Asymmetric reduction of steroidal 20-ketones: Chemical synthesis of corticosteroid derivatives containing and 20α, 21-diol and 17α, 20α, 21-triol side chains

A method is presented for the chemical synthesis of corticosteroid derivatives containing the 20α, 21-diol and 17α, 20α, 21-triol side chains. The ketol side chains of cortisol, corticosterone, 11-deoxycortisol, and 11-deoxycorticosterone were reduced at C-20 with sodium borohydride in a two-phase system consisting of aqueous calcium chloride and an organic phase of chloroform or ethyl acetate. Stereoselectivity of reduction was 92% α-oriented for cortisol and 79% α-oriented for 11-deoxycortisol at ?27°. The 20α-form diminished relative to the 20β-form with increasing temperature. For the 17-deoxy steroids, reduction to the 20α-form was 23% for 11-deoxycorticosterone and 41% for corticosterone. The $\text{20α}\text{20β}$ ratios of 17-deoxy steroids were unchanged between 0° and ?27°. Calcium ions increased the solubility of corticosteroids in the aqueous phase. We propose that calcium ions affect the stereochemistry of reduction by forming a bidentate complex with the side chains of 17α-hydroxy steroids, fixing them in an orientation favorable to 20α-reduction, and by altering the phase partition of the steroids.     

β-sheet to α-helix conversion and thermal stability of β-Galactosidase encapsulated in a nanoporous silica gel

The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (E_β-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (S_β-Gal), E_β-Gal exhibited a higher content of α-helix structure. Heating E_β-Gal up to 75?°C caused a decrease in the content of β-sheet structure and additional augments on E_β-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with S_β-Gal. Steady state fluorescence spectroscopy analysis evidenced an E_β-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for S_β-Gal the denaturation midpoint (Tm) was 59?°C, for E_β-Galit was 48?°C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65?°C and E_β-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of E_β-Gal leading to the loss of thermal stability and enzymatic activity.     

Aggregation and Fragmentation of Phaseolus vulgaris Lectin

The effects of temperature on 6-O-α-maltosyl cyclodextrins (G₂-CDs) production from α- maltosylfluoride (α-G₂F) and cyclodextrins (CDs) by the transfer action of debranching enzymes such as pullulanase and isoamylase were studied.

The amounts of 6-O-α-maltosyl α-cyclodextrin (G₂-α-CD) production by purified pullulanase from Aerobacter aerogenes (A-pullulanase) and from Bacillus acidopullulyticus (B-pullulanase) increased with a rise in temperature, e.g., the amounts at 60°C were about 1.5 times higher than those at 30°C. Initial transfer ratios (G₂-α-CD formed/α-G₂F consumed) of A-pullulanase and B- pullulanase were about 62% and 25% (at 40°C), and about 50% and 15% (at 20°C), respectively. The transfer ratios of both A-pullulanase and B-pullulanase in the reaction using β-CD or γ-CD as acceptor also increased with a rise in temperature.

The transfer ratios were little affected by any change in temperature or any kind of acceptor CDs, in the case of isoamylase, and were about 60%.     

Effect of temperature on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of mealybugs (Hemiptera: Pseudococcidae)

Many species of mealybugs (Hemiptera: Pseudococcidae) are serious pests of economically important crops worldwide. We evaluated the influence of constant temperatures: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34°C on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of various species of mealybugs. No eggs completed their development at 14 and 34°C. Egg-to-adult developmental time significantly decreased from 89.9 days at 16°C to 20.4 days at 32°C. The estimated lower temperature threshold of 10.2°C and 416.6 degree-days were required to complete egg-to-adult development. The mortality of immature stages was maximum at 16 and 32°C and minimum at 28°C. The highest lifetime fecundity was recorded at 28°C and it significantly decreased at 32°C. The longevity of adults was about three times more at 16°C than at 30 and 32°C. The net reproductive rate (R ₀) significantly increased with increased temperatures up to 28°C and significantly decreased at 32°C. The mean generation time (T) significantly decreased with increased temperature up to 30°C, but it significantly increased at 32°C. The intrinsic rate of population increase (r _m ) was highest at 30°C. The finite rate of increase (λ) was significantly greater at 30°C than at other temperatures. These data suggest that S. epius can develop, reproduce and survive in a wide range of temperatures and thus could be regarded a potential biological control agent of mealybugs.     

