Tolerance of cold-acclimated and unacclimated rats to hypoxia at 1.7°C

Cold-acclimated and unacclimated rats were exposed for 4 hours to 6.6 or 4.9% O₂ in N₂ at 1.7°C. Unacclimated rats tolerated both degrees of hypoxia better than cold-acclimated rats. Cold-acclimated rats showed relatively greater increases in serum concentrations of GOT and GPT, aldolase and LDH immediately after exposure to 4.9% O₂ and a greater concentration of GPT at 6.6% O₂ than unacclimated rats. In both groups serum urea nitrogen and plasma corticosterone were elevated. Serum glucose was increased above air flow controls (20.9% O₂) in both groups by 50% at 6.6% O₂ and 100% at 4.9% O₂. Blood lactic acid levels were elevated by about 130%. The cold-acclimated rats had a higher incidence of renal tubular dilatation, fatty changes in striated muscles, and severe hepatic glycogen depletion. All rats in both groups showed myocardial inflammatory foci one day after exposure which persisted for 4 days. Cold-acclimated rats maintained higher body temperatures. The reduced tolerance of the cold-acclimated rats is attributed to an increased metabolism which increased tissue hypoxia.

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Zusammenfassung Kälteakklimatisierte und nicht akklimatisierte Ratten wurden 4 Stunden in 6.6 und 4.9% O₂ im N₂ bei 1, 7°C exponiert. Kälteakklimatisierte Tiere waren gegen beide Hypoxiegrade weniger resistent als nicht akklimatisierte. Sie zeigten einen relativ höheren Anstieg der Serumaktivität von GOT und GPT, Aldolase und LDH unmittelbar nach Uebergang in 4, 9% O₂ und eine höhere Aktivität von GPT bei 6, 6% O₂ als unakklimatisierte Ratten. In beiden Gruppen waren der Serum Harnstoff-N und Plasmakortikosteron erhöht. Serumglukose war über normoxische Kontrollen in beiden Gruppen bei 6, 6% O₂ auf 50% und bei 4, 9% O₂ auf ungefähr 100% erhöht. Die kälteakklimatisierten Ratten wiesen in höherem Masse Dilatationen der Nierentubuli auf, fettige Veränderungen in den gestreiften Muskeln und starke Depletierung des Leberglukogens. Entzündungsherde im Myokard traten nach einem Tag Exponierung auf, die 4 Tage lang bestanden. Kälteakklimatisierte Tiere behielten höhere Körpertemperaturen. Die verminderte Toleranz der kälteakklimatisierten Tiere ist Folge des erhöhten Stoffwechsels, der die Gewebshypoxie verstärkt.

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Resume On a exposé des rats acclimatés et non acclimatés durant quatre heures à des concentrations de 6, 6 et 4, 9% de O₂ dans de l'azote à 1, 7°C. Des bêtes acclimatées au froid se sont avérées moins résistantes aux deux taux d'hypoxie que leurs congénères non acclimatés. Ils présentaient une hausse relativement plus accentuée de l'activité du sérum (GOT, GPT, aldolase et LDH) immédiatement après leur séjour à 4, 9% de O₂ et une activité accrue de GPT à 6, 6% de O₂ par rapport à leurs congénères non acclimatés. De même, l'urée azotée du sérum et la corticostérone du plasma étaient plus élevées chez les deux groupes. Le glucose du sérum, déterminé par rapport à des bêtes de contrôle (20, 9% de O₂), était plus élevé dans les deux groupes de 50% à 6, 6% de O₂ et de 100% pour 4, 9% de O₂. Le taux d'acide lactique du sang a atteint alors 130%. Les rats acclimatés au froid présentaient une plus grande dilatation des tubes rénaux, des modifications lipoidiques dans les muscles striés et un épuisement rapide du glucogène du foie. Après une exposition d'un jour, on a constaté dans le myocarde des foyers d'inflammation qui se sont maintenus durant 4 jours. Les animaux acclimatés au froid présentaient des températures du corps plus élevées. La tolérance réduite des animaux acclimatés au froid est la conséquence d'un métabolisme accentué qui renforce l'hypoxie des tissus.

