Structural studies of starches with different water contents

The proportion of double helices in starches from a series of pea [rb, rug4-b, rug3-a, and lam-c mutants, and the wild type (WT) parental line], potato and maize (normal and low amylose), and wheat (normal) lines, ranged from about 30-50% on a dry weight basis. In relatively dry starch powders, only about half of the double helices were in crystalline order, this proportion being higher for A-type than for B-type starches. Using starch from WT pea as an example, it was found that increasing water content results in an increase in total crystallinity. When the water content was raised to a level similar to that in excess water, the proportion of crystallinity was close to the proportion of double helices (DH). Measuring crystallinity in starches with a high water content is difficult using traditional methods such as x-ray diffraction. A method was developed, therefore, for determining starch structural characteristics in excess water by measuring the enthalpy of gelatinization transition in quasi-equilibrium differential scanning calorimetry (DSC) experiments. It is suggested that DH% = DeltaH(sp)/DeltaH(DH) x 100%, where DeltaH(sp) and DeltaH(DH) represent the specific enthalpies of gelatinisation transition, DeltaH(sp) being measured as J/g dry starch weight and DeltaH(DH) as J/g DH, in starch. Studies on potato and maize starches in excess water and in 0.6M KCl showed, respectively, that DeltaH(DH) was 36.3 and 35.6 J/g for B-type polymorphs and 33.0 and 35.0 J/g for A-type polymorphs. For C-type starches, such as those from pea, intermediate values of DeltaH(DH), related to the proportions A-/B-polymorphs, should be used. The type of crystallinity in starch can be determined by the shift in peak temperature for thermograms in excess water and in excess 0.6M KCl. For B-polymorphs this shift was found to be approximately 2-3 degrees C and for A-polymorphs approximately 7-12 degrees C. The ratio between ordered areas with both A- and B-polymorphs can be determined from the enthalpies of disruption of each area. These enthalpies can be obtained by deconvolution of bimodal thermograms produced by C-type starches in excess 0.6M KCl. This methodical approach can be applied to all starches that give a sharp gelatinisation thermogram in excess water. Using a range of methods, including DSC, it was found that starch granules from the mutant peas are constructed in a similar way to those from the WT, with B-polymorphs in the centre and A-polymorphs at the periphery of all granules. The proportion of A/B-polymorphs, however, differed between the mutants. It was found that in addition to increasing the total crystallinity, increasing the water content within the granules also resulted in an increase in the proportion of B-polymorphs.     

Oxidized and acid thinned starch derivatives of hybrid maize: functional characteristics, wide-angle X-ray diffractometry and thermal properties

Starch isolated from hybrid maize (8535-23) was subjected to oxidation and acid thinning. Proximate analyses revealed that moisture, ash, protein, fat, fibre, and pH reduced after oxidation and acid thinning. Percentage amylose content reduced from 20.42% in native starch to 18.76 and 17.65% in oxidised and acid thinned starch derivatives, respectively. Wide-angle X-ray diffraction patterns indicated strong peaks at 15.9 degrees, 17.2 degrees, 18.8 degrees, and 25.0 degrees 2theta. No significant difference was observed between the X-ray pattern of the native and modified starches. Both swelling power and solubility increased with increase in temperature. Oxidation and acid thinning reduced swelling power and increased solubility starch. At all pHs, both oxidation and acid thinning reduced the swelling capacity of the native starch. Oxidation increased water and oil absorption capacity of the native starch, while both hydrophilic and hydrophobic properties reduced following acid thinning. Least gelation concentration reduced in acid thinned starch but increased in oxidised derivative. Pasting temperature (Tp), peak viscosity (Pv), hot paste viscosity (Hv), and viscosity after 30 min holding at 95 degrees C (H(v30)) reduced following both modifications. However, values for cold paste viscosity (Cv) and setback (SB) reduced in oxidised derivative and increased in acid thinned starch. Light transmittance of the starch pastes reduced with increase in storage days, however, reduction was more pronounced in native and acid thinned starches. Onset temperature (To), peak temperature (Tp) and conclusion temperature (Tc) of gelatinisation reduced in modified starches compared with native hybrid maize starch. Also, gelatinisation enthalpy reduced after oxidation and acid thinning. Enthalpy of regelatinisation increased as days of storage of starch paste increased.     

