The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes

The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 μM roscovitine for 24 h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24 h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 μM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 μM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 μM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.     

The polar lipids of Clostridium psychrophilum, an anaerobic psychrophile

We have examined the polar lipids of Clostridium psychrophilum, a recently characterized psychrophilic Clostridium isolated from an Antarctic microbial mat. Lipids were extracted from cells grown near the optimal growth temperature (+ 5 °C) and at − 5 °C, and analyzed by two-dimensional thin layer chromatography and liquid chromatography coupled with mass spectrometry. The major phospholipids of this species are: cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol. Phosphatidylserine and lyso-phosphatidylethanolamine were found as minor components. The most abundant glycolipids are a monoglycosyldiradylglycerol (MGDRG) and a diglycosyldiradylglycerol (DGDRG). The latter was only seen in cells grown at − 5 °C. An ethanolamine-phosphate derivative of N-acetylglucosaminyldiradylglycerol was seen in cells grown at − 5 °C and an ethanolamine-phosphate derivative of MGDRG was found in cells grown at + 5 °C. All lipids were present in both the all acyl and plasmalogen (alk-1′-enyl acyl) forms with the exception of PS and MGDRG, which were predominantly in the diacyl form. The significance of lipid changes at the two growth temperatures is discussed.     

Partitioning of transpiratory water loss of the desert scorpion, Hadrurus arizonensis (Iuridae)

Terrestrial arthropods lose body water to the environment mainly through transpiration. The aim of this study was to determine the fraction of respiratory losses from total transpiratory water loss in scorpions, as relatively high respiratory losses would indicate a fitness benefit from regulation of gas-exchange rate under stressful desiccating conditions. We measured metabolic rates and water-loss rates of Hadrurus arizonensis (Iuridae) at a range of ecologically-relevant temperatures. Calculation of respiratory water losses was based on increased metabolic and water-loss rates during nocturnal activity (assuming no change in cuticular resistance at a given constant experimental temperature). Respiratory losses accounted for 9.0 ± 1.7% of total transpiratory losses at 25 °C, doubled to 17.9 ± 1.8% at 30 °C and increased to 31.0 ± 2.0% at 35 °C (n = 5, 15 and 15, respectively). Furthermore, the relative importance of respiratory transpiration is likely to be higher at temperatures above 35 °C, which have been recorded even within the burrows of H. arizonensis. Measurements of cuticular lipid melting points do not provide evidence for increased cuticular resistance to water loss at higher temperatures. However, the relatively high fraction of respiratory water losses reported here for H. arizonensis supports the notion of respiratory regulation as an evolved mechanism for conserving scorpion body water stores under stressful conditions.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Phenotypic variation in hatchling Chinese ratsnakes (Zaocys dhumnades) from eggs incubated at constant temperatures

We incubated eggs of the Chinese ratsnake Zaocys dhumnades at four constant temperatures (24, 27, 30 and 30 °C) to examine the effects of incubation temperature on hatching success and hatchling phenotypes. Incubation length increased nonlinearly as temperature decreased, with the mean incubation length being 76.7 d at 24 °C, 57.4 d at 27 °C, 47.3 d at 30 °C, and 44.1 d at 33 °C. Hatching successes were lower at the two extreme temperatures (69% at 24 °C, and 44% at 33 °C) than at the other two moderate temperatures (96% at 27 °C, and 93% at 30 °C). Incubation temperature affected nearly all hatchling traits examined in this study. Incubation of Z. dhumnades eggs at 33 °C resulted in production of smaller hatchlings that characteristically had less-developed carcasses but contained more unutilized yolks. Hatchlings from eggs incubated at 27 and 30 °C did not differ in any examined traits. Taking the rate of embryonic development, hatching success and hatchling phenotypes into account, we conclude that the temperature range optimal for incubation of Z. dhumnades eggs is narrower than the range of 24−33 °C but should be wider than the range of 27−30 °C.     

