Enzymatic and Non-Enzymatic Esterification of long Polyunsaturated Fatty Acids and Lysophosphatidylcholine in Isooctane

Phosphatidylcholine containing large amounts of long polyunsaturated fatty acid, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), was synthesized in isooctane. Immobilized phospholipase A₂ was used as a catalyst. A parallel non-enzymatic esterification reaction was investigated in separate experiments.

The concentrations of lyso-phosphatidylcholine, polyunsaturated fatty acids, water and the enzyme were varied over wide ranges as were the temperature and the reaction time. The type of enzyme, carrier and immobilization procedure were held constant.

The yield of phosphatidylcholine was relatively high (about 21%) when the concentration of polyunsaturated fatty acids was high (300 mg/g of reaction mixture) and the water content was low (below 30% of the dry immobilized enzyme). The highest yield of phosphatidylcholine was found at 80 hours and 75°C. However, at this temperature an extensive non-enzymatic reaction between polyunsaturated fatty acids and lyso-phosphatidylcholine occurred. At 80°C the polyunsaturated fatty acids were partly oxidized. Therefore, a temperature of 45°C to 65°C is probably the optimum temperature for the reaction.     

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The K_m values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.     

Temperature dependence of delayed light emission in spinach chloroplasts

1. Delayed light of chlorophyll emitted at 0.1–3.9 ms after cessation of repetitive flash light was studied at temperatures between +40 and −196 °C in isolated spinach chloroplasts.

2. Induction kinetics of delayed light varied depending on temperature. It was found to be composed of two phases; one was an initial rapid rise followed by a rather fast decline to a low steady state level (fast phase), and the other was a slow increase after the initial rapid rise to the maximum followed by an insignificant slow decrease to a high steady state level (slow phase). The fast phase existed between −175 and 40 °C with the maximum at −40 °C, while the slow phase, between 0 and 40 °C with the maximum at 25 °C.

3. The intensity of delayed light at −175 °C was found to be less than one fiftieth that at 0 °C, and no delayed light emission was observed at −196 °C within experimental accuracy. This is in contrast to the results reported by Tollin, G., Fujimori, E. and Calvin, M. ((1958) Proc. Natl. Acad. Sci. U.S. 44, 1035–1047) in which the intensity of delayed light measured at −170 °C was about a half that at 0 °C.

4. The induction of delayed light measured at −96 °C was found to be significantly suppressed by the preillumination at −196 °C. This finding suggests that the primary photochemical event still survives at −196 °C without emission of delayed light.

5. Decay kinetics of delayed light after the flash excitation revealed the presence of at least two decay components. A slow decay component with a half decay time of several tens of milliseconds was observed at temperatures higher than 0 °C. A fast decay component with a half decay time of about 0.2 ms was observed at temperatures between −120 and 25 °C. The decay rate of this component was slightly retarded on cooling.

6. The System II particles derived from spinach chloroplasts with digitonin treatment showed a temperature dependence of delayed light similar to that of the chloroplasts. System I particles, on the other hand, scarcely emitted the delayed light at any temperature between 40 and −196 °C.     

Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima.

The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80%. Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C.     

Thermal denaturation of the erythrocyte cytoskeleton alters the morphological changes associated with osmotic swelling

1. 1. The shape changes during osmotic swelling of human erythrocytes in a hypotonic medium at room temperature, at 45°C and at the denaturation temperature (49.5°C) of the cytoskeletal protein, spectrin, have been monitored by video microscopy.

2. 2. At room temperature the great majority of cells (which were discoid prior to injection of hypotonic medium) swelled to a spherical shape through an intermediate ellipsoidal form.

3. 3.At 49.5°C (where cells had cupped shapes prior to injection) the transition to the spherical form often involved a stomatocytic rather than ellipsoidal intermediate shape.

4. 4. The cupped form of the cells prior to injection did not account for the high incidence of cells swelling through a stomatocytic intermediate shape at 49.5°C.

5. 5. A theoretical treatment by Svetina and Zeks (1983) attributes the nature of the osmotic swelling transition shape to the difference in area between the outer and inner faces of the membrane. Our results would be consistent with the theoretical predictions if it is assumed that an increase in the area of the inner face of the membrane follows thermal denaturation of the cytoskeleton of cells in hypotonic medium.

Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of the European pond turtle, sexual differentiation of gonads is temperature-dependent. Production of oestrogens appears to play a key role in this phenomenon. Gonadal aromatase activity was measured in embryos incubated at 25°C (masculinizing temperature) and at 30°C (feminizing temperature). At the beginning of the thermosensitive period, the aromatase activity was low at both temperatures but was somewhat higher at 30 than at 25°C. Afterwards, it remained low in differentiating testes at 25°C, whereas it increased in differentiating ovaries at 30°C to form a marked peak when germ cells underwent meiotic prophase. Eggs were shifted either from 25 to 30°C (highly feminizing) or from 30 to 35°C for 6 days at different stages of embryonic development. The 25–35°C shifts performed during the thermosensitive period strongly increased the aromatase activity but were ineffective after this period. The 30–35°C shifts increased the aromatase activity at all stages. Altogether, results indicate that, in differentiating gonads of turtle embryos, temperature acts on the regulation of synthesis (and therefore activity) of cytochrome P-450 aromatase (P-450-aro). The expression of the P-450-aro gene itself could be temperature-dependent. However, temperature could also act upon the expression of another gene involved in P-450-aro regulation.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Thermal denaturation and degradation of schizophyllan

Size exclusion chromatography and low-angle laser light scattering have been used for studying the evolution of schizophyllan polysaccharide during a thermal treatment (t > 100°C) in aerated solution. Thermal denaturation of the native triple helices into single chains is initiated above 135°C and is complete in 10 min at 160°C. Both conformations can coexist in the 130–140°C temperature range. In the presence of oxygen, both forms of the biopolymer undergo severe thermal degradation. The rate of degradation was found to be independent of chain length and conformation. An activation energy of 104 kJ mol⁻¹ was determined. The reaction was base-catalyzed. Analysis of chromatographic patterns indicate that the degradation probably occurs through an ‘all-or-none’ process.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts

1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).

2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (E_a) for the reaction was 48.1 kJ/mol.

3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the E_a for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The E_a was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the E_a decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.

4. 4. Temperature-induced changes in E_a were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.

5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in E_a in the absence of an uncoupler.

6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.

Germination, Radial Growth, and Sporulation of Beauveria bassiana and Metarhizium anisopliae Isolates and Their Virulence to Chilo partellus (Lepidoptera: Pyralidae) at Different Temperatures

The effect of temperature regimes on conidia germination, radial growth and virulence of Beauveriabassiana and Metarhizium anisopliae against the spotted stalk borer Chilo partellus was investigated with Ethiopian isolates. Conidia germination, radial growth and sporulation of all isolates were retarded at 15 and 35°C. A suitable temperature range for the isolates was between 20 to 30°C. Conidia germination was less tolerant of low temperature (15°C) than radial growth. Radial growth and sporulation reacted differently to temperature. At both 25 and 30°C, all isolates induced 100% mortality to C. partellus larvae in six days. The LT₅₀ decreased for the isolates with increasing temperature.     

The back reaction in the primary electron transfer couple of photosystem II of photosynthesis

Changes of C-550, cytochrome b₅₅₉ and fluorescence yield induced in chloroplasts by single saturating flashes were studied at low temperature. A single saturating flash at −196°C was quite ineffective in reducing C-550, oxidizing cytochrome b₅₅₉ or increasing the fluorescence yield, presumably because most of the charge separation induced by the flash was dissipated by a direct back reaction in the primary electron transfer couple. The back reaction, which competes with the dark reduction of the oxidized primary electron donor by a secondary electron donor, becomes increasingly important as the temperature is lowered because of the temperature coefficient of the reaction with the secondary donor. The effect of the back reaction is to lower the quantum yield for the production of stable photochemical products by steady irradiation. Assuming a quantum yield of unity for the photoreduction of C-550 at room temperature, the quantum yield for the reaction is about 0.40 at −100°C and 0.27 at −196°C.     

