Metabolic Activity of Permafrost Bacteria below the Freezing Point

Metabolic activity was measured in the laboratory at temperatures between 5 and −20°C on the basis of incorporation of ¹⁴C-labeled acetate into lipids by samples of a natural population of bacteria from Siberian permafrost (permanently frozen soil). Incorporation followed a sigmoidal pattern similar to growth curves. At all temperatures, the log phase was followed, within 200 to 350 days, by a stationary phase, which was monitored until the 550th day of activity. The minimum doubling times ranged from 1 day (5°C) to 20 days (−10°C) to ca. 160 days (−20°C). The curves reached the stationary phase at different levels, depending on the incubation temperature. We suggest that the stationary phase, which is generally considered to be reached when the availability of nutrients becomes limiting, was brought on under our conditions by the formation of diffusion barriers in the thin layers of unfrozen water known to be present in permafrost soils, the thickness of which depends on temperature.     

Thermal inactivation of Enterococcus faecium: effect of growth temperature and physiological state of microbial cells

AIMS: To provide data on the effects on culture temperature and physiological state of cells on heat resistance of Enterococcus faecium, which may be useful in establishing pasteurization procedures. METHODS AND RESULTS: The heat resistance of this Ent. faecium (ATCC 49624 strain) grown at different temperatures was monitored at various stages of growth. In all cases, the bacterial cells in the logarithmic phase of growth were more heat sensitive. For cells which had entered in the stationary phase, D70 values of 0.53 min at 5 degrees C, 0.74 min at 10 degrees C, 0.83 min at 20 degrees C, 0.79 min at 30 degrees C, 0.63 min at 37 degrees C, 0.48 min at 40 degrees C and 0.41 min at 45 degrees C were found. By extending the incubation times cells were more heat resistant as stationary phase progressed, although a different pattern was observed for cells grown at different temperatures. At the lower temperatures heat resistance increased progressively, reaching D70 values of 1.73 min for cells incubated at 5 degrees C for 50 days and 1.04 min for those grown at 10 degrees C for 16 days. At other temperatures assayed heat resistance became stable for late stationary phase cells, reaching D70 values of 1.05, 1.08 and 1.01 min for cultures incubated at 20, 30 and 37 degrees C. Heat resistance of cells obtained at higher temperatures, 40 and 45 degrees C, was significantly lower, with D70 values of 0.76 and 0.67 min, respectively. Neither the growth temperature nor the growth phase modified the z-values significantly. CONCLUSIONS: D70 values obtained for Ent. faecium (ATCC 49624) varies from 0.33 to 1.73 min as a function of culture temperature and physiological state of cells. However, z values calculated were not significantly influenced by these factors. A mean value of 4.50 +/- 0.39 degrees C was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall results strongly suggest that, to establish heat processing conditions of pasteurized foods ensuring elimination of Ent. faecium, it is advisable to take into account the complex interaction of growth temperature and growth phase of cells acting on bacterial thermal resistance.     

Growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7 on beef extract medium as influenced by package atmosphere and storage temperature.

The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.     

Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetracycline production with sweet potato residue by solid state fermentation

For saving energy in antibiotic production and reducing the amount of agricultural wastes, solid state fermentation was used in this study to produce tetracycline with sweet potato residue by Streptomyces viridifaciens ATCC 11989. It was found that the optimal media for tetracycline production were sweet potato residue 100 g, organic nitrogen (rice bran, wheat bran, or peanut meal) 20 g, (NH(4))(2)SO(4) 2.4 g, KH(2)PO(4) 0.4 g, CaCO(3) 1.8 g, NaCl 0.6 g, MgCl(2) 0.8 g, soluble starch 10 g, methionine 0.2 g, histidine 0.8 g, and monosodium glutamate 1.6 g with initial moisture content 68-72%, and initial pH 5.8-6.0. Each gram of dry weight substrate was inoculated with 1.0 x 10(8) conidia and incubated at 26 degrees C for 5-7 days, producing 4720 mug of total tetracycline equivalent potency. When incubated at 26 degrees C with the initial moisture content 68%, the conidia in solid media germinated on the second day, mycelia grew abundantly on the third day and reached stationary phase on the sixth day. The antibiotic production was consistent with the morphogenesis of S. viridifaciens: activity could be detected on the third day, had the maximal potency on the sixth day, and decreased slightly on the tenth day. (11-3-88 tly).     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22 degrees C and 37 degrees C; production was optimum at 37 degrees C in the stationary phase (14-16 h). A decrease in phospholipase C activity at various storage temperatures (-5 degrees C, 4 degrees C, 37 degrees C) was also observed, although the enzyme was active over a wide range of temperature (5-65 degrees C) and pH (3.5-7.5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Life history of hawthorn spider mite Amphitetranychus viennensis (Acarina: Tetranychidae) on various apple cultivars and at different temperatures

