Variations in myocardial CPK and Na+-K+ ATPase following normo- and hypothermic exposure to dimethyl sulfoxide and glycerol.

Exposure of canine myocardial tissue homogenates to Me₂SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na⁺-K⁺ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me₂SO provides a similar enhancement of Na⁺-K⁺ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me₂SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me₂SO at 0 °C. The results suggest that disruption of the CPK-Na⁺-K⁺ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.     

Effect of temperature on insulin binding and action in cultured human fibroblasts

Insulin stimulation of glycogen synthase activity and insulin binding were measured in fibroblast monolayers at 24, 32, and 37°C. Insulin stimulation of %$\text{I}$ glycogen activity increased with increasing temperature. Maximum response was greater at 37°C than at 32°C, and half maximal stimulation required at 2.0 nM insulin at 37°C vs. 10nM at 32°C. Insulin stimulation of glycogen synthase was greater and somewhat faster at 37°C than at 32°C. No insulin effect was observed at 24°C. ¹²⁵I-insulin binding to monolayers became maximal in 15 min at 37°C, 60 min at 32°C, and 120 min at 24°C. However, insulin binding decreased with increasing temperature, and this decline was due to decreased numbers of receptors. Insulin binding and stimulation of glycogen synthase were comparable at 32°C, with half maxima at 10 nM, indicating no evidence of “spare” receptors. The data indicate that temperature effects on insulin binding and action in fibroblasts are not directly related. The results also suggest that a rate limiting step(s) of insulin action is temperature sensitive, and that this step is not insulin binding.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Lactate Metabolism and Its Effects on Glucose Metabolism in an Excised Neural Tissue

Abstract: Chains of lumbar sympathetic ganglia, excised from 15-day-old chicken embryos, were incubated for 4 h at 36°C in a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO₂–95% O₂. [U-¹⁴C]Glucose and [U-¹⁴C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO₂, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50–70% of the glucose carbon otherwise used for CO₂ production and provided about three times as much carbon for CO₂ as did glucose. The lactate addition increased the total carbon incorporated into CO₂ and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U-¹⁴C]-glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10-fold relative to that of the tissue, the average output of CO₂ from [U-¹⁴C]glucose during a 4-h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate. Consumable substrates other than lactate, such as glutamine and certain amino acids, also affected glucose metabolism.     

Effect of temperature on ion content, ion fluxes and energy metabolism in Chara corallina

Abstract Effects of temperature on the ionic relations and energy metabolism of Chara corallina were investigated. Measurements were made of the ionic content, tracer ion fluxes, and photosynthetic and dark CO₂ fixation in isolated cells, and of O₂ exchange in photosynthesis and respiration in isolated shoot apices. The total intracellular concentration of K⁺, Na⁺ and Cl^? was the same in cells held for 5 days in non-growing medium at 15°C (the growth temperature) as in those held at 25°C or 5°C. The tracer influx in the light of all ions tested (Rb⁺, Na⁺, CH₃NH₃⁺, Cl^? and H₂PO₄^?) was lower at 5°C than at 15°C in experiments in which cells were subjected to 5°C for less than 24 h in toto. The influx at 25°C was greater than that at 15°C for H₂PO^?₄, there was no difference between the two temperatures for Na⁺, while the influx at 25°C was less than that at 15°C for Cl^?, Rb⁺ and CH₃NH₃⁺ For Cl^? and H₂PO^?₄ similar results were found in later experiments with cells grown at 20—23°C. Photosynthetic CO₂ fixation and O₂ evolution, and respiratory O₂ uptake, are greater at 25°C, and lower at 5°C, than they are at the growth temperature of 15°C. In longer-term pretreatments at the different temperatures, tracer Cl^? influx at 15°C and particularly at 25°C were lower than in short-term experiments, while the influx at 5°C was higher. It was concluded from these experiments, and from previous data on H⁺ free energy differences across the plasmalemma, that (1) the maintenance of internal ion concentrations involves a close balancing of influx and efflux of K⁺, Na⁺ and Cl^? at all experimental temperatures; (2) the regulation of the tracer fluxes of the ions is kinetic rather than thermodynamic and (3) that the tracer fluxes at low temperatures are not restricted by the rate at which respiration or photosynthesis can supply energy to them.     

Effects of acute temperature change on the metabolism and swimming ability of juvenile sterlet sturgeon (Acipenser ruthenus,Linnaeus 1758)

The objective of this study was to provide information on changes in the metabolism and swimming ability of juvenile sterlet sturgeon, Acipenser ruthenus, caused by acutely low or high temperatures. Changes in critical swimming speed (U_crit), oxygen consumption rate (MO₂), tail beat frequency (TBF) and tail beat amplitude (TBA) were observed with a Steffensen‐type swimming respirometer, an oxygen electrode and a camera at different swimming speeds at three temperatures: 5°C, 15°C, and 25°C. Fish tested at 5°C and 25°C were maintained at 15°C (near optimal) for one week to simulate conditions below a dam. The U_crit value decreased significantly during acute temperature changes at 5°C and 25°C; U_crit was highest near the optimal temperature. Oxygen consumption rate (MO₂) increased with the swimming speed at 15°C; however, at 25°C and 5°C, the MO₂ decreased with the swimming speed. Both TBA and TBF decreased at 5°C and 25°C compared to values at 15°C. The slopes of the regression lines (TBF/U) at 5°C and 25°C seemed lower compared to 15°C.     

Transfer of ampicillin resistance from Salmonella Typhimurium DT104 to Escherichia coli K12 in food

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin‐resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10^?4 to 10^?5 log₁₀ CFU ml g^?1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10^?6, log₁₀ CFU g^?1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nal^R transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β‐lactamase gene bla_TEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid‐range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic‐resistant genes and the evolution of antibiotic‐resistant bacteria.     

The effects of temperature on swimming performance of juvenile shortnose sturgeon (Acipenser brevirostrum)

The swimming performance of juvenile shortnose sturgeon (～16 cm TL, ～20 g), Acipenser brevirostrum, was quantified with regards to temperature (5 to 25°C) using both increased (U_crit) and fixed velocity (endurance) tests in a laboratory setting. Sturgeons were found to show reduced U_crit values at 5 and 10°C (25.99 and 28.86 cm s^?1 respectively), with performance beginning to plateau at 15°C through 25°C (33.99 cm s^?1). For the endurance protocol, fish were tested at speeds of 35, 40 and 45 cm s^?1 at 5, 15 and 25°C. Performance within a single speed was similar at all temperatures, indicating the usage of anaerobic metabolism to fuel locomotion at these higher velocities. Overall, shortnose sturgeon demonstrated high tolerance towards a wide range of temperatures but showed few differences between performance levels at colder or warmer water conditions.     

Oncolytic measles viruses produced at different scales under serum‐free conditions

Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum‐free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T‐flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated‐batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T‐ and spinner flasks at 37°C (～10⁷ TCID₅₀ mL^?1, where TCID₅₀ is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000‐fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 10⁷ TCID₅₀ mL^?1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large‐scale production.     

PHOSPHOLIPASE ACTIVITY IN SQUID AND FROG AXONS

Evidence for the presence of phosphatide acylhydrolase activity (EC 3.1.1) in centrifuged homogenate supernatants and extracts of squid giant axons and centrifuged homogenate supernatants of frog sciatic nerve bundles is reported. The enzyme was assayed by measurement of the rate of deacylation of [U-¹⁴C]phosphatidyl choline. The deacylation activity in the nerve homogenate supernatants exhibits: a pH maximum at 7.2–7.4 (25°C); a calcium ion maximum at 12–13 mM-CaCl₂(aq); a K_m value of 3.4 × 10^?4 M (25°C); and a temperature maximum at 37°C. The activation energy over the range 8–37°C is 5.7 ± 0.2kcal-mol^?1.     

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [¹²⁵I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [³⁵S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [¹⁴C]glucose incorporation into glycogen measured at 37°C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley-Liss, Inc.     