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A preliminary report presented to the Sixth International Biometeorological Congress, Noordwijk, The Netherlands, 3–9 September 1972.     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

Responses of cattle to the combined exposure to diurnal temperature rhythm (−5 to 25°C) and to simulated high-altitude (4,000 m)

Eight 1/2-year old calves were exposed in a climatized altitude chamber to the following four conditions: 400 and 4,000 m at constant T_a (17°C), 400 and 4,000 m at alternating T_a (–5° to 25°C). Each exposure lasted for 24 h and for the rhythmic conditions included a cold night and warm midday hours, supplemented by infrared heaters. During exposure, hourly measurements were made of heart rate, respiratory rate, rectal and three skin temperatures. Every 3-h blood samples were collected for the determination of 10 blood variables. The following main results were obtained: (a) Altitude alone caused increases in respiratory rate, heart rate, erythrocyte number, haemoglobin, specific gravity of blood and plasma, LDH and all four body temperatures. (b) In the rhythmic exposures, high correlation coefficients were found between ambient temperature on the one hand and skin temperatures (0.88 to 0.94), rectal temperature (–0.43) and respiratory rate (0.49) on the other hand. A change in ambient temperature by 1°C lead, on average, to a change in ear temperature by 1.2°C. (c) in response to falling ambient temperature during the night, rectal temperature and heart rate increased. This was interpreted as indicating a compensatory elevation in meta bolic heat production. At the same time, there was haemoconcentration as shown by elevations in erythrocyte number, haematocrit and haemoglobin. This haemoconcentration might have reflected splenic discharge, possibly supplemented by some loss of water from the plasma. (d) The warm environmental conditions around midday produced mild heat responses in terms of elevated values for respiratory rate, heart rate and body temperatures. (e) It is concluded that the rhythmic temperature with alternating stress of cold and mild heat, especially in combination with high altitude, was a strain on the animals and that they were forced to expend extra energy for combatting altitude- and temperature stress, energy which no longer would be available for productive processes.Presented at the Eight International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Influence of pH and ionic strength on the lysis ofMicrococcus luteus cells by hen lysozyme at low (20°C) and high (physiological, 40°C) temperature

From isoactivity curves (showing activity as a function of pH and ionic strength) it was found that in the pH domain 6.7–8.6 frequently used in experiments involving hen lysozyme, the pH optimum of lysis ofMicrococcus luteus ceils at low ionic strength (0.02–0.05) by the high-temperature form (40°C physiological temperature) was one to two pH units lower than that by the low-temperature form (20°C).116th communication on lysozymes; 115th communication: Harada Y, Lifchitz A, Berthou J, & Jollès P (1981) Acta Cryst. in press.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.     

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Stabilization of pepsin on duolite for the continuous hydrolysis of bovine haemoglobin at pH2 and 40°C

Summary Several methods of pepsin immobilization have been applied in order to achieve the continuous hydrolysis of a 2.5% haemoglobin solution at pH 2 and 40°C. Methods using glutaraldehyde were unsuccesful because of the unstability of the derived enzyme at low pH. Pepsin covalently bounded to a Duolite amine resin by a carbodiimide showed a half life of 15 days during the hydrolysis of haemoglobin in a column reactor. No enzyme activity was detected in the hydrolysates. No accumulation of haem in the column was noticed which could have limited long term studies by plugging the system. Modulation of the degree of hydrolysis was also performed by changing the feeding flow rate of the reactor.     

The role of growth rate, redox-state of the plastoquinone pool and the trans-thylakoid ΔpH in photoacclimation of Chlorella vulgaris to growth irradiance and temperature

The long-term photoacclimation of Chlorella vulgaris Beijer (UTEX 265) to growth irradiance and growth temperature under ambient CO₂ conditions was examined. While cultures grew at a faster rate at 27 than at 5 °C, growth rates appeared to be independent of irradiance. Decreases in light-harvesting polypeptide accumulation, increases in xanthophyll pool size and changes in the epoxidation state of the xanthophyll cycle pigments were correlated linearly with increases in the relative reduction state of Q_A, the primary quinone receptor of photosystem II, when estimated as 1−q_P under steady-state growth conditions. However, we show that there is also a specific temperature-dependent component, in addition to the redox-state of the Q_A, involved in regulating the content and composition of light-harvesting complex II of C. vulgaris. In contrast, modulation of the epoxidation state of the xanthophyll pool in response to increased 1−q_P in cells grown at 5 °C was indistinguishable from that of cells grown at 27 °C, indicating that light and temperature interact in a similar way to regulate xanthophyll cycle activity in C. vulgaris. Because C. vulgaris exhibited a low-light phenotype in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but a high-light phenotype upon addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, we conclude that the plastoquinone pool acts as a sensor regulating the accumulation of light-harvesting polypeptides in C. vulgaris. However, concomitant measurements of non-photochemical fluorescence quenching (q_N) and the epoxidation state of the xanthophyll pool appear to indicate that, in addition to the redox-state of the plastoquinone pool, the trans-thylakoid ΔpH may also contribute to sensing changes in irradiance and temperature that would lead to over-excitation of the photosynthetic apparatus. We suggest that sink capacity as reflected in photosynthate utilization and cell growth ultimately regulate photoacclimation in C. vulgaris. Received: 17 April 2000 / Accepted: 23 May 2000     

Diversity in the ability of Agaricus bisporus wild isolates to fruit at high temperature (25°C)

The button mushroom Agaricus bisporus commercially cultivated requires 16-19 °C during the fruiting period. Wild strains are also present in natural habitat, and in light of their wide range of geographic distribution reported, from boreal region to tropical region, questions on the development adaptation to temperature arose. Isolates from various geographic areas were screened for their ability to fruit at higher temperature (FHT ability) than commercial cultivars. The FHT trait discriminated at the varietal rank. Agaricus bisporus var. eurotetrasporus was unable to develop any sporophores whilst A. bisporus var. burnettii adapted perfectly to 25 °C for fruiting, suggesting that the FHT ability is a fixed trait in these varieties. In contrast, FHT ability of A. bisporus var. bisporus appeared variable and correlated neither with climate/microclimate nor with habitat. However, FHT ability taken as a whole appeared higher in North American populations than in European ones. Some A. bisporus var. bisporus isolates revealed a good potential for cultivation at 25 °C.     

Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life?

Bacterial microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review focuses on the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance of extremely halophilic eubacteria; the focus is on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50°C. If all three conditions are required for optimal growth, these microorganisms are termed "poly-extremophiles". However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. Therefore the question is: do the combined extreme growth conditions of the recently isolated poly-extremophiles, i.e., anaerobic halophilic alkalithermophiles, approach a physico-chemical boundary for life? These poly-extremophiles are of interest, as their adaptive mechanisms give insight into organisms' abilities to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms.     

Experimental forest soil warming: response of autotrophic and heterotrophic soil respiration to a short-term 10°C temperature rise

We warmed the top soil of a mature coniferous forest stand by means of heating cables on control and trenched plots within 24 h by 10°C at 1 cm soil depth (9°C at 5 cm depth) and measured the effect on the autotrophic (R_A) and heterotrophic (R_H) component of total soil CO₂ efflux (R_S). The short time frame of warming enabled us to exclude confounding fluctuations in soil moisture and carbon (C) flow from the canopy. The results of the field study were backed up by a lab soil incubation experiment. During the first 12 h of warming, R_A strongly responded to soil warming; The Q ₁₀ values were 5.61 and 6.29 for 1 and 5 cm soil depth temperature. The Q ₁₀ values for R_A were almost twice as high as the Q ₁₀ values of R_H (3.04 and 3.53). Q ₁₀ values above 5 are above reasonable plant physiological values for root respiration. We see interactions of roots, mycorrhizae and heterotrophic microbes, combined with fast substrate supply to the rhizosphere as an explanation for the high short-term temperature response of R_A. When calculated over the whole duration (24 h) of the field soil-warming experiment, temperature sensitivities of R_A and R_H were similar (no significant difference at P < 0.05); Q ₁₀ values were 3.16 and 3.96 for R_A and 2.94 and 3.35 for R_H calculated with soil temperatures at 1 and 5 cm soil depth, respectively. Laboratory incubation showed that different soil moisture contents of trenched and control plots affected rates of R_H, but did not affect the temperature sensitivity of R_H. We conclude that a single parameter is sufficient to describe the temperature sensitivity of R_S in soil C models which operate on larger temporal and spatial scales. The strong short-term response of R_A may be of relevance in soils suspected to experience increasingly strong diurnal temperature variations.     