Bovine oocyte vitrification using the Cryotop method: Effect of cumulus cells and vitrification protocol on survival and subsequent development

The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG + 15% Me₂SO + 0.5 M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG + 11.4% trehalose in three steps or 40% EG + 11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% vs. 91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited significantly higher developmental competence than those vitrified at the MII stage (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage and blastocyst rate among three protocols (86.0% vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than non-vitrified control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage rate or blastocyst development (P > 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes.     

Longevity and temperature response of pollen as affected by elevated growth temperature and carbon dioxide in peanut and grain sorghum

It is important to understand the effects of environmental conditions during plant growth on longevity and temperature response of pollen. Objectives of this study were to determine the influence of growth temperature and/or carbon dioxide (CO₂) concentration on pollen longevity and temperature response of peanut and grain sorghum pollen. Plants were grown at daytime maximum/nighttime minimum temperatures of 32/22, 36/26, 40/30 and 44/34 °C at ambient (350 μmol mol⁻¹) and at elevated (700 μmol mol⁻¹) CO₂ from emergence to maturity. At flowering, pollen longevity was estimated by measuring in vitro pollen germination at different time intervals after anther dehiscence. Temperature response of pollen was measured by germinating pollen on artificial growth medium at temperatures ranging from 12 to 48 °C in incubators at 4 °C intervals. Elevated growth temperature decreased pollen germination percentage in both crop species. Sorghum pollen had shorter longevity than peanut pollen. There was no influence of CO₂ on pollen longevity. Pollen longevity of sorghum at 36/26 °C was about 2 h shorter than at 32/22 °C. There was no effect of growth temperature or CO₂ on cardinal temperatures (T_min, T_opt, and T_max) of pollen in both crop species. The T_min, T_opt, and T_max identified at different growth temperatures and CO₂ levels were similar at 14.9, 30.1, and 45.6 °C, respectively for peanut pollen. The corresponding values for sorghum pollen were 17.2, 29.4, and 41.7 °C. In conclusion, pollen longevity and pollen germination percentage was decreased by growth at elevated temperature, and pollen developed at elevated temperature and/or elevated CO₂ did not have greater temperature tolerance.     

Relatively low upper threshold temperature in lizards from cool habitats

We used the slender forest skink (Scincella modesta) as a model animal to test for the hypothesis that the upper threshold of incubation temperature is relatively low in lizards using shaded (and thus, cool) habitats. Eight gravid females were collected in early May 2005 from a population in Hangzhou, Zhejiang (eastern China). All females laid a single clutch of 7–13 eggs between mid-May and early June. Eggs were incubated at 24, 28 and 30 (±0.2) °C. None of eggs incubated at 30 °C hatched. Eggs incubated at 24 and 28 °C differed in incubation length but not in hatching success. The incubation length at 24 and 28 °C averaged 22.3 and 20.3 days, respectively. Hatchlings from eggs incubated at 24 and 28 °C did not differ in all examined morphological traits, but hatchlings from eggs incubated at 28 °C performed apparently worse in the racetrack than did their counterparts from eggs incubated at 24 °C. The temperature of 28 °C is close to the upper thermal threshold for successful embryonic development in S. modesta. Compared to other oviparous lizards using open (and thus, warm) habitats, the upper thermal threshold and the range of optimal temperatures for embryonic development are both lower in S. modesta. Our study supports the previous conclusion that species living in thermally different habitats may differ in the upper thermal threshold and the range of optimal temperatures for embryonic development.     

Effect of temperature on sporulation of Neozygites floridana isolates from different climates and their virulence against the tomato red spider mite, Tetranychus evansi

The fungal pathogen Neozygites floridana Weiser and Muma has been evaluated as a classical biological candidate for introduction into Africa against the invasive tomato red spider mite Tetranychus evansi Baker and Pritchard. In this study, the effect of temperature on sporulation, germination and virulence of three isolates of N. floridana collected from T. evansi in three climatically distinct regions of Brazil and Argentina was determined. Six constant temperatures of 13 °C, 17 °C, 21 °C, 25 °C, 29 °C and 33 °C were tested for their effect on the ability of the three fungal isolates to sporulate, germinate and kill the mites. Six alternating-temperature regimes of 17-13 °C, 21-13 °C, 29-13 °C, 33-13 °C, 33-23 °C, 33-28 °C under a 12 h photophase were also tested to estimate virulence of the three isolates against T. evansi. The Vipos isolate discharged more conidia than isolates from Recife or Piracicaba at all temperatures and sporulation was strongly temperature dependent. Optimal sporulation rates were observed at 25 °C while optimal germination rates were observed at 25 °C and 29 °C. At 29 °C, the shortest mean survival time of T. evansi (3.16 days, 95% CI of 3.05-3.27) was observed for the isolate from Vipos, while the longest LT₅₀ (3.47 days, 95% CI 3.34-3.59) was observed for the isolate from Piracicaba. Mortality of mites increased as the differences between alternating day and night temperatures increased from 8 °C (21-13 °C), to 10 °C (33-23 °C), to 16 °C (29-13 °C), with smallest and highest temperature differences of 4 °C (17-13 °C) and 20 °C (33-13 °C), both producing low mortalities. The overall results suggest that the Vipos isolate is better adapted to a wider range of temperatures than the other isolates tested.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

Influence of dietary protein levels and water temperature on growth,body composition and nutrient utilization of Cirrhinus mrigala (Hamilton, 1822) fry

The experiment was conducted to determine the protein requirement of Cirrhinus mrigala fry at three different water temperatures, 28, 30 and 32 °C, and to investigate the influence of dietary protein levels and water temperatures on weight gain, body composition, food and nutrient utilization. The 36% protein concentration in the diet yielded highest weight gain at all temperatures from 28 to 32 °C. However, daily protein intake was lower at 28 °C than at 30 and 32 °C. The diet with 36% dietary protein and 32.17% carbohydrate produced the best weight gain and gross conversion efficiency (GCE) at 30 °C. The protein-sparing effect was observed at temperatures 30 and 32 °C in diets 1 and 2, having carbohydrate levels 37.36–44.91%. Highest protein efficiency ratio (PER) was recorded at 28% dietary protein level, while it was lowest at 40% dietary protein level in all three temperatures. Lipid in the body of fishes increased at the end of the experiment in comparison to the initial body lipid. The body protein was found to be related to daily protein intake at 30 and 32 °C. Significant difference (P<0.05) was also noted between temperatures, the hepatosomatic index (HSI) and viscerosomatic index (VSI). The diet with 36% dietary protein and 32.17% carbohydrate was observed to be the best diet for C. mrigala fry at 28–32 °C.     

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO₂-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M_r of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k_cat (3.10 ± 0.04 s⁻¹) and K_m (0.32 ± 0.03 mg ml⁻¹) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k_cat at both 37 and 5 °C, and a low E_a (6.99 kJ mol⁻¹) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD₅₀ was 8.7 μg ml⁻¹ (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO₂-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO₂.     

Activation of transgene expression in skeletal muscle by focused ultrasound

To correlate thermal dose from focused ultrasound (FUS) with gene expression and tissue injury, a temperature plateau strategy was employed. Plasmids encoding luciferase gene under the control of hsp70B promoter were transfected into the right gastrocnemius muscle in a rat via electroporation. One day after transfection, hind limbs were treated with 3.3-MHz focused ultrasound, using one of four different temperature plateaus with spatial-peak time-average focal temperatures (T_SPTA) of 46 °C, 48 °C, 51 °C and 62 °C. The treatment duration at the plateau temperature was varied from 0 to 30 s. Gene expression was analyzed in vivo one day following FUS treatment, and H&E staining was employed to assess tissue injury. Gene activation and tissue damage correlated closely with thermal dose. The highest level of gene activation was induced by FUS at T_SPTA = 51 °C for 20 s, which was found to be statistically equivalent to that produced by water-bath hyperthermia.     

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.     

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80 °C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135 ± 6% and 193 ± 8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50 °C and about 45 °C respectively, while 65 °C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.