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT₅₀) – the net photosynthetic rate at an ambient CO₂ of 33 Pa (P_n33), the dependencies of P_n to light and to CO₂ and the room temperature chlorophyll a fluorescence were recorded under optimal conditions. Herbaceous plants acquired freezing tolerances to temperatures between ?10 and ?15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non-senescing leaves P_n33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around ?10°C Brassica survived down to ?24°C, but P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts. Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative P_n33.     

A comparative study of development and demographic parameters of <Emphasis Type="Italic">Tetranychus merganser</Emphasis> and <Emphasis Type="Italic">Tetranychus kanzawai</Emphasis> (Acari: Tetranychidae) at different temperatures

We investigated the effect of temperature on development and demographic parameters such as the intrinsic rate of natural increase (r _m) of the two spider mite species Tetranychus merganser Boudreaux and T. kanzawai Kishida at eleven constant temperatures ranging from 15 to 40°C at intervals of 2.5°C. Both male and female T. merganser and T. kanzawai completed development from egg to adult at temperatures ranging from 15 to 37.5°C. The longest developmental duration of immature stages was found at 15°C and the shortest developmental duration was found at 35°C for both species. Using linear and non-linear developmental rate models, the lower thermal thresholds for egg-to-adult (female and male) and egg-to-egg development were estimated as 12.2–12.3°C for T. merganser and as 10.8°C for T. kanzawai. The highest developmental rates were observed at around 35°C, whereas the upper developmental thresholds were around 40°C for both species. In fact, at 40°C, a few eggs of either species hatched, but no larvae reached the next stage. The r _m-values of T. merganser ranged from 0.072 (15°C) to 0.411 day⁻¹ (35°C), whereas those of T. kanzawai ranged from 0.104 (15°C) to 0.399 (30°C). The r _m-values were higher for T. kanzawai than for T. merganser at temperatures from 15 to 30°C, but not at 35°C (0.348 day⁻¹). Total fecundity of T. merganser was also higher than that of T. kanzawai at 35°C. These results indicate that higher temperatures favor T. merganser more than T. kanzawai.     

Temperature responses of carbon mineralization in conifer forest soils from different regional climates incubated under standard laboratory conditions

¹⁴C‐labelled straw was mixed with soils collected from seven coniferous forests located on a climatic gradient in Western Europe ranging from boreal to Mediterranean conditions. The soils were incubated in the laboratory at 4°, 10°, 16°, 23° and 30 °C with constant moisture over 550 days. The temperature coefficient (Q₁₀) for straw carbon mineralization decreased with increasing incubation temperatures. This was a characteristic of all the soils with a difference of two Q₁₀ units between the 4–10° and the 23? 30 °C temperature ranges. It was also found that the magnitude of the temperature response function was related to the period of soil incubation. Initial temperature responses of microbial communities were different to those shown after a long period of laboratory incubation and may have reflected shifts in microbial species composition in response to changes in the temperature regime. The rapid exhaustion of the labile fractions of the decomposing material at higher temperatures could also lead to underestimation of the temperature sensitivity of soils unless estimated for carbon pools of similar qualities. Finally, the thermal optima for the organic soil horizons (Of and Oh) were lower than 30 °C even after 550 days of incubation. It was concluded that these responses could not be attributed to microbial physiological adaptations, but rather to the rates at which recalcitrant microbial secondary products were formed at higher temperatures. The implication of these variable temperature responses of soil materials is discussed in relation to modelling potential effects of global warming.     