Effects of annealing on the polymorphic structure of starches from sweet potatoes (Ayamurasaki and Sunnyred cultivars) grown at various soil temperatures

Starches extracted from the sweet potato cultivars Sunnyred and Ayamurasaki grown at 15 or 33 degrees C (soil temperature) were annealed in excess water (3 mg starch/mL water) for different times (1, 4, 8 or 10h) at the temperatures 2-3 degrees K below the onset melting temperature. The structures of annealed starches, as well as their gelatinisation (melting) properties, were studied using high-sensitivity differential scanning calorimetry (HSDSC). In excess water, the single endothermic peak shifted to higher temperatures, while the melting (gelatinisation) enthalpy changed only very slightly, if any. The elevation of gelatinisation temperature was associated with increasing order/thickness of the crystalline lamellae. The only DSC endotherm identified in 0.6 M KCl for Sunnyred starch grown at 33 degrees C was attributed to A-type polymorphic structure. The multiple endothermic forms observed by DSC performed in 0.6M KCl for annealed starches from both cultivars grown at 15 degrees C provided evidence of a complex C-type (A- plus B-type) polymorphic structure of crystalline lamellae. The A:B-ratio of two polymorphic forms increased upon annealing due to partial transformation of B- to A-polymorph, which was time dependent. Long heating periods facilitated the maximal transformation of B- to A-polymorph associated with limited A:B ratio.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Breakdown kinetics of the tri-chromium(III) oxo acetate cluster ([Cr(3)O(OAc)(6)]+) with some ligands of biological interest

Kinetics for the breakdown of the trinuclear chromium acetate cluster, [Cr(3)O(OAc)(6)](+), with a series of monoprotic and diprotic ligands in weakly acidic aqueous media (pH approximately 4 or approximately 5) have been investigated spectrophotometrically at 40-60 degrees C. The results point to an ion-pair equilibrium as the first step followed by associative interchange mechanism forming the mononuclear product of the reaction. Pseudo-first-order rates were determined from absorbance data and associated activation parameters were calculated using the Eyring equation. Enthalpy and entropy terms of the reactions (e.g., histidine, DeltaH(double dagger) = 75 +/- 15 kJ mol(-1), DeltaS(double dagger) = -130 +/- 25 J K(-1) mol(-1); lactic acid, DeltaH(double dagger) = 66 +/- 13 kJ mol(-1), DeltaS(double dagger) = -155 +/- 30 J K(-1) mol(-1); glycine, DeltaH(double dagger) = 31 +/- 6 kJ mol(-1), DeltaS(double dagger) = -225 +/- 45 J K(-1) mol(-1)) are consistent with an associative interchange (I(a)) mechanism, and produce a linear isokinetic plot (slope = 50 degrees C). Rates and activation parameters are comparable to those of substitution reactions of the chromium(III) hexaaqua cation. Other ligands studied included malonic acid and the amino acid, aspartic acid. Observed rates are faster than water exchange rates, but typically slower than anion substitution rates, and indicate that trinuclear chromium(III) clusters are expected to be kinetically stable in neutral to slightly acidic conditions.     

Succinyl and acetyl starch derivatives of a hybrid maize: physicochemical characteristics and retrogradation properties monitored by differential scanning calorimetry

Starch isolated from a hybrid maize (8535-23) was chemically modified by succinylation and acetylation. No pronounced difference was observed between the X-ray pattern of native starch and modified starch samples, and the samples gave the characteristic A pattern of cereal starches. Onset temperature (T(o)), peak temperature (T(p)), concluding temperature (T(c)) and enthalpy of gelatinisation (DeltaH), reduced after succinylation and acetylation, but gelatinisation temperature range increased following starch modifications. Modifications reduced starch retrogradation.     

The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.

This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.     

New starches from traditional Chinese medicine (TCM)--Chinese yam (Dioscorea opposita Thunb.) cultivars

The starches separated from two different Dioscorea opposita Thunb. cultivars were investigated for morphological, thermal and crystal properties. The shape of starch granules separated from different D. opposita Thunb. cultivars varied from round to oval or irregular. The surface of the starch granules appeared to be smooth without any fissures. The average particle diameter of starches from different D. opposita Thunb. cultivars was 40.3 and 38.7mum for D. 47 and D. SXY starch, respectively. The transition temperatures (T(o), T(p) and T(c)) and enthalpy of gelatinization (DeltaH(gel)) were determined using differential scanning calorimetry (DSC). The D. SXY starch showed the lower T(o) (74.2 degrees C) and the broader R (12.4). T(p) and T(c) of starch from D. 47 were higher than that of D. SXY starch. DeltaH(gel) values (11.37J/g) of D. 47 was higher than that of D. SXY starch (10.78J/g). The crystal type of starches separated from two different D. opposita cultivars was a typical C-type pattern. The degree of crystallinity of two D. opposita cultivars starches was about 45.9% and 31.5%, respectively.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and -2 degrees C in different concentrations of methanol and sucrose

