Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

Thermal adaptability of large white pigs in the tropical environment

Twenty-four Large White weaners-twelve males and twelve females, were randomly divided into three groups of eight (four males and four females per two separate pens) and were assigned to three groups of two pens each for the males and the females weaners.One group of two pens was without wallow facility (control), one other group was provided with wallows (wet) and the third group was in air-conditioned room (cold). Twice a day, the respiratory rate and the rectal temperatures were measured early in the mornings at 8.00–9.00 hrs (A.M.) and late in the early evenings at 16.00–18.00 hrs.The mean respiratory frequency (A.M) ranged from 7 to 9; 6–12 and 8–13 breaths per minute for the cold, wet and control respectively while the mean respiratory frequency (P.M.) ranged from 6 to 9, 10 to 17 and 13 to 19 breaths per minute for the cold, wet and control respectively.The mean rectal temperatures (A.M.) ranged very slightly from 38.54° to 39.12°C; 38.50° to 39.05°C and 38.61°C to 39°C for the cold, wet as control respectively while the mean rectal temperatures (P.M.) ranged from 39.00° to 39.22°C; 38.97° to 39.29°C and 39.28° to 39.55°C for the cold, wet and control respectively.The animals were maintained for another seven to ten weeks and were slaughtered. The slaughter characteristics did not indicate an appreciable thermal stress except for the reproductive organs which showed weight increase indicating reduced efficiency of the thermally stressed animals as is the case in the tropical environment.     

Frozen storage stimulates the formation of embryo-like structures and elongating shoots in explants from mature Larix decidua and L. x eurolepsis trees

Branches were collected from a Larix decidua and a L. x eurolepis tree, both 36 years old, in mid-winter. These branches were placed in freezers at –5°C, –10°C, and –18°C. Primordial shoot explants were excised after 2 to 9 months of frozen storage. The material remained viable at all three temperatures for at least 9 months. The frozen storage stimulated formation of embryo-like structures that were capable of forming shoots with elongated stems.Abbreviations 1/2 LM half strength Litvay medium     

Plasminogen activator: Morphological evidence of binding, internalization and delivery to lysosomes in 3T3 mouse fibroblasts

Summary Using a direct conjugate of urokinase and ferritin, the binding has been followed at the plasma membrane and the internalization of urokinase into BALB/C-3T3 fibroblasts, cultured in plasminogen-free conditions. At 0° C, the conjugate was observed bound on both coated and uncoated cell surface regions as singlets, and small and large clusters. No binding was observed in the presence of excess native urokinase. The binding was impaired by preincubation of the conjugate with a competitive inhibitor of the catalytic site, suggesting an interaction between the receptor and the catalytic site of the enzyme.Within 1 min at 37° C, urokinase clustered on coated regions of the plasma membrane. At 5 min after warming, ferritin was found on deeply indented coated pits and in both coated and uncoated vesicles close to the cell surface. By 10 min at 37° C, ferritin particles were present in uncoated endosomes and in multivesicular bodies in the Golgi area. Within 10 min, the receptors on the surface strongly decreased. New receptors were observed on the membrane after 20 min at 37° C. At this time, ferritin was observed both in endosomes or multivesicular bodies and in vesicles close to the plasma membrane.     

Effect of incubation temperature on green sturgeon embryos, <Emphasis Type="Italic">Acipenser medirostris</Emphasis>

Regulation of river flow and the amount of winter rainfall are the major factors affecting the water temperature of the spawning grounds, for green sturgeon in the Klamath River. During the primary spawning period of green sturgeon, mid-April to June, the water temperature may vary from 8 to 21°C. To estimate the potential implications of this modified thermal regime, we examined the survival and development in three progeny groups of green sturgeon embryos from zygote to hatch, at constant incubation temperatures (11–26°C). Temperatures 23–26°C affected cleavage and gastrulation and all died before hatch. Temperatures 17.5–22°C were suboptimal as an increasing number of embryos developed abnormally and hatching success decreased at 20.5–22°C, although the tolerance to these temperatures varied between progenies. The lower temperature limit was not evident from this study, although hatching rate decreased at 11°C and hatched embryos were shorter, compared to 14°C. The mean total length of hatched embryos decreased with increasing temperature, although their wet and dry weight remained relatively constant. We concluded that temperatures 17–18°C may be the upper limit of the thermal optima for green sturgeon embryos, and that the river thermal regime during dry years may affect green sturgeon reproduction.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060–5010 mg/kg dry wt of culture) at ambient temperature (16–29°C) in daylight than at ambient temperature (18–23 °C) in darkness or at controlled temperatures of 11 °C, 20 °C or 25 °C in darkness. Yields at 25 °C were low. Incubation at 11 °C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 °C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 °C (2460-21 360 mg/kg), 1 strain at 11 °C (6570 mg/kg). Yields at a temperature oscillating daily from 10–20 °C were less than at 15 °C. One of the 5 strains produced appreciable amounts of a-zearalenol (1645 mg/kg at 20°C) and 2 of nivalenol (340 and 499 mg/kg at 20 °C).     

