Metabolic and hormonal responses in theophylline-increased cold resistance in males

The effects of theophylline (a phosphodiesterase inhibitor-adenosine receptor antagonist) and substrate feeding (Ensure, 250 kcal/235 ml) on cold resistance were studied in seminude males undertaking submaximal (50% maximum O2 consumption), intermittent (34% of total time) exercise in the cold (-5 to 15 degrees C, individually adjusted) for 3 h. Each subject (n = 7) served as his own control and was tested on a weekly schedule. Under control treatment, rectal temperature (Tre) decreased by 0.9 degrees C to approximately 36.2 degrees C after cold exposure, whereas under theophylline and Ensure, the decrease of Tre was only 0.4 degrees C, indicating a significant increase (P less than 0.05) in cold resistance (50% better than control). The plasma concentration of theophylline was 4.8-5.9 micrograms/ml and was positively correlated with plasma concentration of free fatty acids. Plasma norepinephrine (NE) increased significantly during cold exposure; the absolute concentration was significantly higher after theophylline pretreatment. The plasma concentrations of glucose, epinephrine, cortisol, and adenosine 3',5'-cyclic monophosphate did not change and the changes of free thyroxine and triiodothyronine were minor. Together, the effectiveness of theophylline + Ensure in acutely increasing cold resistance may be due to increased substrate availability for thermogenesis, part of which, through theophylline's potentiation of both sympathetic release of NE and NE-stimulated lipolysis and part of which, through supplementary feeding of Ensure.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Spermatogenesis in Hydra oligactis. II. How temperature controls the reciprocity of sexual and asexual reproduction

Hydra oligactis undergo two mutually exclusive modes of reproduction: at warm temperatures (18-22 degrees C) animals reproduce asexually by budding, while at cold temperatures (10-12 degrees C) gamete differentiation occurs. Using a monoclonal antibody which is specific for cells of the sperm lineage, it was discovered that under conditions where sperm differentiation does not occur (18-22 degrees C), cells continually enter the sperm pathway but progression down the pathway is prematurely halted, effectively blocking the production of sperm. To elucidate the mechanism by which completion of sperm differentiation is controlled, the cell cycle times of interstitial cells entering the sperm pathway at both the restrictive (18 degrees C) and permissive (10 degrees C) temperatures were examined. It was envisaged that at the restrictive temperature the cell cycle times of committed cells would lengthen as they proceeded down the pathway, leading to dilution and eventual loss of cells at later stages of sperm differentiation. This did not occur. Although cells of the sperm lineage were found overall to divide more slowly at 18 degrees C than at 10 degrees C, at both temperatures the cell cycle times shortened as cells proceeded further down the pathway, making a dilution mechanism untenable. The effect of high temperature on the survival of cells was then tested by subjecting animals to a heat shock. Within 12 hr of the increase in temperature, the total number of sperm lineage interstitial cells dropped 10-fold while the total numbers of epithelial and somatic interstitial cells remained virtually unchanged. A distinct consequence of this cell loss was the disappearance of cells furthest down the sperm pathway. It is proposed that as cells move down the sperm pathway, they become increasingly sensitive to high temperature which adversely affects their survival; the higher the temperature, the earlier in the pathway cells die. The lethal effect is abolished by lowering the temperature, allowing sperm differentiation to continue to completion. The possible adaptive advantages of temperature controlling gametogenesis are discussed.     

Functional status of cryopreserved rat liver mitochondria

Mitochondria from the rat liver have been frozen down to -196 degrees C under protection of dimethylsulfoxide, after which they were subjected to periodical increase and decrease in temperature (cycling) in the ranges of -105 degrees C to -196 degrees C or -135 degrees to -196 degrees C. There have been observed some non-lethal damages of mitochondria which were revealed in the decrease in the respiratory control and in the ADP/O ratio after the cycling. The damage rate increased with the increase of the number of temperature decrease-increase cycles. The damages were also greater in the case when the upper limit of cycling temperature was higher than the glass transition temperature of the freezing medium (Tg = -126 degrees C). The non-lethal damages of mitochondria are explained by the influence of electric fields, appearing in the frozen sample during the temperature increase or decrease.     