The hatching performance of common carp (Cyprinus carpio L.) embryos was examined after 12-72-h storage at 4 and -2 degrees C using different concentrations of sucrose (0.1, 0.25, 0.5 and 1.0 M or 3.42, 8.55, 17.10 and 34.2%), methanol (MeOH) (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 M or 1.6, 3.2, 4.8, 6.4, 8.0, 9.6 and 11.2%), or varying concentrations of methanol in 0.5 M (17.10%) sucrose. For sucrose, 0.5 M (17.10%) showed the maximum survival (41+/-1% (12 h) to 11+/-1.5% (72 h)) at 4 degrees C. No survival was observed at -2 degrees C with any concentration of sucrose. At both temperatures employed, hatching was higher with mixed combination of methanol (1.5 M or 4.8%) and 0.5 M (17.10%) sucrose (4 degrees C: 41+/-1.5% (12 h), 38+/-1.2% (72 h); -2 degrees C: 33+/-1.7% (12 h), 28+/-1.2% (72 h)) compared to methanol alone (4 degrees C: 38+/-1.5% (12 h), 35+/-2.5% (72 h); -2 degrees C: 31+/-2.5% (12 h), 25+/-2% (72 h)). The combination of 1.5 M (4.8%) methanol and 0.5 M (17.10%) sucrose produced the best results among all the concentrations tested at both temperatures.     

Novel direct access to enantiomerization barriers from peak profiles in enantioselective dynamic chromatography: enantiomerization of dialkyl-1,3-allenedicarboxylates

The axially chiral allenes dimethyl-1,3-allenedicarboxylate 1 and diethyl-1,3-allenedicarboxylate 2 show characteristic plateau formation during enantioselective GC separation on the chiral stationary liquid phase Chirasil-beta-Dex. The elution profiles, obtained from temperature-dependent dynamic GC (DGC) experiments (1: 100-140 degrees C; 2: 110-150 degrees C) were evaluated with the recently derived approximation function (AF) k1(approx) = f(t(R)(A),t(R)(B),w(h)(A),h(plateau), N) to yield the enantiomerization rate constant directly k(1). These values were compared with those obtained by computer-aided simulation with ChromWin. The Eyring activation parameters of the experimental interconversion profiles were determined to be: DeltaG(#)(298.15 K) = 103.6 +/- 0.9 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.4 kJ mol(-1), DeltaS(#) = -198 +/- 7 J K(1) mol(-1) for dimethyl-1,3-allenedicarboxylate 1, and DeltaG(#)(298.15 K) = 103.5 +/- 1.1 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.5 kJ mol(-1), DeltaS(#) = -197 +/- 9 J K(-1) mol(-1) for diethyl-1,3-allenedicarboxylate 2. The approximation function (AF) presented here allows the fast determination of rate constants k(1) and activation barriers of enantiomerization DeltaG(#) from chromatographic parameters without extensive computer simulation.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)     

A calorimetric study of 3d metal ions-acyclovir interactions. The 2-hydroxyethoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting the coordination through N(7)

The equilibrium constants and enthalpic values of metal acyclovir complexes have been determined by calorimetry for Co(II) (log K=0.96+/-0.05, DeltaH (kJ/mol)=-19.7+/-1.3), Ni(II) (log K=1.39+/-0.03, DeltaH (kJ/mol)=-21.5+/-1.0), Cu(II) (log K=1.83+/-0.03, DeltaH (kJ/mol)=-23.2+/-0.8) and Zn(II) (log K=0.71+/-0.06, DeltaH (kJ/mol)=-18.6+/-1.5). The equilibrium constants are similar to those of the divalent ions with guanosine and 2,9-dimethylpurine. By comparison with previous thermodynamic data, it can be shown that the 2-hydroxyethoxymethyl group promotes coordination through N(7) versus N(1) of the guanine ring for 3d metal ions. These results reveal that the 2-hydroxyethoxymethyl group placed on the purine ring of guanine in acyclovir causes a greater effect than that of the 9-methyl in purines and similar to or greater than that of the ribose moiety in guanosine. The 2-hydroxyethyoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting a similar coordination chemistry (with very close log K and DeltaH values) for acyclovir, deoxy-guanosine and guanosine with divalent metals.     

Human coronary vascular smooth muscle and endothelium-dependent responses after storage at -75 degrees C.

In the present study, we investigated whether an established method of cryostorage at -75 degrees C in the presence of dimethyl sulfoxide (Me2SO) and fetal calf serum (FCS) could preserve the vascular and endothelial responses of isolated human coronary arteries. A total of 123 ring segments (4-5 mm in length) of epicardial coronary arteries were isolated within 1 to 2 h from hearts of four patients receiving a cardiac transplant. Thirty-nine coronary ring segments were studied immediately upon cleaning of surrounding tissues, while 84 similarly cleaned segments were stored at -75 degrees C for 7 to 10 days prior to in vitro reactivity studies. In the freshly isolated coronary arteries, addition of prostaglandin F2 alpha, endothelin (ET-1), or acetylcholine consistently produced a dose-dependent contraction, reaching a maximum contractile force of 9.6 +/- 0.7, 4.5 +/- 0.5, and 3.1 +/- 0.5 g (M +/- SEM), respectively, while histamine, thrombin and substance P consistently produced an endothelium-dependent relaxation (EDR) with a maximum of -89 +/- 2.8, -85 +/- 5.0, and -72 +/- 3.5%, respectively. Isoproterenol produced an endothelium-independent relaxation (-82 +/- 4.5%). Cryostorage of human coronary arteries at -75 degrees C without cryoprotectant resulted in a complete loss of the contractile response. In contrast, addition of Me2SO and FCS in the cryostorage medium significantly preserved the contractile responses, although they were decreased (1.9 +/- 0.3, 1.5 +/- 0.3, and 0.6 +/- 0.1 g to PGF2 alpha, ET-1, and acetylcholine, respectively) when compared to the fresh controls. The maximum EDR to histamine, thrombin, and substance P in the cryostored coronaries were also reduced to -40 +/- 5.6, -21 +/- 3.3, and -47 +/- 4.7%, respectively, and the isoproterenol-induced relaxation was reduced to -62 +/- 4.1%. These results suggest that although the cryostorage method described in the present report provided only limited preservation of human coronary arteries, significant vascular smooth muscle and endothelial-dependent functions were retained. Thus, it is possible that further refinement of the present cryostorage methodology may provide better preservation of functionally viable human blood vessels.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.     

Liquid storage of miniature boar semen.

The effects of liquid storage at 15 degrees C on the fertilizing ability of miniature pig semen were investigated. Characterization of ejaculated semen from 3 miniature boars was carried out. Semen volume and pH were similar among these boars. In one of the boars, sperm motility was slightly low, and sperm concentration and total number of sperm were significantly lower than in the others (P < 0.01). Seminal plasma of the semen was substituted with various extenders (Kiev, Androhep, BTS and Modena) by centrifugation and semen was stored for 7 days at 15 degrees C. Sperm motility was estimated daily at 37 degrees C. For complete substitution of seminal plasma, Modena was significantly more efficient than the other extenders (P < 0.001) in retaining sperm motility. Semen from each of the 3 miniature boars that had been stored for 5 to 7 days at 15 degrees C in Modena was used for artificial insemination of 15 miniature sows. The farrowing rates were 100, 100 and 60%, and litter sizes were 6.4 +/- 1.5, 5.8 +/- 0.8 and 5.0 +/- 1.0 for each boar semen, respectively. The boar that sired the smallest farrowing rate was the same one that showed lower seminal quality with respect to sperm motility, sperm concentration and total number of sperm. These results suggest that miniature boar semen can be stored for at least 5 days at 15 degrees C by the substitution of seminal plasma with Modena extender.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

XRD studies of chitin-based polyurethane elastomers

Chitin-based polyurethane elastomers (PUEs) were synthesized by step growth polymerization techniques using poly(epsilon-caprolactone) (PCL) varying diisocyanate and chain extender structures. The viscosity average molecular weight (M(v)) of chitin was deduced from the intrinsic viscosity and found; M(v)=6.067 x 10(5). The conventional spectroscopic characterization of the samples with FTIR, (1)H NMR and (13)C NMR were in accordance with proposed PUEs structure. The crystalline behavior of the synthesized polymers were investigated by X-ray diffraction (XRD), differential scanning calorimetery (DSC) and loss tangent curves (tan delta peaks). The observed patterns of the crystalline peaks for the lower angle for chitin in the 2theta range were indexed as 9.39 degrees, 19.72 degrees, 20.73 degrees, 23.41 degrees and 26.39 degrees. Results showed that crystallinity of the synthesized PUEs samples was affected by varying the structure of the diisocyanate and chain extender. Crystallinity decreased from aliphatic to aromatic characters of the diisocyanates used in the final PU. The presence of chitin also favors the formation of more ordered structure, as higher peak intensities was obtained from the PU extended with chitin than 1,4-butane diol (BDO). The value of peak enthalpy (DeltaH) of chitin was found to be 47.13 J g(-1). The higher DeltaH value of 46.35 J g(-1) was found in the samples extended with chitin than BDO (39.73 J g(-1)).