Leptin,ghrelin, and energy metabolism of the spawning burbot (Lota lota,L.)

The aim of this study was to investigate the energy metabolism of the burbot (Lota lota, n=38) before, during, and after spawning, which represents the greatest annual metabolic demand for the species. A decrease in body mass, relative weight of the livers, and glycogen concentration of the livers was observed toward the end of spawning. The prespawning period was characterized by high rates of liver glycogenolysis and lipid mobilization. Also, plasma triiodothyronine and sex steroid levels were high before reproduction. During spawning, liver lipolysis was reduced and muscle glycogenolysis stimulated. The levels of triiodothyronine and sex steroids decreased. After reproduction, liver glycogenolysis was suppressed and the rate of gluconeogenesis increased. Thyroid hormone levels were elevated after spawning. Leptin protein and a ghrelin-immunoreactive peptide were detected in burbot plasma. Their concentrations were relatively low before and during reproduction but increased after spawning. The functions of leptin and the ghrelin-immunoreactive peptide in the physiology of the burbot are not consistent with the models of their function in mammals.     

Liver and plasma lipids of spawning burbot

Levels of liver and plasma lipids of the burbot Lota lota were highest before reproduction and decreased during and after spawning. Triacylglycerols accounted for 95% of the liver lipids. High concentrations of leptin-immunoreactive peptide were present in the burbot liver, suggesting that it may be secreted by this organ.     

Preservation of rat bone marrow with dimethylsulphoxide at -150 degrees C.

The authors tested preserving properties of three concentrations of dimethylsulphoxide (15%, 10% and 7.5%) in preservation of rat bone marrow cells at -150 degrees C. Cells of rat bone marrow were frozen at 1 degree C/min to -20 degrees C, 5 degrees C/min to -80 degrees C and then placed directly at -150 degrees C and held at such temperature for 6 months. Vitality of cells was checked monthly for a period of 6 months by means of several vitality tests with dyes (eosin and trypane blue), autoradiography and erythrophagocytosis. It was found that cells capable of cleavage could be equally preserved at such low temperature with all the three DMSO concentrations while mature cells (granulocytes, reticular cells) revealed considerably higher erythrophagocytic activity when preserved at 15% DMSO and lower activity at 10% and 7.5% DMSO.     

Breeding of yeast strains able to grow at 42 degrees C

Saccharomyces cerevisiae strain 2-39/10A is able to ferment alcohol at 42 degrees C. The ability of various yeast strains, including 2-39/10A, to grow at high temperatures was compared. The strain 2-39/10A was able to grow at 42 degrees C and the high temperature growth was found to be governed by more than one gene. The yeast strains that can grow at 42 degrees C were bred by crossing the haploid strains, which are inherently unable to grow at high temperatures.     

Ultrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30 degrees C and 37 degrees C: a time course study by TEM, SEM and RIA

Isolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37 degrees C) and 30 degrees C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30 degrees C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37 degrees C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P less than 0.01) in livers perfused at 30 degrees C (50.8 +/- 7.7% vs 38 +/- 7.7%, 37 degrees C), but the net T3 output (3.16 +/- 1.04 micrograms) and the conversion of T4 to T3 (4 +/- 0.62%) were significantly higher (P less than 0.001) in livers perfused at 37 degrees C in comparison to livers perfused at 30 degrees C (1.61 +/- 0.84 micrograms and 1.68 +/- 0.76%, respectively). In conclusion, at 30 degrees C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Structure of membrane lipids and physico-biochemical properties of the plasma membrane from Thermoplasma acidophilum, adapted to growth at 37 degrees C.

Thermoplasma acidophilum, a mycoplasma-like organism, grows optimally at 56 degrees C and pH2. The low temperature extreme of growth is 37 degrees C. The plasma membrane of cells grown at 37 degrees C was isolated and characterized physicobiochemically. Membrane lipids which comprise 25% of the membrane dry weight consist mainly of two repetitively methyl-branched C40 side chains that were ether-linked to two glycerol molecules. The lipid structures were elucidated by combined gas chromatography-mass spectroscopy, direct probe mass spectroscopy and 13C NMR. 37 degrees C-grown cells contained lipids with 42% more pentane cyclization than the 56 degrees C-grown cells. In 37 degrees C-grown cells, phospholipid and serine content decreased by about 10% each, carbohydrate content increased by 5%. EPR studies demonstrated an increase in membrane lipid fluidity of 37 degrees C-grown cells with an upper transition temperature at 35 degrees C which was shifted down by 10 degrees C compared with cells grown at 56 degrees C. Membrane-bound ATPase activities also indicated similar changes upon adaptation. There is a close correlation between membrane fluidity and physiological functioning of this membrane-bound enzyme.     