Temperature-dependent Development of Four Egg Parasitoid Trichogramma Species (Hymenoptera: Trichogrammatidae)

Trichogramma species have been successfully utilized for biocontrol of several lepidopteran pests worldwide. The development, survival and progeny production of two Kenyan species' Trichogramma bournieri Pintureau & Babault and Trichogramma sp. nr. mwanzai Schulten & Feijen (collected from Kenya), Trichogramma evanescens Westwood (Germany) and Trichogramma chilonis Ishii (India) was studied at four constant temperatures (13, 18, 25 and 34°C) with the 011 aim of assessing the relative biotic potential of the two native species for 011 biocontrol of Helicoverpa armigera and Plutella xylostella in Kenya. The study was conducted at the Institute 011 for Biological Control (BBA), Darmstadt, Germany. The Trichogramma species tested showed variations 011 in fertility, developmental time, percent emergence, progeny production and sex ratio 011 at the four temperature regimes. Fertility decreased as temperature increased from 25 to 34°C. 011 T. chilonis and T. evanescens completed development at all temperatures tested, but T. 011 bournieri and T. sp. nr. mwanzai failed to complete development at 13°C. The developmental 011 period for all the species decreased as the temperature increased. The duration of development from 011 oviposition to adult emergence varied from 8 days to 12 weeks shorter at 34°C than at 011 13°C for T. chilonis and T. evanescens . For the various temperatures tested, a linear model was 011 satisfactory for egg to adult development at P = 0.05 for T. chilonis and T. evanescens . The 011 lower temperature thresholds for development and duration in degree-days were 8.83°C and 188 for 011 T. chilonis and 9.23°C and 192 for T. evanescens , respectively. For all temperatures tested, 011 T. sp. nr. mwanzai had the highest preimaginal survivorship. Adult emergence was lower at 13°C and 34°C than at 011 18 and 25°C. The highest fertility (mean ±SE) (50.37 ±2.32 adult 011 female -1 ) and progeny production (44.03 ±2.02 adult female -1 ) was recorded at 25°C for 011 T. evanescens . Sex ratio was biased towards female at all temperatures in T. bournieri and T. chilonis . 011 At all temperatures tested, T . sp. nr. mwanzai produced more males than females. For all species tested, 011 favourable parasitism was between 18 and 25°C. The results from this study will be useful for mass rearing purposes as well as for future field release programmes.     

Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature

1. 1. Myosin and its subfragment-1 (Sl) from carp acclimated to 10°C showed higher actin-activated Mg²⁺-ATPase activity and lower thermostability than their counterparts from carp acclimated to 30°C. Accordingly, filament velocity for the 10°C-acclimated carp myosin was higher at any measuring temperatures from 3 to 23°C than that for the 30°C-acclimated carp myosin.

2. 2. Three types of cDNA clones encoding myosin heavy chains were isolated from thermally acclimated carp. The 10 and 30°C types were predominating in carp acclimated to 10 and 30°C, respectively, whereas the intermediate type was found as a minor component in the 10°C-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 30°C types.

3. 3. The three types of myosin rod all showed a typical coiled-coil structure of -helices. DSC scans demonstrated that myosin rod prepared from carp acclimated to 10°C had a lower thermostability than that from carp acclimated to 30°C, showing that low thermostability in cold-acclimated carp myosin prevails over the entire molecule.

4. 4. cDNA clones encoding myosin alkali light chains were isolated from thermally acclimated carp. Northern blot analysis showed that the ratios of LC3/LC1 mRNAs were significantly higher (3.92) in the 30°C- than 10°C-acclimated (3.10) carp.