High Temperatures and Net CO2 Uptake, Growth, and Stem Damage for the Hemiepiphytic Cactus Hylocereus undatus1

Hylocereus undatus, which is native to tropical forests experiencing moderate temperatures, would not be expected to tolerate the extremely high temperatures that can be tolerated by cacti native to deserts. Nevertheless, total daily net CO₂ uptake by this hemiepiphytic cactus, which is widely cultivated for its fruits, was optimal at day/night air temperatures of 30/20°C, temperatures that are higher than those optimal for daily net CO₂ uptake by cacti native to arid and semiarid areas. Exposure to 35/25°C for 30 weeks led to lower net CO₂ uptake than at 10 weeks; exposure to 40/30°C led to considerable necrosis visible on the stems at 6 weeks and nearly complete browning of the stems by 19 weeks. Dry mass gain over 31 weeks was greatest for plants at 30/20°C, with root growth being especially noteworthy and root dry mass gain representing an increasing percentage of plant dry mass gain as day/night air temperatures were increased. Viability of chlorenchyma cells, assayed by the uptake of the vital stain neutral red into the central vacuoles, was decreased 50 percent by a one‐hour treatment at 55°C compared with an average of 64°C for 18 species of cacti native to deserts. The lower high‐temperature tolerance for H. undatus reflected its low high‐temperature acclimation of only 1.4°C as growth temperatures were raised by 10°C compared with an average acclimation of 5.3°C for the other 18 species of cacti. Thus, this tropical hemiepiphytic cactus is not adapted to day/night air temperatures above ca 40/30°C, although its net CO₂ uptake is optimal at the relatively high day/night air temperatures of 30/20°C.     

Relationships of Secondary Structure,Microstructure, and Mechanical Properties of Heat-induced Gel of Soy 11S Golobulin

Secondary structure, microstructure, and mechanical properties of heat induced 11S globulin gel were studied to discover their relationships. Heat-induced 11S globulin gel at 80, 90, and 95°C were comprised of 7.2, 16.6, and 23.8% of α-helix; 19.4, 19.5, and 27.5% of β-sheet, and 73.5, 64.5, and 48.6%, of random coil, respectively. This indicated the gel formed at higher temperatures contained more α-helix and less random coil structures. Micrographs of gels heated at 90 and 95°C had a more extended and integral matrix. Gel strength of heat-induced gels at 90 and 95°C were significantly greater than that of 80°C. These data indicated that the increase in α-helix of heat-induced 11S globulin gel have facilitated the establishment of a good gel matrix.     

Linking isoprene with plant thermotolerance, antioxidants and monoterpene emissions

The purpose of the present study was to test the possible plant thermotolerance role of isoprene and to study its relationship with non-enzymatic antioxidants and terpene emissions. The gas exchange, chlorophyll fluorescence, extent of photo- and oxidative stress, leaf damage, mechanisms of photo- and antioxidant protection, and terpene emission were measured in leaves of Quercus ilex seedlings exposed to a ramp of temperatures of 5 °C steps from 25 to 50 °C growing with and without isoprene (10 µL L⁻¹) fumigation. The results showed that isoprene actually conferred thermotolerance (shifted the decrease of net photosynthetic rates from 35 to 45 °C, increased F_v/F_m at 50 °C from 0.38 to 0.65, and decreased the leaf area damaged from 27 to 15%), that it precluded or delayed the enhancement of the antioxidant non-enzymatic defence conferred by α-tocopherol, ascorbic acid or β-carotene consumption in response to increasing temperatures, and that it decreased by approximately 70% the emissions of monoterpenes at the highest temperatures. This suggests that there are inducible mechanisms triggered by the initial stages of thermal damage that up-regulate these antioxidant compounds at high temperatures and that these mechanisms are somehow suppressed in the presence of exogenous isoprene, which seems to already exert an antioxidant-like behaviour.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Physiological flexibility: the key to success and survival for Antarctic fairy shrimps in highly fluctuating extreme environments

1. The anostracan fairy shrimp Branchinecta gaini inhabits one of the most hostile environments on earth, living in pools and lakes in Antarctica. Between January 2002 and January 2003 temperatures in two pools where B. gaini are extremely abundant on Adelaide Island ranged from ?18.6 to ?15.7 °C in winter, to 19.4 to 17.1 °C in summer, whilst air temperatures ranged from ?34 to 6.3 °C. 2. Branchinecta gaini survives winter as cysts, but endures large summer temperature fluctuations as adults. Cysts froze between ?24.4 and ?25.7 °C. In experiments adults survived 0–10 °C with no mortality for 1 week, 25 °C for nearly 48 h with 50% mortality, and at 32 °C complete mortality occurred in <1 h. 3. Oxygen consumption (M?O₂) in B. gaini approximately doubled for every 10 °C temperature rise (Q₁₀ = 2.04) up to 20 °C where it reached a peak. Females had, on average 19% higher M?O₂ than males. Females also had greater metabolic scopes, (maximum–minimum M?O₂ across temperatures was ×3.6 for females, ×3.1 for males). 4. Ventilation frequency increased linearly with temperature, and did not decline at 25 °C, indicating animals were ‘trying’ progressively harder to supply oxygen to tissues, and oxygen deficiency was the probable cause of death. Females had a higher ventilation frequency than males (8.6–17.1% higher) and they also exhibited greater scope to raise ventilation frequency (×2.4 for females versus ×1.5 for males). 5. Great metabolic flexibility allows B. gaini to exploit extreme, highly fluctuating environments, and larger ventilatory and respiratory scopes allow females to survive higher temperatures than males. Because of this flexibility their prospects for coping with physical environmental change are high.     

Estimating reaction norms for predictive population parameters,age specific mortality,and mean longevity in temperature‐dependent cohorts of Culex quinquefasciatus Say (Diptera: Culicidae)

Culex quinquefasciatus plays a major role in the transmission of important parasites and viruses throughout the world. Because temperature is an important limiting factor on growth and longevity of all mosquito species, estimating the reaction norms provides very important basic information for understanding both plasticity and individual variations of the population. In the present study, Cx. quinquefasciatus were maintained at five different constant temperatures (15°, 20°, 23°, 27°, and 30°C) for two subsequent generations. Reproductive population parameters in blood‐fed mated females and longevities of virgin and blood‐fed mated adults reared at different temperatures were compared for the two generations. Longevity increased as temperature decreased within a range of 15° to 30°C for the unmated adults, and 15° to 27°C for the mated and blood‐fed adults. Generation times were as long as 124.07 and 106.76 days for two subsequent generations reared at 15°C, and the highest intrinsic rate of increase (r_m) values were estimated at 0.22 and 0.18, respectively, from the cohorts reared at 27°C. For survival rates, reproductive rates (R₀), and r_m values, 30°C was found to be a critical temperature for this species. These cohorts produced the smallest amount of eggs (R₀= 5.06), r_m values decreasing across generations (from 0.11 to 0.06), and the survival rates from egg to adult were found to be insufficient (16.1 and 10.8%). Additionally, the rate of exponential increase with age and age specific mortalities (b) were calculated for the virgin cohorts. Age specific mortality rates increased as temperature decreased. The increase in mortality rates started to accelerate at 27°C and was more pronounced at 30°C, for both females and males. We estimated the coefficients of variation for the b values in which females have smaller coefficients than those of the males at all temperatures.     

Structural versatility of peptides from Cα,α-dialkylated glycines. I. A conformational energy computation and x-ray diffraction study of homo-peptides from Cα,α-diethylglycine

Conformational energy computations on a derivative and a homo-dipeptide of C^α,α-diethylglycine were performed. In both cases the N- and C-terminal groups are blocked as acetamido and methylamido moieties, respectively. It was found that the C^α,α-diethylglycine residues are conformationally restricted and that the minimum energy conformation corresponds to the fully extended C₅ structure when the N? C^α? C′ bond angle is smaller than 108° (as experimentally observed). The results of the theoretical analysis are in agreement with the crystal-state structural propensity of the complete series of N-trifluoroacetylated homo-peptides of this C^α,α-dialkylated residue from monomer to pentamer, determined by x-ray diffraction and also described in this work. Interestingly, for the first time, a crystallographically planar peptide backbone was observed (in the protected tripeptide). A comparison with peptides of C^α,α-dimethylglycine, C^α-methyl, C^α-ethylglycine, and C^α,α-di-n-propylglycine indicates that the fully extended conformation becomes more stable than the helical structures when both amino acid side-chain C^β atoms are substituted.     

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6?mM isopropyl β-D-1-thiogalactopyranoside for 9?h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ～10?mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu²⁺, Mn²⁺, Zn²⁺, and Al³⁺ ions. Furthermore, the K_M and k_cat values for purified Aaβ-agarase were ～0.02?mM and ～45/s, respectively. These kinetic values were up to approximately 15–100-fold lower than the K_M values reported for other agarases and approximately 7–30-fold higher than the k_cat/K_M values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.     

Supercooling ability and cold hardiness of the pollen beetle Meligethes aeneus

Supercooling point (SCP) and cold‐hardiness of the pollen beetle Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae) were investigated. Mature eggs from the oviduct were supercooled on average to ?28.0 °C and from oilseed rape buds to ?24.4 °C; first instars were supercooled to ?21.0 °C and second instars to ?16.8 °C. Despite their high supercooling ability, none of the eggs survived 24 h exposure to ?2.5 °C. The supercooling ability of adults varied significantly among feeding and non‐feeding beetles: high SCPs prevailed during the whole warm period, being about ?12 °C; low values of SCP of ?20 °C dominated in non‐feeding beetles. In spring and autumn, beetles displayed the same acclimation efficiency: after 1 week of exposure at 2.0 °C with no access to food their SCPs were depressed equally by about 3 °C. Meligethes aeneus beetles have a different response to low temperatures depending on the season. The lowest tolerance was found in reproductively active beetles after emergence from overwintering sites; the time needed to kill 50% of individuals (Ltime₅₀) was 56.2 h at ?7 °C and the lower lethal temperature needed to kill 50% (Ltemp₅₀) after 24 h exposure was ?8.6 °C. Cold hardiness increased from midsummer to midwinter; Ltime₅₀ was 80 h in August, 182.8 h in September, and 418.1 h in January. Lethal temperature after 24 h exposure was ?9.1 °C in August and ?9.8 °C in September. In February, after diapause, the beetles started to loose their cold tolerance, and Ltemp₅₀ was slightly increased to ?9.5 °C. Hibernating beetles tolerated long exposure at ?7 °C well, but mortality was high after short exposure if the temperature dropped below ?9 °C for 24 h. Despite the season, the beetles died at temperatures well above their mean SCP; consequently, SCP is not a suitable index for cold hardiness of M. aeneus.     

Light and temperature dependence of the emission of cyclic and acyclic monoterpenes from holm oak (Quercus ilex L.) leaves

In a laboratory study, we investigated the monoterpene emissions from Quercus ilex, an evergreen sclerophyllous Mediterranean oak species whose emissions are light dependent. We examined the light and temperature responses of individual monoterpenes emitted from leaves under various conditions, the effect of heat stress on emissions, and the emission-onset during leaf development. Emission rate increased 10-fold during leaf growth, with slight changes in the composition. At 30 °C and saturating light, the monoterpene emission rate from mature leaves averaged 4·1 nmol m^–2 s^–1, of which α-pinene, sabinene and β-pinene accounted for 85%. The light dependence of emission was similar for all monoterpenes: it resembled the light saturation curve of CO₂ assimilation, although monoterpene emission continued in the dark. Temperature dependence differed among emitted compounds: most of them exhibited an exponential increase up to 35 °C, a maximum at 42 °C, and a slight decline at higher temperatures. However, the two acyclic isomers cis-β-ocimene and trans-β-ocimene were hardly detected below 35 °C, but their emission rates increased above this temperature as the emission rates of other compounds fell, so that total emission of monoterpenes exponentially increased from 5 to 45 °C. The ratio between ocimene isomers and other compounds increased with both absolute temperature and time of heat exposure. The light dependence of emission was insensitive to the temperature at which it was measured, and vice versa the temperature dependence was insensitive to the light regime. The results demonstrated that none of the models currently applied to simulate isoprene or monoterpene emissions correctly predicts the short-term effects of light and temperature on Q. ilex emissions. The percentage of fixed carbon lost immediately as monoterpenes ranged between 0·1 and 6·0% depending on temperature, but rose up to 20% when leaves were continuously exposed to temperatures between 40 and 45 °C.