Growth,growth efficiency,and assimilation efficiency of the Tahoe sucker in cyclic and constant temperature

Synopsis Tahoe sucker, Catostomus tahoensis, were fed at three ration levels (starvation, 50% of repletion, and repletion) at three constant and cyclic temperature regimes (4–12°, 8°, 8–18°, 13°, and 13°–23°, 18° C) to examine growth rate and gross growth efficiencies. Growth rates increased with increasing temperature and ration level. Growth rates were not different between cyclic temperatures and the constant temperature equivalent to the mean of the cycle. Growth efficiencies were similar for cyclic and constant temperature regimes. Maintenance rations increased from 0.9% of the initial wet weight per day at low temperatures to 2.0 and 1.7% at intermediate and high temperatures, respectively. Assimilation efficiencies (not measured at low temperatures) did not differ between constant and cyclic temperatures. Tahoe sucker growth rates and assimilation efficiencies may not be enhanced in small streams because of this species' inability to mediate temperature cycles through behavioral thermoregulation.     

Factors affecting oviposition and fecundity in the grain miteAcarus siro L. (Acarina:Acaridae), especially temperature and relative humidity

Oviposition and fecundity in the grain miteAcarus siro were studied at 5–30°C and 62.5–90% RH. At and above 20°C, 80% RH, mating and oviposition occurred soon after emergence, but at lower temperatures and humidities egg laying was progressively delayed from one to several days. Females needed to mate repeatedly in order to achieve maximum egg production, optimum conditions for which were 15°C, 90% RH, where total output per female averaged 435 with a maximum of 858. Oviposition rates were highest at higher temperatures, the mean daily rate at 20 and 25°C, 90% RH, rising to maximum levels of 28/29 eggs per female per day on day six.Oviposition followed clearly defined patterns, favourable conditions producing rapid increases in the mean daily oviposition rate to high peak levels reached at an early stage in the oviposition period. Less favourable conditions resulted in reduced outputs and lower, more uniform rates of egg laying. The mean oviposition period, varying with humidity, fell from 72–122 days at 5°C to 9–13 days at 30°C and the mean incubation period from 42–70 days at 5°C to 3–4 days at 30°C. Egg viability increased with increasing humidity but was little affected by temperature and unaffected by age of the female at time of oviposition.Males tended to live longer than females at most conditions; longevity—depending on humidity—averaging 13–15 days at 30°C and 129–175 days at 5°C. Adult life for females averaged 12–19 days at 30°C and 88–169 days at 5°C. An index of suitability, calculated from egg number, viability and duration of the egg stage and oviposition period, indicated that the most favourable conditions for oviposition and hatching were 20–25°C and 80–90% RH.     

The impact of temperature on activity and consumption rate of moth eggs by Blattisocius tarsalis (Acari: Ascidae)

The activity of adult female Blattisocius tarsalis was investigated at temperatures of 7–13°C. At 7°C only 33% of the mites walked and these individuals all walked less than 1.5 cm min–1. All mites walked at 9°C and at 12°C all were highly active, walking more than 1.5 cm min–1. Daily food consumption rates of adult female B. tarsalis offered young eggs of Ephestia kuehniella as food were investigated at temperatures of 9–27°C. Food consumption below 13°C was very low. At temperatures of 13–19°C the mean number of fully ingested eggs had a level of between 0.5 and 1.0 egg in 24 h. At temperatures of 21–27°C the mean number of fully ingested eggs was above 1.0 egg in 24 h. The mean number of destroyed eggs per female at temperatures above 13°C ranged from three to five eggs per day. © Rapid Science Ltd. 1998     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Microhabitat segregation and physiological differences in co-occurring tiger beetle species,Cicindela oregona and Cicindela tranquebarica

Summary The daily movements of two co-occurring tiger beetle species were monitored in conjunction with changes in microclimate along streams in Northeast Arizona. Cicindela oregona and C. tranquebarica temporarily segregated across areas of beach exhibiting different microclimates. C. oregona progressively moved from the dry upper beach to the wet stream edge as beach temperatures increased and humidity decreased. The actively foraged throughout the day in this moist habitat at air temperatures between 25 and 38°C. C. tranquebarica remained on the dry, upper portions of the beach and shuttled between sun and shade at air temperatures above 35°C. Only when stream edge temperatures exceeded 30°C was tranquebarica found in this subhabitat. Both species exhibited physiological tolerances in the laboratory that were consistent with their microhabitat preferences in the field. Although both species had similar high lethal temperatures (47–48°C) in saturated air, oregona died at lower temperatures (39–43°C) than tranquebarica (46–47°C) under dry (0% RH) conditions. C. oregona was considerably more active than tranquebarica at body temperatures below 30°C and exhibited higher levels of active metabolism between 25 and 40°C. In addition, C. tranquebarica exhibited significantly lower water loss rates than oregona at 30, 35 and 40°C.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase

The CO₂ evolution of intact potato tubers (Solanum tuberosum, L., var. Bintje) was analyzed during a 10-day period of their warm (25 ± 2°C) or cold (5 ± 1°C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO₂ evolution rates were analyzed at 20°C, to reflect their possible capacities. The 20°C CO₂ production declined from 13 to 8 mg kg^–1 h^–1 after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg^–1 h^–1. In contrast, 20°C CO₂ evolution did not change after the first day of cold storage, increased up to 14.5 mg kg^–1 h^–1 after 2 days, and decreased to about 12 mg kg^–1 h^–1 after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O₂ min^–1 mg^–1 attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.     

