Subthreshold changes in excitable membranes of Cucurbits pepo L. stem cells during cooling-induced action-potential generation

The nature of subthreshold changes in excitable plasma membranes has been investigated in stem parenchyma cells of Cucurbita pepo L. during action-potential generation induced by gradual cooling (from 23 to 10 ° C). The character of the subthreshold depolarization of excitable cells is shown to be mainly defined by a decrease in the activity of the plasma-membrane electrogenie pump (H⁺-ATPase). In its turn, the pump activity is controlled by thermal changes in the structure of the membrane lipid matrix. Based on the results obtained, a sequence of subthreshold changes has been suggested in which thermally induced structural rearrangements of membrane lipids play the role of trigger.Abbreviations AP action potential - DCCD N,N-dicyclohexil-carbodiimide - E_m membrane potential - I_e/I_m ratio of pyrene excimer/monomer fluorescence intensities     

Chlortetracycline analysis of boar spermatozoa after incubation,flow cytometric sorting,cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 μg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38°C, 4 hr), flow sorting, cooling (to 15 or 5°C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5°C (30.4, 48.5, 21.1%) than in those cooled to 15°C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively). Mol. Reprod. Dev. 46:408–418, 1997. © 1997 Wiley-Liss, Inc.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diet and cooling interactions on physiological responses of grazing dairy cows, milk production and composition

The objective of this trial was to evaluate the effects of diet and cooling in the holding pen before milking on rectal temperature, respiration rate and milk production and composition. Fifty-eight lactating Holstein cows were used in a factorial split-plot design, at Rafaela Experimental Station from 12 January to 3 March 2003. The treatments were combinations of two diets: control (CD) and balanced (BD) with two levels of cooling before milking: none (NSF) and a sprinkler and fans (SF). Forage:concentrate ratios for CD and BD were 81:19 and 68:32, respectively. Cows were milked twice daily. Milk production was recorded daily, and milk composition (fat, protein, lactose and urea) was analysed twice a week. The physiological data were recorded once a week, before the cattle entered the holding pen and after milking, in the afternoon. Average maximum weekly temperature humidity index was 75.4 and ranged from 61.4 to 83. There were highly significant effects of cooling on physiological responses. Milk production was affected by diet and cooling, with no interaction; the highest and lowest production of milk was 22.42 and 20.07 l/cow per day, for BD+SF and CD+NSF, respectively. Protein was affected by diet, and was higher for BD (3.17 vs. 3.08%). There were interaction effects on milk fat at the 8% level, the highest concentration being 3.65% for BD+NFS. It was concluded that under grazing conditions, cooling by sprinkler and fans before milking improves the comfort of dairy cows, and that the effects on milk production and composition are enhanced when diets are specially formulated for heat-stress periods.     

Arousal and re-feeding rapidly restores digestive tract morphology following aestivation in green-striped burrowing frogs

During aestivation, the gut of the green-striped burrowing frog, Cyclorana alboguttata undergoes significant morphological down-regulation. Despite the potential impact such changes might have on the re-feeding efficiency of these animals following aestivation, they appear to be as efficient at digesting their first meals as active, non-aestivating animals. Such efficiency might come about by the rapid restoration of intestinal morphology with both arousal from aestivation and the initial stages of re-feeding. Consequently, this study sought to determine what morphological changes to the intestine accompany arousal and re-feeding following 3 months of aestivation. Arousal from aestivation alone had a marked impact on many morphological parameters, including small and large intestine masses, small intestinal length, LF heights, enterocyte cross-sectional area and microvilli height and density. In addition, the onset of re-feeding was correlated with an immediate reversal of many morphological parameters affected by 3 months of aestivation. Those parameters that had not returned to control levels within 36 h of feeding generally had returned to control values by the completion of digestion (i.e. defecation of the meal). Re-feeding was also associated with several changes in enterocyte morphology including the incorporation in intracytoplasmic lipid droplets and the return of enterocyte nuclear material to the 'active' euchromatin state. In conclusion, morphological changes to the gut of aestivating frogs which occur during aestivation are transitory and rapidly reversible with both arousal from aestivation and re-feeding. The proximate causes behind these transitions and their functional significance are discussed.     

Respiratory cooling in rattlesnakes

We used infrared thermography to study respiratory cooling in the rattlesnakes (Viperidae: Crotalinae) and to partition the effects of air temperature, humidity, and activity levels on head-body temperature differences. We observed a single, cooled region centered around the mouth and nasal capsule that extended across the pit membrane at air temperatures above 20 degrees C. Both head and body temperatures of rattlesnakes increased linearly with air temperature. Head-body temperature differentials also increased with air temperature, but declined significantly at higher relative humidities. Rattling rattlesnakes exhibited significantly greater head-body temperature differentials than did resting rattlesnakes. We suggest that respiratory cooling may provide a thermal buffer for the thermoreceptive pit organs at high air temperatures, but caution that this adaptive hypothesis must be tested with direct neural or behavioral assays.     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Cloth color preference under the influence of face cooling

1. We studied the effect of face cooling accompanied by a fall of tympanic temperature on color preference in 8 women at an ambient temperature of 28°C.2. The face only was cooled by blowing 15°C air at 6 m/s on the face only. Controls received either no wind at all or a wind (24°C, 6 m/s) on the body and appendages only.3. Most subjects preferred warmer colors after facial cooling than after no facial cooling.4. The facial cooling and decrease in tympanic temperature may indicate an increase in load error between core temperature and set-point.     

Convective water exchanges during differential cooling and heating: implications for dissolved constituent transport

Convective water exchange patterns, determined from water temperature variations, were examined in the Minky Creek embayment of Guntersville Reservoir, Alabama (USA), during the month of September, 1990. During periods of differential cooling, cooler water originating from shallow stations moved as an underflow current toward the center of the embayment, while warmer water moved as an overflow current toward the shore. During periods of differential heating, convective exchange was much more shallow, occurring in the upper 3 m of the water column. As warmer water from shallow regions moved out as an overrflow current, this water was replaced by a return flow of cooler water originating from the pelagic epilimnion. Wind speed appeared to influence convective exchange patterns during differential heating phases by affecting the depth of the surface mixed layer. The potential for convective exchanges during periods of differential cooling and heating occurred on 54% and 74% of the days, respectively, in September. This high potential for horizontal water exchanges in Guntersville Reservoir has strong implications for the lateral transport of dissolved constituents.     

5’-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo

Background

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5’-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport.

Results

Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice.

Conclusions

Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.