parents. The B/Leningrad/14/55 reassortant was slightly more attenuated than its cold-adapted parent in ferrets. These studies extend knowledge of the properties of viruses used to prepare experimental live influenza B human vaccines.

Animals↗

Cold shock injury and ecological costs of rapid cold hardening in the grain aphid Sitobion avenae (Hemiptera: Aphididae).

The ability of first instar nymphs and newly moulted pre-reproductive adults of the grain aphid S. avenae to rapidly cold harden was investigated. When nymphs reared at 20 degrees C were transferred directly to -8 degrees C for 3 h, there was 18% survival. This exposure was selected as the discriminating temperature. Maximum increases in survival were achieved by acclimating nymphs for 2 h at 0 degrees C and adults for 3 h at 0 degrees C, resulting in survival of 83% and 68%, respectively. Cooling nymphs from 10 to 0 degrees C at different rates (1, 0.1 and 0.05 degrees C min(-1)) also increased cold hardiness, with the slowest rate of 0.05 degrees C min(-1) conferring the highest survival following exposure to the discriminating temperature. Adult aphids also expressed a rapid cold hardening response but to a lesser extent, with survival increasing from 16% to 68% following 3 h at 0 degrees C. There were no 'ecological costs' associated with rapid cold hardening in terms of development, longevity or fecundity. The data support the hypothesis that rapid cold hardening can be induced during the cooling phase of natural diurnal temperature cycles, allowing insects to track daily changes in environmental temperatures.

Acclimatization↗

Cold-stable and cold-adapted microtubules.

Most mammalian microtubules disassemble at low temperature, but some are cold stable. This probably has little to do with a need for cold-stable microtubules, but reflects that certain populations of microtubules must be stabilized for specific functions. There are several routes by which to achieve cold stability. Factors that interact with microtubules, such as microtubule-associated proteins, STOPs (stable tubule only polypeptides), histones, and possibly capping factors, are involved. Specific tubulin isotypes and posttranslational modifications might also be of importance. More permanent stable microtubules can be achieved by bundling factors, associations to membranes, as well as by assembly of microtubule doublets and triplets. This is, however, not the explanation for cold adaptation of microtubules from poikilothermic animals, that is, animals that must have all their microtubules adapted to low temperatures. All evidence so far suggests that cold adaptation is intrinsic to the tubulins, but it is unknown whether it depends on different amino acid sequences or posttranslational modifications.

Adaptation, Physiological↗

Cold- and ouabain-resistance of renal Na,K-ATPase in cold-exposed and hibernating jerboas (Jaculus orientalis).

The temperature dependence and the ouabain sensitivity of Na,K-ATPase was examined in the nephron of normal, cold-exposed, and hibernating jerboas. The transport and hydrolytic activity of renal Na,K-ATPase displayed similar temperature dependence in rats and normal jerboas. Cold-resistance of Na,K-ATPase appeared in cold-exposed jerboas and further increased during hibernation. Three subpopulations of Na,K-ATPase displaying very high (Ki approximately 10(-13) M), high (Ki approximately 10(-9) M) and low sensitivity to ouabain (Ki approximately 10(-6) M) were detected in the thick ascending limb and collecting duct of jerboas. In thick ascending limbs, the subpopulation of very high sensitivity to ouabain disappeared in cold-exposed animals, which accounted for the previously reported decrease in Na,K-ATPase activity. In collecting ducts of cold-exposed animals, the subpopulation of very high sensitivity to ouabain also disappeared, but the resulting decrease in activity was overbalanced by the appearance of the subpopulation of high sensitivity.

Animals↗

Cold-shock response and cold-shock proteins.

Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupt temperature downshift. Cold-shock proteins are synthesized to overcome the deleterious effects of cold shock. CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, function and regulation at the level of transcription, translation and mRNA stability. Homologues of CspA are present in a number of bacteria. Widespread distribution, ancient origin, involvement in the protein translational machinery of the cell and the existence of multiple families in many organisms suggest that these proteins are indispensable for survival during cold-shock acclimation and that they are probably also important for growth under optimal conditions.

Adaptation, Biological↗

Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis.

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold-inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a DeltaHik33 mutant using the DNA microarray technique. In wild-type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold-repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold-regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.

Bacterial Proteins↗

A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

Abscisic Acid↗

Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes.

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold-recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold-recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.

Anaphase↗

Ineffectiveness of oral terfenadine in natural colds: evidence against histamine as a mediator of common cold symptoms.

The role of histamine in the pathogenesis of infectious rhinitis is unclear, as is the efficacy of antihistaminic drugs in the treatment of the common cold. This study evaluated the short-term efficacy of oral terfenadine (Seldane) in the treatment of the common cold. Over a 5-week period, the authors recruited 250 adults who had developed cold symptoms within 6 to 48 hours prior to enrollment. Volunteers had a primary complaint of runny or stuffy nose; at least one other respiratory symptom; no fever or exudative pharyngitis; and no history of atopy, sinusitis, or use of cold preparations within 1 week of enrollment. Out of the eligible subjects, 126 were randomly assigned terfenadine (60 mg), and 124 received placebo. Volunteers self-administered either terfenadine or placebo twice a day on Days 1, 2 and 3, and a final dose on the morning of Day 4. They also recorded the severity of their clinical symptoms (runny nose, sniffles, sneezing, postnasal drip, cough and sore throat) on symptom cards. Both groups reported similar severity scores throughout the treatment period. Average symptom burdens declined at almost identical rates for both groups. Terfenadine was well tolerated and had a low incidence of side effects. According to subject evaluation, terfenadine was no more effective than placebo. The mean +/- SD score of global efficacy was 2.2 +/- 1.1 in the terfenadine group and 2.1 +/- 1.3 in the placebo group (P = NS). Slightly fewer terfenadine recipients (41%) than placebo recipients (48%) said they would use the study medication again for treating cold symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Coping with the cold: the cold shock response in the Gram-positive soil bacterium Bacillus subtilis.

All organisms examined to date, respond to a sudden change in environmental temperature with a specific cascade of adaptation reactions that, in some cases, have been identified and monitored at the molecular level. According to the type of temperature change, this response has been termed heat shock response (HSR) or cold shock response (CSR). During the HSR, a specialized sigma factor has been shown to play a central regulatory role in controlling expression of genes predominantly required to cope with heat-induced alteration of protein conformation. In contrast, after cold shock, nucleic acid structure and proteins interacting with the biological information molecules DNA and RNA appear to play a major cellular role. Currently, no cold-specific sigma factor has been identified. Therefore, unlike the HSR, the CSR appears to be organized as a complex stimulon rather than resembling a regulon. This review has been designed to draw a refined picture of our current understanding of the CSR in Bacillus subtilis. Important processes such as temperature sensing, membrane adaptation, modification of the translation apparatus, as well as nucleoid reorganization and some metabolic aspects, are discussed in brief. Special emphasis is placed on recent findings concerning the nucleic acid binding cold shock proteins, which play a fundamental role, not only during cold shock adaptation but also under optimal growth conditions.

Adaptation, Physiological↗

Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid.

Commelina communis stomata closed within 1 h of transferring intact plants from 27 degrees C to 7 degrees C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl(2) by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm(3)) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.

Abscisic Acid↗