Development of sweating ability in winter- and summer-born Friesian calves aged 1 to 6 weeks

Sweating rate, rectal and skin temperatures and respiration rate were measured at weekly intervals from 7 days of age (for 4 weeks in Experiment 1; 6 weeks in Experiment 2) in winter- and summer-born Friesian calves exposed to a temperature of 39°C dry bulb and 32°C wet bulb in a climate chamber. Four calves were studied in each season in both experiments. In Experiment 1, ambient temperatures were from 3° to 9°C higher in early summer than in late winter. During each 39°C exposure, sweating rate increased from basal levels of 40–90 to plateau levels of 120–300 g/m² per h after 90–120 min. The increase in sweating rate with age was most pronounced in winter-born calves, but summer-born calves had higher values at 1 week of age (167±52.4 vs 94.4±30.1 g/m² per h). Seasonal differences in ambient temperature were greater in Experiment 2 (11° to 17°C). In this case summer-born calves had higher sweating rates at each age (plateau values of 220–320 g/m² per h), and showed a more rapid increase in sweating rate during each 39°C exposure than winter-born calves (plateau values of 100–250 g/m² per h). The results demonstrate major changes in sweating competence during the first 4–6 weeks of life in Friesian calves, a quite pronounced effect of season (ambient temperature) on the levels of sweating achieved, and indicate that low sweating rates in newborn calves are a contributing factor in deaths due to hyperthermia in semi-arid grazing areas.     

Effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridomas during batch culture

In order to investigate the effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridoma cells, cells in different growth stages during a batch culture were fixed and stored at 4 and -20 °C, respectively. Flow cytometric analysis indicates that both fixation temperatures can be used in monitoring the changes in intracellular antibody content of the cells during a batch culture. However, it is better to fix and store the cells at -20 °C than 4 °C with regard to preservation of intracellular antibody and storage stability.     

The effect of LAB 173711 and ethephon on time of flowering and cold hardiness of peach flower buds

Peach flowers are often killed during bloom by spring frosts. LAB 173711, a compound with abscisic (ABA)-like activity, and ethephon delayed flowering in peach trees. In greenhouse experiments, LAB 173711, at concentrations of 10^–3–10^–2 M, was most effective in delaying bloom when applied after a 5°C cold storage period, rather than before the dormancy breaking treatment. In contrast, ethephon delayed bloom most effectively when applied before 5°C cold storage; ethephon caused flower bud abscission when treatments were made after the chilling requirement had been satisfied. In field experiments, ethephon delayed flowering by 6–7 days, which reduced bud injury after a spring frost during bloom. No flower bud injury was found on ethephon-treated trees after temperatures of –4.3°C; whereas without ethephon 25% of the flower buds were frost damaged. LAB 173711 delayed the time to 50% bloom by 2–3 days. However, this was not long enough to avoid low-temperature injury to the flower buds.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Lipid formation in the oleaginous mould Entomophthora exitalis grown in continuous culture: effects of growth rate,temperature and dissolved oxygen tension on polyunsaturated fatty acids

Summary The oleaginous fungus Entomophthora exitalis was grown in continuous culture at a constant dilution rate (0.04 h^–1) and over a range of temperatures (20–30° C). As the growth temperature was decreased from 30 to 20° C the percentage of polyunsaturated fatty acids (PUFA) increased proportionally from 18 to 27% (w/w) of the total fatty acids. The increase in unsaturation was as a result of an increased proportion of n-6 PUFA (particularly arachidonic acid) in the phospholipid and sphingo- plus glycolipid fractions. The triacylglycerol fraction of lipids displayed a negligible change. The proportion of phospholipids within the extracted lipid increased between 26 and 20° C without any change in the lipid content of the fungus. Although the changes in lipid unsaturation correlated, at first inspection, to the culture dissolved O₂ tension (DOT), growth of the fungus at a constant dilution rate and temperature (22° C) over a range of DOT values failed to influence lipid unsaturation. Thus temperature is the principal regulation factor of the degree of unsaturation in the lipids of this organism. Offprint requests to: C. Ratledge     

Purification and Stability during Storage of Phoshoenolpyruvate Carboxylase from Leaves of Amaranthus hypochondriacus,a NAD-ME Type C4 Plant

A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg^–1(protein) s^–1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40–60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, –20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen.