Reorganization of dynamic self-assemblies of cellulose diacetate in solution: dynamical critical-like fluctuations in the lower critical solution temperature system

Dynamics of cellulose diacetate (CDA, the total degree of substitution (TDS) = 2.44) in dimethylacetamide (DMAc) in dilute solution was investigated at 2, 10, 20, 30, 40, 49.7, and 61.5 degrees C through dynamic light scattering in the quiescent state. The following three facts were made clear. First, CDA existed in three types of structures in the polar solvent, DMAc; one is a single CDA chain, and the others are dynamic structures, or self-assemblies, which were formed temporarily and locally by the solvent-mediated hydrogen bonding between the intermolecular C-6 position hydroxyls of the anhydroglucose units in the CDA backbone. Second, CDA showed a nature of low-temperature solubility in DMAc, that is, CDA is expected to dissolve molecularly below -12 degrees C but to take a phase separation above 65 degrees C, where two structures such as collapses of a single CDA chain and an aggregate appear. Third, a reorganization in the dynamic structures was detected at the temperature T = 33.8 degrees C. At this temperature, two dynamic structures showed the discontinuity in their correlation lengths, whereas the single CDA realized an uncorrelated chain state in the dynamical sense. In view of the low-temperature solubility of CDA in DMAc, this abnormal behavior around T was explained by dynamical critical-like fluctuations if T were treated as a kind of lower critical solution temperature (LCST) in the CDA/DMAc system. Here, the self-assemblies arise as the dynamical fluctuations under the spinodal decomposition situation and the competition between the hydrogen bonding (HB) and the hydrophobic interaction (HPhI) makes the conformation of CDA chains change drastically. In this scheme, the solvent-mediated HB and HPhI play important roles in the structure reorganization of cellulose derivatives in strong electronegative solvents, though HB and HPhI cooperate with the inherent chain helicality.     

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.     

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 1. Thermodynamic analysis.

We have investigated the spin-state equilibrium of adrenal mitochondrial P450scc (cholesterol-side-chain-cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 degrees C to -20 degrees C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are delta HA = 0.7 kJ.mol-1 and delta SA = 5J.K-1.mol-1 at low temperatures and delta HB = -42 kJ.mol-1 and delta SB = -152 J.K-1.mol-1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH. The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 +/- 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.     

Crystallization behavior and thermal properties of melt-crystallized poly[(R)-3-hydroxybutyric acid-co-6-hydroxyhexanoic acid] films.

Melt-crystallized films of poly[(R)-3-hydroxybutyric acid-co-10mol% 6-hydroxyhexanoic acid] (P[(R)-3HB-co-6HH]) were prepared by isothermal crystallization at various temperatures for 3 days, and subsequently stored at room temperature after the films formed well-developed and volume-filled spherulites. The lamellar morphologies and properties of melt-crystallized films were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, transmission electron microscopy, and atomic force microscopy. The melting endotherm of P[(R)-3HB-co-6HH] films was composed of a broad peak starting around room temperature and of a sharper peak starting above the isothermal crystallization temperature. The stacking of flat-on lamellae with lamellar periodicity of 8-10 nm was detected on the surface of P[(R)-3HB-co6HH] films after the primary crystallization at 110 degrees C. On storage at room temperature above the Tg (-5 degrees C) of copolyester, thin crystals of 1-4 nm thickness appeared on the surface of P[(R)-3HB-co-6HH] films crystallized at 110 degrees C. These results suggest that long sequences of (R)-3HB units in a random copolyester form relatively thick P[(R)-3HB] crystalline lamellae during the primary crystallization process at a given crystallization temperature, while shorter sequences of (R)-3HB units, which are incapable of crystallizing at a given crystallization temperature, form relatively thin crystalline lamellae during the subsequent crystallization process at room temperature.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.     

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: effect of alkylamine chains

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of +/- 0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB changes from -6.5 to -8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50 degrees C. These observations and our analyses of delta HB and delta SB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50 degrees C is delta HB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for delta HB is not available.     

Temperature and salt effects on proteolytic function of turnip mosaic potyvirus nuclear inclusion protein a exhibiting a low-temperature optimum activity

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease responsible for processing the viral polyprotein into functional proteins. The NIa protease exhibits an unusual optimum proteolytic activity at about 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K(m) revealed that K(m) decreases more drastically than k(cat) as temperature decreases. The thermodynamic analysis showed that the decrease of K(m) is driven entropically, suggesting a possibility that the substrate binding might need a large entropy cost. The secondary structure of the NIa protease was significantly perturbed at temperatures between 20 and 40 degrees C and the protease was unfolded at very low concentrations of guanidine hydrochloride with a transition midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl(2) and MgCl(2) stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the salt ions was due to the lowering of K(m). The salt ions promoted the structural rigidity as evidenced in the higher resistance to the heat-induced unfolding in the presence of the salt ions. The increase in rigidity may lead to the lowering of K(m) possibly by reducing the entropic cost for substrate binding. Taken together, these results suggest that the NIa protease is highly flexible in structure and the low-temperature optimum activity might possibly be attributed to lowered entropy cost for substrate binding at lower temperatures.     

Temperature and pH dependent changes of immunoglobulin G structure.

1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

Cryoenzymology of the hammerhead ribozyme.

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.     

Cryopreservation and transport of mouse spermatozoa at -79 degrees C.

In the present study, 2 experiments were carried out. In experiment 1, mouse spermatozoa were frozen and stored in an ultra-low temperature freezer maintained at -79 degrees C, from 1 week to 8 months. In vitro fertilization rates of the frozen-thawed sperm after 1 week and 4 months of storage were high at 71 and 71%, respectively. These values did not differ significantly from the value (73%) of the control stored at -196 degrees C. In contrast, the 8-month storage rate was significantly lower at 51%. In experiment 2, frozen spermatozoa were transported in a Styrofoam box packed in dry ice from Hokkaido to Tokyo. In vitro fertilization rate of frozen-thawed sperm after transport at -79 degrees C was high at 88%, which was not significantly different from that (84%) of the transported control at -190 degrees C. After transferring two-cell embryos derived from frozen spermatozoa to recipients, 37-62% of the embryos developed into offspring in both experiments. These results indicate that mouse spermatozoa can survive cryopreservation in an ultra-low temperature freezer (-79 degrees C) for up to 4 months and transport at -79 degrees C.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Thawing dog spermatozoa in just-boiled water: submersion time and effect on sperm quality compared to thawing in water at 70 degrees C

Dog spermatozoa have better quality after thawing in water at 70-75 degrees C instead of 35-38 degrees C. The aim of Experiment 1 was to determine the time needed to thaw 0.5 mL straws in just-boiled (98 degrees C) water and that of Experiment 2 to determine whether thawing frozen dog spermatozoa in just-boiled water will result in better quality than thawing in water at 70 degrees C. Prior to freezing the straws of Experiment 1, a Type J thermocouple with wire diameters of 0.08 mm (Osiris Technical Systems, Centurion, South Africa) was placed in the center of each of ninety-three 0.5 mL straws (IMV Technologies, L'Aigle, France) filled with extender (Biladyl* with 0.5%, v/v of Equex STM paste**) and 54 filled with extender plus 200 x 10(6)spermatozoa/mL (Minitüb, Germany (*) and Nova Chemical Sales, MA (**)). Thirty straws with extender were thawed in water at 70 degrees C and the others in just-boiled water. Temperatures inside straws were recorded 10 times/s during warming. Two ejaculates were then collected from each of eight dogs and one from each of three others. Extended ejaculates from the same dog were pooled, frozen 8 cm above liquid nitrogen, and 2 straws from each of the 11 batches thawed in water at 70 degrees C for 8s and 2 in just-boiled water for 6.5s. Sperm morphology and viability were assessed on eosin-nigrosin smears made after thawing and the percentage progressively motile spermatozoa was estimated immediately, 1, 2 and 3h after thawing. The optimal submersion time in just-boiled water was 6.5s for both sperm concentrations, resulting in average temperatures of 23.6+/-1.5 degrees C (+/-S.E.M.) and 24.9+/-1.6 degrees C inside straws with extender or extender plus spermatozoa (P=0.6). The temperature inside straws thawed in water at 70 degrees C was 13.6+/-1.7 degrees C after 8s. Apart from a 1.5% higher (P<0.05) mean percentage motile sperm 2h after thawing, thawing dog spermatozoa in just-boiled (98 degrees C) water holds no benefit over thawing in water at 70 degrees C, which is easier to do.     

Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Swelling and dissolution of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide.

Bombyx mori silk fibers were dissolved in N-methyl morpholine N-oxide (MMNO), an organic cyclic amine oxide used for the solvent spinning of regenerated cellulosic fibers. The commercial MMNO monohydrate used in this study as a solvent for silk is a hygroscopic compound crystalline at room temperature, which becomes an active solvent after melting at 76 degrees C. The degree of hydration of MMNO was checked by DSC measurements. The solvation power of MMNO towards silk fibroin drastically decreased at a water content > or = 20-21% w/w. Dissolution of silk required both thermal and mechanical energy. The optimum temperature was 100 degrees C. At lower temperatures dissolution proceeded very slowly. At higher temperatures, rapid depolymerization of silk fibroin occurred. The value of the Flory-Huggins interaction parameter chi for the MMNO-H2O-silk fibroin system was -8.5, suggesting that dissolution is a thermodynamically favored process. The extent of degradation of silk fibroin was assessed by measuring the intrinsic viscosity and determining the amino acid composition of silk after regeneration with an aqueous methanol solution, which was effective in removing the solvent and coagulating silk. Regenerated silk fibroin membranes were characterized by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. The prevailing molecular conformation of silk fibroin chains was the beta-sheet structure, as shown by the intense amide I-III bands at 1704, 1627, 1515, 1260, and 1230 cm(-1). The value of the I1260/I1230 intensity ratio (crystallinity index) was 0.68, comparable to that of the fibers. The DSC thermogram was characteristic of a silk fibroin material with unoriented beta-sheet crystalline structure, with an intense decomposition endotherm at 294 degrees C. The SEM examination of fractured surfaces showed the presence of a dense microstructure with a very fine texture formed by densely packed roundish particles of about 100-200 nm diameter.     

Influence of structural modifications on the phase behavior of semi-synthetic cerebroside sulfate

Cerebroside sulfate (galactosylceramide I3-sulfate) containing alpha-hydroxy lignoceric acid (C24:0h-CBS), nervonic acid (C24:1-CBS), or hexacosanoic acid (C26:0-CBS) was prepared by a semi-synthetic procedure and studied by differential scanning calorimetry. The phase behavior of these species in 2 M KCl was compared to that of shorter chain length hydroxy and non-hydroxy fatty acid species reported earlier. All three of the new lipids undergo metastable phase behavior similar but not identical to the other species. In addition, the metastable phase behavior of all of the non-hydroxy fatty acid species was found to be more complex than previously thought, with several phases of high transition temperatures and enthalpies possible. Fatty acid hydroxylation inhibits the transition from the metastable to some of the more stable phases. It also significantly increases the phase transition temperatures of both the metastable and stable phases indicating that it contributes to the hydrogen bonding network formed between the lipid molecules and helps overcome the lateral repulsive effect of the negatively charged sulfate. The C-15 cis double bond significantly lowers the temperature and enthalpy of the phase transition indicating that it increases the lateral separation of the lipid molecules and decreases the intermolecular hydrogen bonding interactions. However, it does not prevent formation of a more stable phase. By comparing the effect of various structural modifications reported here and earlier it could be concluded that fatty acid chain length has little effect on the phase transition temperature and enthalpy. This suggests that the forces between the lipid molecules may be dominated by head group interactions rather than interactions between the lipid chains. However, fatty acid chain length has a significant effect on the tendency of the hydroxy fatty acid species to form the more stable phase. The ease of formation of the stable phase increases with increase in chain length. Thus an increase in chain length helps overcome the kinetic barrier to stable phase formation presented by hydroxylation of the fatty acid.     

Effect of freezing temperature,at which straws were plunged into liquid nitrogen,on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.