The influence of gelatin polypeptides,potassium, calcium and osmolality on the hypothermic perfusion of rabbit hearts

After a period of perfusion at 37 °C with a standard perfusate, rabbit hearts were cooled to +10 °C and perfused at this temperature for 5 hr with a variety of solutions. The hearts were then rewarmed to 37 °C and perfused again with the standard perfusate to assess heart function. The effects on subsequent normothermic function of including gelatin polypeptides (Haemaccel) and of increasing the osmolality and the concentrations of K⁺ and Ca²⁺ in the solutions used for hypothermic perfusion were studied. The best results were achieved with a noncolloidal electrolyte solution containing 20 mm K⁺ and 4.8 mm Ca²⁺ which gave an average maximum percentage recovery of function of 57.9 ± 7.1%. The addition of sufficient mannitol to raise the osmolality from 300 mOsm/Kg to 420 mOsm/Kg improved (but not significantly) the maximum percentage recovery of function to 61.2 ± 8.5%     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

Differential cerebral hypothermia

Differential cerebral hypothermia was induced in these experiments by isolating the cerebral circulation in the halothane-anesthetized goat. The brain was perfused through isolated cerebral branches of the internal maxillary artery using a height-adjusted reservoir system which provided a constant inflow pressure. Cerebral blood flow (CBF) and cerebral O₂ metabolic rate (CMRO₂) were measured continuously as brain temperatures were decreased from 38 to 28, 18 and 8 °C and during rewarming. Arterial blood gases were maintained constant. During hypothermia CBF decreased at brain temperatures of 28 °C and did decrease further at 18 or 8 °C. CMRO₂ decreased linearly from 38 to 8 °C and was 7% control levels at 8 °C. CBF and CMRO₂ returned to control levels upon rewarming. Cerebral lactate metabolism did not change significantly during hypothermia or rewarming. Evoked cortical potentials were abolished at 8 °C but recovered upon rewarming. These results indicate that if adequate brain perfusion is maintained during hypothermia and rewarming, recovery of CBF, metabolism, and brain neural activity can be obtained.     

Influence of the time interval after venepuncture and the storage temperature on the uptake of 14C-serotonin by human blood platelets

The active uptake of ¹⁴C-5-HT into human platelets at 37°C was studied at various times (10–130 min) and at various storage temperatures (4°, 22°, 37°C) after venepuncture. 5-HT uptake was significantly influenced by both variables. There was no direct correlation between 5-HT uptake and storage temperatures, storage time and changes in the pH of PRP, resp. The apparent K_m-value for the 5-HT uptake (0.5μM) remained constant. However, the K_i-values obtained for different uptake inhibitors at the different experimental conditions indicate the need for exact standardization.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

A Cold Acclimation Protein with Refolding Activity on Frozen Denatured Enzymes

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH₄)₂SO₄ precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at -20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10^-10, 4.35×10^-9, 8.98×10^-12, and 3.05×10^-11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.     

The influence of differentiation-inducing agents on plasma membrane surface characteristics of THP-1 cells

Undifferentiated THP-1 cells from Cell Culture Collection of the Institute of Cytology, RAS (St. Petersburg), are characterized by weak expression of Toll-like receptor-4 (TLR4) on the cell surface (up to 2%) and by almost undetectable expression of CD14 and CD11b receptors. Differentiation agent phorbol-12-myristate-13-acetate independently of its concentration (2 × 10⁻⁷ M or 10⁻⁸ M) and incubation time (24 or 48 h) did not initiate CD11b surface expression and did not change the parameter S_app (0.605 ± 0.005 at 37°C) reflecting the cell membrane viscosity. Differentiation of THP-1 cells induced by another differentiation agent, 1α,25-dihydroxyvitamin D₃, caused expression of CD14 (up to 70–80%) and CD11b (up to 15–20%) receptors, again without changes in plasma membrane viscosity. The rate constants of the reduction of 5- and 16-doxyl-stearic acids by THP-1 cells were in the range of 6–8 × 10⁻³ s⁻¹ at 37°C. During cell differentiation significant changes in cell electrophoretic mobility (EM, μm s⁻¹ V⁻¹ cm) were observed. Mean value of EM for undifferentiated THP-1 cells was −1.332 ± 0.011, whereas for phorbol-12-myristate-13-acetate- and 1α,25-dihydroxyvitamin D₃-treated cells it was −1.432 ± 0.030 and −1.212 ± 0.016, respectively.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.