Development duration and reproduction rate of hawthorn spider mite Amphitetranychus viennensis (Zacher) were carried out on five different apple cultivars (Amasya (local cultivar), Golden Delicious, Granny Smith, Starking Delicious and Starkrimson Delicious) at 25 degrees C, 65 +/- 10% RH and 16:8 L:D. In addition, the same parameters were determined on Golden Delicious leaves at three constant temperatures (20, 30 and 35 degrees C, 65 +/- 10% RH and 16:8 L:D) in the laboratory. A. viennensis showed a better performance on Golden Delicious than on the other apple cultivars. This was mainly due to a short development time (10.7 days), high daily egg production (5.2 eggs/female/day) and early reproduction peak. The highest intrinsic rate of natural increase (rm) was determined on the variety Golden Delicious (rm = 0.247/day), while the lowest one was observed on the variety Starking Delicious (rm = 0.215/day). The developmental periods of A. viennensis varied from 7.4 to 18.8 days at 35 and 20 degrees C for females, while it varied from 7.9 to 17.2 days at 30 and 20 degrees C for males. The development threshold of the eggs and pre-adult stages were 9.72 and 9.07 degrees C, total effective temperature was 72.99 and 185.18 degree-days, respectively. The mean generation time (To) of the population ranged from 16.13 days at 30 degrees C to 29.15 days at 20 degrees C. The net reproductive rate (R0) increased from 54.33 female/female at 20 degrees C to 78.34 female/female at 25 degrees C, and decreased to 75.71 female/female at 30 degrees C. The highest intrinsic rate of increase (rm) was reached at 30 C (rm = 0.268/day), the lowest one at 20 degrees C (rm = 0.136/day).     

Effect of thermoperiod on diapause intensity in Pyrrhocoris apterus (Heteroptera Pyrrhocoridae)

The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction.     

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.     

Modeling the growth of Enterococcus faecium in bologna sausage.

A study to set up mathematical models which allow the prediction of Enterococcus faecium growth in bologna sausage (mortadella) was carried out. Growth curves were obtained at different temperatures (5, 6, 12, 15, 25, 32, 35, 37, 42, 46, 50, 52, and 55 degrees C). The Gompertz and logistic models, modified by Zwietering, were found to fit with the representation of experimental curves. The variations of the parameters A (i.e., the asymptotic value reached by the relative population during the stationary growth phase), mu m (i.e., the maximum specific growth rate during the exponential growth phase), and lambda (i.e., the lag time) with temperature were then modeled. The variation of A with temperature can be described by an empirical polynomial model, whereas the variation of mu m and lambda can be described by the Ratkowsky model modified by Zwietering and the Adair model, respectively. Data processing of these models has shown that the minimum growth temperature for E. faecium is 0.1 degrees C, the maximum growth temperature is 53.4 degrees C, and the optimal growth temperature is 42 to 45 degrees C.     

Social versus individual behaviour: a comparative approach to thermal behaviour of the honeybee (Apis mellifera L.) and the American cockroach (Periplaneta americana L.)