Studies of alpha- and betaL-crystallin complex formation in solution at 60 degrees C

Studies of molecular mechanisms of chaperone-like activity of alpha-crystallin became an active field of research over last years. However, fine interactions between alpha-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between alpha- and betaL-crystallins was studied with thermal denaturation of betaL-crystallin at 60 degrees C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography and electrophoresis. A mixed solution of alpha- and betaL-crystallins in concentrations about 10 mg/ml incubated at 60 degrees C was found to contain their soluble complexes with mean radius of gyration approximately 14 nm, mean molecular weight approximately 4000 kDA and maximal size approximately 40 nm. In pure betaL-crystallin solution, complexes were not observed at 60 degrees C. In SAXS studies, transitions in the alpha-crystallin quaternary structure at 60 degrees C were shown to occur and result in a double increase of the molecular weight. It suggests that during the temperature-induced denaturation of betaL-crystallin it binds with modified alpha-crystallin or, alternatively, alpha-betaL-crystallin complexation and alpha-crystallin modifications are concurrent. Estimates of the alpha-betaL-crystallin dimensions and relative contents of alpha- and betaL-crystallins in the complex suggest that several alpha-crystallin molecules are involved in complex formation.     

Influence of caffeine on metabolic responses of men at rest in 28 and 5 degrees C

Cold stress and caffeine ingestion are each reported to increase plasma catecholamines, free fatty acid (FFA) concentrations, and energy metabolism. This study examined the possible interaction of these two metabolic challenges in four double-blind counterbalanced trials. Young adult men (n = 6) ingested caffeine (5 mg/kg) or placebo (dextrose, 5 mg/kg) and rested for 2 h in 28 or 5 degrees C air. Cold stress alone elevated (P less than 0.05) plasma norepinephrine, metabolism (O2 consumption, VO2), and respiratory exchange ratio (RER). Caffeine alone increased (P less than 0.05) plasma epinephrine and FFA but not RER. When the two challenges were combined (caffeine plus 5 degrees C for 2 h) norepinephrine and epinephrine were increased (P less than 0.05) as was FFA. However, VO2, RER, and skin and rectal temperatures were not different from the responses observed at 5 degrees C after placebo ingestion. The data suggest that caffeine selectively increases plasma epinephrine, whereas cold air increases norepinephrine. During the cold exposure, increasing epinephrine and FFA above normal levels did not appear to influence the metabolic or thermal responses to the cold stress. In fact the increase in RER suggested a greater carbohydrate oxidation.     

Effects of vitamin E on fowl semen storage at 4 degrees C

Vitamin E was assayed for either in chicken spermatozoa or seminal plasma. Effects of vitamin E on the motility and fertilizing ability of chicken semen stored for 24 hours at 4 degrees C were also studied. A mean of 0.25 mug vitamin E 10 (9) cells was found in spermatozoa and 0.074 mug in seminal plasma. When the medium for in vitro storage of semen was supplemented with vitamin E the motility of spermatozoa was not affected. However, vitamin E improved the fertilizing ability of semen stored for 24 hours at 4 degrees C, especially at the dose of 8 mug/ml of semen diluent.     

Freeze-fracturing in ultrahigh vacuum at -196 degrees C

Conventional freeze-etching is carried out in a vacuum of approximately 10(-6) torr and at a specimen temperature of -100 degrees C. The relatively poor topographic resolution of most freeze-etch replicas, and the lack of complementarity of morphological details in double replicas have been thought to be caused by structural distortions during fracturing, and radiation damage during replication. Both phenomena can be reduced by lowering the specimen temperature. To prevent condensation of residual gases (especially H2O) on the fracture faces at lower specimen temperature, an improved vacuum is required. Therefore, an ultrahigh vacuum freeze-fracture apparatus has been developed which allows fracturing and Pt/C-shadowing of specimens at -196 degrees C while maintaining a vacuum of 10(-9) torr. It consists of a modified Balzers BA 350 ultrahigh vacuum (UHV) unit, equipped with an airlock which enables the input of nonhoar-frosted specimens directly into the evacuated bell jar. A comparison of the paracrystalline plasmalemma structure in yeast cells portrayed by the conventional technique and by UHV-freeze-fracturing at -196 degrees C shows the improved topographic resolution which has been achieved with the new technique. The improvement is explained by less structural distortions during fracturing at lower temperatures. The particles of the paracrystalline regions on the P face are more regularly arranged and exhibit a craterlike substructure which corresponds with a ringlike depression in the E face. The optical diffraction patterns of these paracrystalline regions demonstrate the improvement of the structural record by showing well-defined third- and fourth-order spots.     

Biliary excretion of [14C]taurocholate by rainbow trout (Salmo gairdneri) is stimulated at warmer acclimation temperature

Sexually immature Shasta strain rainbow trout (Salmo gairdneri) of either sex were acclimated at 10, 14 or 18 degrees C for at least 4 weeks and plasma pharmacokinetics and biliary excretion of i.p. injected [14C]taurocholate (TC) examined in spinally transected or free-swimming fish, respectively. Plasma elimination half-lives but not absorption rate constants for [14C]TC (10 mumol/kg) were about two-fold reduced in 18 as compared to 10 or 14 degrees C acclimated fish. Distribution of [14C]TC to tissues other than plasma, liver, bile and small intestine was not different in 10, 14 or 18 degrees C acclimated free-swimming fish at 1 or 4 hr post-injection. Biliary excretion of [14C]TC (7.5-10 mumol/kg) at 1 hr post-injection was significantly higher in 14 and 18 as compared to 10 degrees C acclimated fish.     