Effect of the holding time at 15 °C prior to cryopreservation,the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8 h), thawing rate (37 °C for 20 s or 70 °C for 8 s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24 h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8 s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20 s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4 h post-thawing.     

A comparison of the drying of chymotrypsin bound to bead cellulose by lyophilisation and in air at 25 to 70°C

Summary The effect was studied of the method of drying chymotrypsin attached to bead cellulose on its chemical and physical characteristics. These characteristics are not deteriorated when replacing the commonly used lyophilisation by fluid drying in the air stream. The preparations saved essentially the same proteolytic activity as well as stability even when increasing the drying air temperature to 70°C.     

Thermophilic bacteria that tolerate a wide temperature and pH range colonize the Soldhar (95 °C) and Ringigad (80 °C) hot springs of Uttarakhand,India

Twenty-eight bacterial cultures, isolated from hot springs in Uttarakhand, were characterized with particular reference to their wide temperature and pH tolerance and production of enzymes in the thermophilic range. All the bacterial isolates were observed as Gram-positive or variable rods in varied arrangement. Bacterial isolates exhibited tolerance to a wide temperature range (20–80 °C), from mesophilic (+11° to +45 °C) to thermophilic (+46 ° to +75 °C); few almost reached the hyperthermophilic range (+76 °C). The isolates also tolerated a wide pH range (4–14) and moderate salt concentration. The optimum growth of the bacterial isolates was observed at 55 °C and 7 pH. Out of 28 isolates, 25 produced lipase, 25 amylase, 24 cellulase, 22 protease and 13 xylanase at 55 and 65 °C. Tolerance to a wide temperature and pH range and the production of enzymes in a thermophilic temperature range can be considered as indicators of ecological competence of these bacterial isolates for colonizing the high temperature environment. On the basis of 16S rDNA similarity, 20 bacterial isolates belonged to Bacillus licheniformis, five to Paenibacillus ehimensis and one each to Bacillus sonorensis, B. tequilensis, and Staphylococcus epidermidis. Besides variation in phenotypic characters, strains of B. licheniformis and P. ehimensis showed varying 16S rDNA similarity between 97–99 % and 95–99 %, respectively. Consideration of temperature preferences in classifying microorganisms on the basis of their minimum, maximum, and optimum growth requirements is also discussed. The study has ecological relevance in the context of colonization of high temperature environments by thermophilic bacteria.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

Sequences downstream of the start codon and their relations to G + C content and optimal growth temperature in prokaryotic genomes

The mechanism of translation initiation is responsible for shaping the mRNA sequences downstream of the start codon. However, this region has not been systematically analyzed in prokaryotes. We used sequence logos and statistic methods to analyze the patterns of overrepresented sequences in this region for 125 species of bacteria and 23 species of archaea. The specific positions are compared to the first 33 amino acids in the proteins. At the 2nd amino acid position, Lys, Ser or Thr is highly overrepresented for 68% to 84% of the genomes examined and Ala is highly overrepresented for 57% of the genomes. Overrepresentation of Lys2 is negatively correlated with the G + C content and overrepresentation of Ser2 or Thr2 is positively correlated with the G + C content of genomes. Ile at the 4th to the 8th positions were found to be overrepresented for 91% of the genomes analyzed and this seemed to be conserved for both bacteria and archaea. Organisms growing at high temperatures have relatively low extent of nucleotides bias at 5′ termini of open reading frames (ORFs). The extent of overrepresenting A and underrepresenting G at ORF 5′ termini is reduced in thermophiles and hyperthermophiles for both archaea and bacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.