     

Optimization of storage conditions for adequate shelf-life of 'pesta' formulation of Fusarium oxysporum 'foxy 2', a potential mycoherbicide for Striga: Effects of temperature, granule size and water activity

An adequate shelf-life of mycoherbicidial products is an essential requirement for their acceptance and commercialization. Therefore, attempts were made to study the effects of temperature, granule size, and water activity (R.H./100) on the viability of the encapsulated propagules of Fusarium oxysporum 'Foxy 2' in 'Pesta' granules during storage. 'Pesta' granules were made with different inocula of Foxy 2, including: microconidia; mixture of mycelia and microconidia; fresh and dried chlamydospore-rich biomass. Two sizes of each granular preparation (0.5-2 and 0.25-0.5 mm) were stored in the refrigerator at 4°C as well as at room temperature (21±3°C) for 1 year. Additional samples were also stored at water activities (a_w) of 0.12 and 0.41 at 25°C. Regardless of the type of formulated propagules and the granule size, all samples stored at 4°C maintained a significantly higher viability compared to those kept under room temperature. At 4°C, the 'Pesta' preparations with the larger granule size (0.5-2 mm) maintained more viable propagules than those with the smaller one (0.25-0.5 mm) in case of microconidia, mycelia plus microconidia and fresh chlamydospore inoculum after 1 year of storage. Granule size did not affect the viability of the dried chlamydospores. At 25°C, shelf-life of all 'Pesta' granules was significantly prolonged when stored at a low water activity of 0.12 compared to the storage at 0.41 a_w. The results of the combined effect of water activity and temperature also revealed clearly that all formulated propagules in 'Pesta' granules retained a significantly higher viability when stored at 0.62 a_w and 4°C than at 0.12 a_w and 25°C, indicating the most pronounced effect of storage temperature.     

Geographic variation in field body temperature of sceloporus lizards

1. Using data from the literature, I assessed how broad climatic patterns affected field body temperatures (T_b’s) of lizards in the genus Sceloporus.

2. Sceloporus at temperate latitudes had mean T_b’s of 35°C throughout their elevational range. This pattern is associated with “tropical” temperatures that extend into high north latitudes during the summer and the relatively low elevations occupied by the lizards.

3. At tropical latitudes, mean T_b declined from 35°C at low elevations to 31°C at high elevations. This pattern is associated with low seasonal variation in temperature at tropical latitudes and the relatively high elevations occupied by the lizards.     

Structure and thermal stability of the extracellular fragment of human transferrin receptor at extracellular and endosomal pH

Fourier transform infrared spectroscopy has been used to study the solution structure and thermal stability of the extracellular fragment of human transferrin receptor (tfRt) at extracellular and endosomal pH. At extracellular pH tfRt is composed of 56% -helix, 19% β-sheet and 14% turns. Upon acidification to endosomal pH the -helical content of the protein is reduced and the β-sheet content increased by nearly 10%. At extracellular pH, the midpoint temperature of thermal denaturation (T_m) for the loss of secondary and tertiary structure, and the formation of aggregated structures, is 71°C. At endosomal pH this temperature is reduced by ≈ 15°C. The apparent entropies of thermal denaturation indicate that the native structure of tfRt at endosomal pH is far more flexible than at extracellular pH.     

The temperature dependence of the deformation of bone

The deformation characteristics of compact bone have been measured as a function of temperature in the range from −58 to 90°C. At temperatures below 25°C elastic, anelastic and plastic contributions to the strain could be distinguished. The Young's modulus increased as the temperature was reduced, while the nonelastic component was approximately independent of temperature. An interpretation of these results is made in terms of an idealised hydroxyapatite-collagen composite. At temperatures higher than 50°C the deformation was compounded by non-equilibrium recovery and an irreversible change in the structure of bone.