Effect of methylxanthines on production of cellulases by Penicillium echinulatum

AIM: In this work, the effect of supplementing liquid cellulase production media (CPM) with methylxanthines (aminophylline, caffeine and theophylline), with and without the addition of glucose, on the secretion of cellulases by Penicillium echinulatum strain 2HH (wild-type) and the derived mutant strain 9A02S1 was studied. METHODS AND RESULTS: When compared with unsupplemented CPM, both strains produced higher beta-glucosidase and filter paper activities (FPAs) in CPM supplemented with 1 micromol l(-1) of caffeine but lower activities with 5 micromol l(-1) of caffeine. With theophylline only, strain 9A02S1 produced higher beta-glucosidase and FPAs, while aminophylline produced no effect on the cellulase activity of either strain. Supplementation of CPM with 0.5% (w/v) of glucose plus caffeine resulted in higher beta-glucosidase and FPAs being produced by strain 2HH, but not strain 9A02S1, than in CPM supplemented with 0.5% (w/v) of glucose only. CONCLUSIONS: These results indicate that different concentrations of caffeine and theophylline can increase the beta-glucosidase and FPAs produced by P. echinulatum strains 2HH and 9A02S1. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that some methylxanthines, in adequate concentration, can be used as media components to increase cellulase production.     

Effect of temperature on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The influence of temperature on the extraction kinetics of Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum was examined in this study. The extraction of CyA from mycelia was performed in a 2-L stirred, baffled vessel using 30% v/v aqueous methanol. The temperature range used was from 5 to 45 degrees C. A linear relationship was found between the extraction yield of CyA and temperature. As the temperature increased, the yield of CyA increased with a maximum CyA yield of 18.3% obtained at 45 degrees C, which is 21.3% higher than the yield at 25 degrees C. The activation energy for the extraction of CyA from T. inflatum was found to be 36.7 kJ/mol, which indicates that the extraction of CyA from T. inflatum is controlled by both solubilization of CyA and diffusion of CyA through the solid phase of mycelia. The overall mass transfer coefficient, k(L)a(S), was found to increase from 1.02 x 10(-3) to 1.34 x 10(-2) s(-1) as the temperature increased from 5 to 45 degrees C. The effective diffusivity of CyA in the solid matrix of mycelia was found to increase from 1.05 x 10(-15) to 1.43 x 10(-14) m(2)/s as the temperature increased from 5 to 45 degrees C. A mathematical diffusion model was developed and was used to fit the experimental kinetic data of CyA extraction and determination of CyA effective diffusivities at different temperatures. This is the first time CyA diffusivities as a function of extraction temperature are reported in the literature.     

Effects of cold on microvascular fluid movement in the cat limb

We investigated the effects of low temperatures down to approximately 5 degrees C on postcapillary resistance (Rv) and isogravimetric capillary pressure (Pci) in the isolated constant-flow-perfused cat hindlimb to see if a low-temperature-induced increase in Rv and decrease in Pci could lead to an increase in filtration pressure and edema formation. A low-viscosity perfusate (20% cat plasma, 80% albumin-electrolyte solution; viscosity approximately 1 cP) was used. Isoproterenol (10(-7) M) was added to vasodilate the limb and achieve normal microvascular permeability. Rv and Pci were estimated from the slope and zero-flow intercept, respectively, of the straight-line fit to the isogravimetric venous pressure vs. flow data. Rv and Pci were determined in each experiment at an initial 37 degrees C control, at a lowered temperature (30, 23, 15, or 5-10 degrees C), and then at 37 degrees C again. The ratio of Rv at the low temperatures relative to the initial 37 degrees C control increased almost linearly as temperature was reduced. The increase was 3.4 times control at the lowest temperature. Pci decreased significantly from control only in the lowest temperature group where the change was -5.4 mmHg. Analysis of our data with the low-viscosity perfusate shows that the limb can become edematous if temperature is lowered to approximately 5 degrees C unless venous pressure (Pv) is lowered to venous collapse and flow reduced to less than approximately 20 ml.min-1.100g-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein tyrosine phosphorylation during prolonged in vitro incubation of ejaculated bovine spermatozoa is regulated by the oxidative state of the medium

Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18-20 degrees C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37 degrees C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M(r) 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.     

Changes in pH in the cytoplasm of rat thymocytes after treatment with the mitogen concanavalin A and protein kinase C activator phorbol ester

The intracellular pH (pHi) of rat thymocytes has been measured with the fluorescent probe 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein, both in the resting cells and under mitogenic stimulation. Concanavalin A (Con A) has been found to increase pHi from 7.16 +/- 0.02 to 7.30 +/- 0.02 during the first minutes after addition; the phorbol ester TPA raised pHi to 7.25 +/- 0.02. The Con A- and TPA-induced rise of pHi is due to activation of Na+/H+ exchange since it was abolished by amiloride, an inhibitor of Na+/H+ antiport, or in a low-Na+ medium. The elevation of intracellular cAMP level, decrease of cellular ATP, or the lowering of the temperature from 37 degrees down to 25 degrees C inhibited the pHi rise induced by Con A or TPA.     

Structural and functional modifications of the manganese cluster in Ca(2+)-depleted S1 and S2 states: electron paramagnetic resonance and X-ray absorption spectroscopy studies.

The effect of extraction of weakly bound Ca2+ by low-pH treatment on the O2-evolving apparatus was studied by use of low-temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. In low-pH-treated PSII membranes, an S2 EPR multiline signal with modified line shape was induced by illumination at 0 degrees C, but its signal amplitude decreased upon lowering the excitation temperature with concomitant oxidation of cytochrome (cyt) b-559 in place of Mn. The half-inhibition temperature for formation of the modified multiline signal was found at -33 degrees C, which was much higher than that for formation of the normal S2 state in untreated control membranes. Signal IIf was normally induced down to -30 degrees C, but its dependence on excitation temperature was different from that for modified S2. This was interpreted as indicating that the low-temperature blockage of modified S2 formation is due to the incapability of electron abstraction from the Mn cluster. The Mn K-edge of X-ray absorption near-edge structure (XANES) spectrum shifted to lower energy by 0.8 eV after low-pH treatment, but the shift was reversed by addition of Ca2+. Upon illumination at 0 degrees C of treated membranes, the K-edge energy was up-shifted by 0.8 eV, but was not upon illumination at 210 K. These results were interpreted as indicating that extraction of weakly bound Ca2+ by low-pH treatment gives rise to structural and functional modulations of the Mn cluster.     

Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.     

Hyperpolarizing effect of aminophylline, theophylline, and cAMP on rat diaphragm fibers

We studied the effect of aminophylline and theophylline (0.1-2 mM) on the resting membrane potential (Vm) of rat diaphragm fibers in vitro (25 degrees C). The main findings are the following. 1) Aminophylline and theophylline hyperpolarize the fibers in a dose-dependent manner. This effect is present with 0.1 and 0.25 mM of aminophylline and theophylline, respectively, and the maximum effect is reached with 1 mM of the drug (approximately 5-8 mV in comparison to the normal values). This effect is reversible by washing out the preparation with normal solution. 2) Dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 2 mM) produces a similar increment in the Vm. 3) The hyperpolarizing action observed in the presence of aminophylline, theophylline, and DBcAMP is suppressed by 5 X 10(-4) M ouabain or by lowering the bath temperature to 5 degrees C. These results suggest that the xanthines may directly or indirectly stimulate a Na-K pump. Two possibilities may be considered: 1) an electrogenic effect of the Na-K pump and 2) a reduction in the extracellular K+ concentration in the solution contacting the external side of the cell as a consequence of the activity of the Na-K pump. Alternative mechanisms such as a reduction in Na permeability or an increment in K permeability might collaborate in the hyperpolarizing effect of the drugs tested.     

The effect of chlorpromazine on the temperature and osmotic sensitivity of erythrocytes]

The effect of chlorpromazine on the development of cold shock in erythrocytes exposed to sodium chloride was shown to depend on the tonicity of the medium in which the cells were cooled from 37 degrees C down to 0 degrees C as well as on the amphipate concentration. After cooling of erythrocytes in a NaCl (0.75-1.5 M)-containing medium with chlorpromazine (7 x 10(-5) M, 2.1 x 10(-4) M and 3.5 x 10(-4) M) the hypertonic cold shock was inhibited, the protective effect of the amphipate being less pronounced at its increasing concentrations. After cooling of cells under conditions of moderate hypertonicity (0.3-0.6 M NaCl) no modifying effect of chlorpromazine on the sensitivity of erythrocytes to the temperature decrease from 37 degrees C down to 0 degrees C was manifested. However, under iso- and hypertonic conditions chlorpromazine used at 2.1 x 10(-4) M and 3.5 x 10(-4) M stimulated the cold shock development in erythrocytes. A sharp increase in the medium tonicity (up to 1.8-3.0 M and higher) the cells underwent isothermal hemolysis which was more expressed at 0 degrees C than at 37 degrees C. These data suggest that chlorpromazine significantly activates the hemolytic process at low temperatures.     

Effect of temperature on life table parameters of predatory thrips Scolothrips longicornis (Thysanoptera: Thripidae) fed on twospotted spider mites (Acari: Tetranychidae)

Environmental variables such as temperature are important factors that affect the efficiency of biological control agents. This study examined the effect of temperature on the sex ratio, longevity, oviposition periods, fecundity and life table parameters of the predatory thrips Scolothrips longicornis Priesner (Thysanoptera: Thripidae) fed on twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), at six constant temperatures: 15, 20, 26, 30, 35, and 37 degrees C. Approximately 75% of the progeny were female, except at 37 degrees C, at which temperature the proportion of males increased. Adult longevity as well as the preoviposition, oviposition, and postoviposition periods decreased significantly with temperature. Thus, adults lived for approximately 5 wk at 15 degrees C and < 1 wk at 37 degrees C with preoviposition, oviposition, and postoviposition periods ranging from 6.4 to 0.4, 24.4-3.1, and 7-0.8 d between the two temperature extremes, respectively. The maximum (56.48 eggs) and minimum (11.69 eggs) value of total fecundity was recorded at 26 and 37 degrees C, respectively. The intrinsic rate of natural increase (r(m)) of S. longicornis increased linearly with increasing temperature from 0.056 d(-1) at 15 degrees C to 0.310 d(-1) at 35 degrees C. The lower temperature threshold for the population increase of S. longicornis was estimated to be around 5 degrees C. The data suggest that the upper temperature threshold for the predatory thrips is approximately 37 degrees C. The results showed that populations of S. longicornis are able to develop at a broad range of temperatures and that this predator is well adapted to the high temperatures that occur in the Mediterranean region.     

温度对棉大卷叶螟生长发育和繁殖的影响

本文利用生命表方法评价了温度对棉大卷叶螟生长发育和繁殖的影响.结果表明：在17 ℃～26 ℃温度范围内,棉大卷叶螟的发育历期随温度升高而显著缩短;当温度超过32 ℃时,其发育历期延长.棉大卷叶螟的世代起点发育温度为12.08 ℃,有效积温为436.2日·度.在17 ℃～35 ℃温度范围内,棉大卷叶螟的存活率在26 ℃下最高,其他温度下的存活率随温度的升高或降低而降低.在17 ℃～35 ℃之间,棉大卷叶螟实验种群的内禀增长率、世代净增殖率、周限增长率分别在29、26和32 ℃下最大,其值分别为0.1268、415.65和1.185.世代平均历期随着温度的升高而缩短,在17 ℃下最长,为89.11 d;在35 ℃下最短,为28.68 d.     

A spin label study of rat brain membranes. Effects of temperature and divalent cations.

Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.     

Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer.

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20 degrees C to 35 degrees C. Polymer domain size at 20 degrees C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S-CO reaction rate decreased with increasing temperature from 20 degrees C to 35 degrees C, and increased with decreasing domain size. In comparison, Hb A-CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S-CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.