To study the relationship between the individual and social thermoregulatory behaviour, we used honeybee workers and American cockroaches. Single insects or groups of 10-20 individuals were placed in a temperature gradient chamber, and their thermal preference was recorded for 48 h under natural summer photoperiod. Single bees showed diurnal changes in selected ambient temperature, which culminated at 14:00 reaching 34+/-2 degrees C, and then slowly decreased, reaching a nocturnal minimum of 28+/-2 degrees C at 04:00. In contrast, the zenith of temperature selected by groups of bees (31+/-1 degrees C) was reached at 04:00 and the nadir (29+/-2 degrees C) was recorded at 14:00. Groups of bees clustered together during the night time, and dispersed during intense day time activity. Such changes were absent in groups of cockroaches. Cockroaches selected an ambient temperature of 30+/-1 degrees C both during day and night. In conclusion, there is a striking analogy in the diurnal thermal behaviour between a colony of bees and mammals. During their nychthemeral rest phase, both of them select higher temperatures than during the activity phase and, simultaneously, they reduce their overall surface area of heat loss to conserve metabolic heat. Therefore, the colony behaves as a homeothermic superorganism. In contrast, a single bee, isolated from the colony, utilizes a heterothermic strategy to save energy for a morning warm up.     

Fish protein hydrolysis by a psychrotrophic marine bacterium isolated from the gut of hake (Merluccius hubbsi)

The aims of this study were to identify a psychrotrophic bacterium, strain CR41, producing a cold adapted protease during growth at low temperatures and to evaluate the ability of the cells to hydrolyze hake fish protein. The strain was isolated from the intestinal tract of hake collected from the San Jorge Gulf (Patagonia, Argentina) and it was identified as Pseudoalteromonas. Growth and fish protein hydrolysis were determined using an aerated simple mineral medium plus 10% fish protein concentrate. Proteolytic activity was measured at 7 and 22 degrees C during culture in the concentrate. Protease production started in the exponential growth phase and reached a maximum during stationary phase. Protease activity at 7 degrees C was lower than at 22 degrees C. After 8 h of incubation, the percentage of hydrolyzed protein was 84% at 7 degrees C and 95% at 22 degrees C. Electrophoresis detection showed that degradation of muscle hake proteins was complete at both temperatures, and in gelatin zymograms extracellular activity showed two proteolytic bands with apparent molecular masses of approximately 31.6 and 62 kDa.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Cryoanalgesia: electrophysiology at different temperatures

Somatosensory evoked potentials (SEP) and sensory conduction velocity (SCV) were measured in rabbit sciatic nerves following graded cold lesioning. The SEP disappeared when injury was induced at temperatures below -60 degrees C, but returned on day 41+/-4 (mean+/-SD). SEP returned on day 56+/-11 days when the lesion was induced at 100 to -180 degrees C. The SEP latency was prolonged after creating lesions at -100 to -180 degrees C, compared with both the sham operated and the -20 degrees C groups. These experiments suggest the cryolesions produced at temperatures between -60 and -100 degrees C are most suitable for altering the electrophysiological conduction of the nerve, and may result in suitable post-operative analgesia.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Parasitism capacity of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) reared under different temperatures on Bonagota salubricola (Meyrick) (Lepidoptera: Tortricidae) eggs

The parasitism capacity of Trichogramma pretiosum Riley strain bonagota on Bonagota salubricola (Meyrick) eggs was studied under the temperatures of 18, 20, 22, 25, 28, 30 and 32 degrees C. The number of days with parasitism, accumulated parasitism, total number of eggs parasitized per female and parasitoid longevity was evaluated. In the first 24h, parasitism ranged from 1.6 (32 degrees C) to 8.8 (22 degrees C) eggs of B. salubricola. Accumulated egg parasitism of B. salubricola reached 80% in 1st to 4th day at 20 degrees C to 32 degrees C, respectively, and in the 7th day at 18 degrees C. Temperatures from 18 degrees C to 22 degrees C were the best suited for the total eggs parasitized for female, resulting in 35.4 and 24.6 eggs/male respectively. T. pretiosum female longevity ranged from 7.8 to 2.5 days, at 18 degrees C and 32 degrees C, respectively. The results showed that T. pretiosum strain bonagota is better adapted to temperatures from 18 degrees C to 22 degrees C.     

Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel

AIMS: This study investigated the in vitro effects of water activity (a(w); 0.85-0.987) and temperature (10-40 degrees C) on growth and ochratoxin A (OTA) production by two strains of Aspergillus carbonarius isolated from wine grapes from three different European countries and Israel on a synthetic grape juice medium representative of mid-veraison (total of eight strains). METHODS AND RESULTS: The synthetic grape juice medium was modified with glycerol or glucose and experiments carried out for up to 56 days for growth and 25 days for OTA production. The lag phase prior to growth, growth rates and ochratoxin production were quantified. Statistical comparisons were made of all factors and multiple regression analysis used to obtain surface response curves of a(w) x temperature for the eight strains and optimum growth and OTA production by A. carbonarius. The lag phase increased from <1 day at 25-35 degrees C and 0.98 a(w) to >20 days at marginal temperatures and water availabilities. Generally, most A. carbonarius strains grew optimally at 30-35 degrees C, regardless of solute used to modify a(w), with no growth at <15 degrees C. The optimum a(w) for growth varied from 0.93 to 0.987 depending on the strain, with the widest a(w) tolerance at 25-30 degrees C. There was no direct relationship among growth, environmental factors and country of origin of individual strains. Optimum conditions for OTA production varied with strain. Some strains produced optimal OTA at 15-20 degrees C and 0.95-98 a(w). The maximum OTA produced after 10 days was about 0.6-0.7 microg g(-1), with a mean production over all eight strains of 0.2 microg g(-1) at optimum environmental conditions. CONCLUSIONS: This work demonstrates that optimum conditions for OTA production are very different from those for growth. While growth rates differed significantly between strains, integration of the OTA production data suggests possible benefits for use of the information on a regional basis. SIGNIFICANCE AND IMPACT OF THE STUDY: Very little detailed information has previously been available on the ecology of A. carbonarius. This knowledge is critical in the development and prediction of the risk models of contamination of grapes and grape products by this species under fluctuating and interacting environmental parameters.     

Cryomicroscopic observations of cattle embryos during freezing and thawing

A cryomicroscope was used to observe changes in the appearance of day 6 1 2 to 7 1 2 cattle embryos during cooling and warming in 1.4M glycerol/PBS. Embryos were cooled at various rates between 0.2 and 25 degrees C/min to temperatures between -25 and -60 degrees C and then cooled rapidly ( approximately 250 degrees C/min) to temperatures below -140 degrees C. The volume of the embryos calculated from the cross-sectional area during slow cooling decreased at -25 degrees C to about 50% of the isotonic volume. Fracture planes could be observed in the extracellular ice matrix surrounding the embryos after rapid cooling to approximately -140 degrees C. The fracture planes often touched the zona pellucida and sometimes caused cracks in the zona. Cracks in the zona pellucida were observed more often after rapid cooling from temperatures between -20 to -35 degrees C (9 13 ) than from temperatures between -36 to -60 degrees C (2 7 ). When embryos were warmed rapidly ( approximately 250 degrees C/min) from temperatures below -140 degrees C, no change was observed in the appearance of either the embryo or its surroundings except the melting of the extracellular ice. However, when embryos were warmed slowly (2 or 5 degrees C/min), a series of events was observed; first, at approximately -70 degrees C the cytoplasm and the extracellular space gradually darkened and reached maximum darkness at approximately -55 degrees C. Then, on continued slow warming, the dark material gradually disappeared and finally the large extracellular ice crystals melted.     

The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture.

Haemonchus contortus eggs were cultured in intact fecal pellets at various temperatures (5-35 degrees C) for 22 days. Temperature and relative humidity were kept constant throughout the incubation period. Nl larval development occurred at 5 degrees C; peak third-stage larval recovery occurred at 20 degrees C. Egg mortality was an age-dependent phenomenon, whereas larval mortality remained constant irrespective of larval age. Development was characterized by a minimum development time followed by a transition to the next stage which occurred at a constant rate. All rates were temperature dependent. The minimum development times reported here are much less than those previously reported. Based on these results a mathematical model was used to describe the demography of the free-living stages of H. contortus at various temperatures.     

Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.