Properties of single cardiac Na channels at 35 degrees C

Single Na channel currents were recorded in cell-attached patches of mouse ventricular myocytes with an improved patch clamp technique. Using patch pipettes with a pore diameter in the range of 200 nm, seals with a resistance of up to 4 T omega were obtained. Under those conditions, total noise could be reduced to levels as low as 0.590 pA rms at 20 kHz band width. At this band width, properties of single- channel Na currents were studied at 35 degrees C. Six out of a total of 23 patches with teraohm seals contained channel activity and five of these patches contained one and only one active channel. Amplitude histograms excluding transition points showed heterogenous distributions of levels. In one patch, part of the openings was approximately Gaussian distributed at different potentials yielding a slope conductance of 27 pS. The respective peak open probability at -10 mV was 0.26. The mean open time was determined at voltages between -60 and -10 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise to be approximately 40 microseconds at -60 mV and 50-74 microseconds between -50 and -10 mV. It is concluded that single cardiac Na channels open at 35 degrees C frequently with multiple levels and with open times in the range of several tens of microseconds.     

Short-term storage of rabbit embryos at 4 degrees C

A simple method for storing preimplantation mammalian embryos was tested under conditions which could be easily maintained inside an ordinary refrigerator set at 4 degrees C. No significant loss of viability occurred when rabbit embryos were stored at 4 degrees C for 7 days and either cultured in vitro at 37 degrees C or transferred to recipient does. Significant losses occurred when embryos were stored for 10 days or longer before culture at 37 degrees C (P < .01). Stored embryos transferred to recipients had a significantly longer average gestation period than embryos transferred without cold storage (P < .05).     

Cross-bridge kinetics modeled from myoplasmic [Ca2+] and LV pressure at 17 degrees C and after 37 degrees C and 17 degrees C ischemia

We modeled changes in contractile element kinetics derived from the cyclic relationship between myoplasmic [Ca(2+)], measured by indo 1 fluorescence, and left ventricular pressure (LVP). We estimated model rate constants of the Ca(2+) affinity for troponin C (TnC) on actin (A) filament (TnCA) and actin and myosin (M) cross-bridge (A x M) cycling in intact guinea pig hearts during baseline 37 degrees C perfusion and evaluated changes at 1) 20 min 17 degrees C pressure, 2) 30-min reperfusion (RP) after 30-min 37 degrees C global ischemia during 37 degrees C RP, and 3) 30-min RP after 240-min 17 degrees C global ischemia during 37 degrees C RP. At 17 degrees C perfusion versus 37 degrees C perfusion, the model predicted: A x M binding was less sensitive; A x M dissociation was slower; Ca(2+) was less likely to bind to TnCA with A x M present; and Ca(2+) and TnCA binding was less sensitive in the absence of A x M. Model results were consistent with a cold-induced fall in heart rate from 260 beats/min (37 degrees C) to 33 beats/min (17 degrees C), increased diastolic LVP, and increased phasic Ca(2+). On RP after 37 degrees C ischemia vs. 37 degrees C perfusion, the model predicted the following: A x M binding was less sensitive; A x M dissociation was slower; and Ca(2+) was less likely to bind to TnCA in the absence of A. M. Model results were consistent with reduced myofilament responsiveness to [Ca(2+)] and diastolic contracture on 37 degrees C RP. In contrast, after cold ischemia versus 37 degrees C perfusion, A x M association and dissociation rates, and Ca(2+) and TnCA association rates, returned to preischemic values, whereas the dissociation rate of Ca(2+) from A x M was ninefold faster. This cardiac muscle kinetic model predicted a better-restored relationship between Ca(2+) and cross-bridge function on RP after an eightfold longer period of 17 degrees C than 37 degrees C ischemia.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.     

Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.     

Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4 degrees and 8 degrees C

AIM: To establish whether or not carvacrol and cinnamic acid delay microbial spoilage of fresh-cut fruit. METHODS AND RESULTS: Dipping of fresh-cut kiwifruit in carvacrol solutions at 5-15 mM reduced total viable counts from 6.6 to < 2 log cfu g-1 for 21 d at 4 degrees C; however, undesirable colour and odour changes were also observed. Treatment with 1 mM of carvacrol or cinnamic acid reduced viable counts on kiwifruit by 4 and 1.5 log cfu g-1 for 5 d at 4 degrees C and 8 degrees C, respectively. Treatment of fresh-cut honeydew melon with 1 mM of carvacrol or cinnamic acid extended the lag phase of the microbial flora from less than 1 d in the untreated controls to 3 d at 8 degrees C and 5 d at 4 degrees C. Viable counts on the treated melon were 6 log cfu g-1 lower on Day 3 at 8 degrees C and 4 log cfu g-1 lower on Day 5 at 4 degrees C, compared with the untreated controls. IMPACT OF THE STUDY: Treatment with 1 mM of carvacrol or cinnamic acid delays spoilage of fresh-cut kiwifruit and honeydew melon at chill temperatures without adverse sensory consequences.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.