Seasonal variation in resting metabolic rate and body temperature of streaked tenrecs, Hemicentetes nigriceps and H. Semispinosus (Insectivora: Tenrecidae)

Annual variations in resting metabolic rate (RMR) and body temperature (Tb) were measured in the streaked tenrecs Hemicentetes nigiceps and H. semispinosus. RMR in non-reproducing, non-torpid adults was lower than predicted by the Kleiber (1961) curve, supporting Genoud's (1990) hypothesis on BMR in hibernating mammals. Both species demonstrated a reduction in RMR and Tb during the austral winter and these changes were independent of body mass. H. semispinosus frequently roused from torpor to forage whereas H. nigriceps remained dormant throughout the winter, This contrast in activity patterns may result from altitudinal differences in species distributions. H. nigriceps may be an obligate hibernator whereas H. semispinosus, living at lower, warmer altitudes, may be a facultative hibernator. It is proposed that the ability of H. semispinosus to remain active during favourable climatic conditions allows winter breeding and increases annual reproductive output.     

COLD RESISTANCE OF THE BRAIN DURING HIBERNATION: THE ROLE OF STEARYL CoA DESATURASE IN BRAIN AND LIVER AS THE SOURCE FOR MONOENES

Brain and liver stearyl CoA desaturase activity and its associated microsomal electron transport chain was investigated in both the warm-adapted and hibernating hamster. It was shown that the activity of this enzyme in brain was essentially the same in both the warm-adapted and hibernating hamster. In liver an 8-fold increase in desaturase activity was observed for the hibernator without corresponding increases in the activity of the microsomal electron transport chain. It is concluded that the increase of monoenes in brain that contributes to the lipid adaptation probably results from peripheral production of these fatty acids.     

23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.     

Inhibition of succinate oxidation and K+ transport in mitochondria during hibernation

Respiration of liver mitochondria of ground squirrels changes with physiological state. The inhibition of respiration at the level of dehydrogenases occurs during hibernation which is spontaneously removed during arousal. The main mechanism causing a decrease in respiration during hibernation seems to be the inhibition of succinate oxidation, induced by oxaloacetic acid. This is evidenced by the removal of the inhibition by glutamic and isocitric acids. A close correlation between the changes of K+ transport in mitochondria and of the physiological state of hibernator is observed. During hibernation the K+ transport rate decreases 3 times and during arousal it increases 1.5-fold in comparison with the active animals. The K+ content in mitochondria of hibernating and active ground squirrels is the same, whereas during arousal it increases 2-fold.     

中国达乌尔黄鼠的夏季诱发冬眠——对有否冬眠触发物质的探讨

用中国北方草原地区的季节性冬眠动物达乌尔黄鼠,经1981和1984两年的工作,重复了Dawe(1969)注射冷藏的冬眠动物血清,诱发夏季活泼黄鼠冬眠的原始实验。实验成功地实现了在非冬眠季节诱发达乌尔黄鼠冬眠,发现禁食在人工诱发冬眠中起重要作用,却不能证实血源性冬眠触发物(HIT)的存在。     

Metabolic consequences of limited substrate anion permeability in brown fat mitochondria from a hibernator,the golden hamster

Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4–5°C), brown fat is probably able to combust fatty acids totally.     

Osmotic and metabolic responses to dehydration and urea-loading in a dormant,terrestrially hibernating frog

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69–79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea. Decreased hydration was accompanied by a marked reduction in the resting rate of oxygen consumption, which was inversely correlated with plasma osmolality and urea concentration. In a separate experiment, resting rates of oxygen consumption in fully hydrated frogs receiving injections of saline or saline containing urea did not differ initially; however, upon dehydration, metabolic rates decreased sooner in the urea-loaded frogs than in control frogs. Our findings suggest an important role for urea, acting in concert with dehydration, in the metabolic regulation and energy conservation of hibernating R. sylvatica.     

Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal

The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters ($\text{Cricetus cricetus}$) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

Nuclear magnetic resonance in the investigation of cerebral tumors and cerebral edema: a clue to the cellular alterations that may affect the distribution of water

The understanding of tumor-associated cerebral edema involves an elucidation of the mechanisms involved in the altered distribution of water in the vicinity of cells. Changes in cellular macromolecules such as intracellular proteins, extracellular matrix components, and cell-membrane proteins may alter the water interactions in and around cells. The technique of pulsed nuclear magnetic resonance (NMR) gives a measure of the relaxation properties of protons in water molecules in such systems. The T1 and T2 relaxation times are increased in cerebral tumors and peritumoral tissue compared with normal brain. The in vitro study of cerebral tumors requires a tumor model that possesses the properties of the actual tumor under study. The C6 astrocytoma cell line has many of the properties of glioblastoma multiforme. An NMR study of C6 astrocytoma cells grown in monolayer, as spheroids of varying sizes and when implanted into rat hosts, has been undertaken. Results show that T1 and T2 relaxation times are not a static feature of the tumor cells but may reflect changing microenvironments that result from the contribution of a number of interacting factors present in the growing tumor.     

Studies on the physical state of water in living cells and model systems. II. NMR relaxation times of water protons in aqueous solutions of gelatin and oxygen-containing polymers which reduce the solvency of water for NA+, sugars, and free amino acids

This communication reports our study of the NMR relaxation times, T1 and T2 of water protons in aqueous solutions of bovine serum albumin, gelatin, polyvinylpyrrolidone, poly(ethylene oxide), and polyvinylmethylether over a wide concentration range. In contrast to solutions of gelatin and bovine serum albumin, the T1/T2 ratio of the three synthetic polymers are close to unity over the entire range studied. When combined with earlier-reported data of water made "non-solvent" to Na salts, the present data provided the basis for calculating the T1 and T2 as well as the rotational correlation time tau c of the "non-solvent" water. It was shown that only a modest increase by a factor of about 3 of tau c is enough to produce water that is "non-solvent" for Na citrate and sulfate. The new data reconciles NMR data of living cells with the theory of the cell water given in the association-induction hypothesis. The variability of tau c of "non-solvent" water also offers explanations of apparently conflicting conclusions on the physical state of cell water from dielectric measurements.     

19F NMR measurements of the rotational mobility of proteins in vivo.

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.     

Influence of inhibitors of cytoskeleton proteins on water exchange of wheat roots under the after-action of water stress

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water-Holding Capacity (WHC). The effective coefficient of water self-diffusion (D(eff)), spin-spin relaxation times (T(2)) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.     

Identification of experimentally induced colitis by in vitro nuclear magnetic resonance

The present study determined whether in vitro nuclear magnetic resonance could be used to assess experimentally induced colitis in rats. Acute colitis was induced in 6 Sprague-Dawley rats by acetic acid enema, while 6 control animals received saline enemas. All animals were sacrificed 24 hours post-enema, and NMR relaxation times, T1 and T2, of colonic samples were determined on a 10 MHz spin analyzer (RADX, Houston, TX). Colonic water content was determined on the same samples by desiccation. Colitis animals showed significantly higher T1 and T2 relaxation times and tissue water content than controls. T1 and T2 times correlated significantly with tissue water content. Twelve additional animals were studied histologically, six of which received acetic acid enemas and showed extensive transmural colitis. Our results suggest that in vivo proton NMR might be a useful means of non-invasively assessing the degree of colonic inflammation.     

Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel

In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active “winter” ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the “summer” ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.     

Relaxation of water protons in highly concentrated aqueous protein systems studied by 1H NMR spectroscopy

Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exchange times between bound and bulk water proton groups (tauex) have been estimated for the range of the highest concentrations (30%-46%). The obtained values are of the order of milliseconds assuring that the slow exchange condition is fulfilled in the most concentrated samples.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

Cold resistance of the brain during hibernation: I. K+ transport in cerebral cortex slices

The brains of the hibernating hamsters and 13-lined ground squirrels maintain Na⁺ and K⁺ at the same concentrations as in the awake state. The ability of slices of the cerebral cortex when incubated in vitro to accumulate or retain K⁺ is similar in the awake hamster and rat at both 38 and 5 ° C. On the other hand, slices of cerebral cortex from the hibernating hamster retained slightly more K⁺ at 5 °C than did those of awake hamster or rat. It was concluded that the cerebral cortex of the awake hamster is probably not cold resistant with respect to the maintenance of cation balance. Further, the cold resistance that exists in the cerebral cortex of the hibernating hamster is largely destroyed when the brain is disrupted by slicing.