Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated (11)S-globulin (0 < C(H(2)O) < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment (Delta(N)(D)C(p)), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Delta(N)(D)C(p) in native protein, like the increment DeltaC(p) in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C(p) values for hydrated denatured proteins with the C(p) values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T(D), and thermal effect, DeltaQ(D)) and that of glass transition temperature, T(g), for (11)S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.     

Respiration and photosynthesis of bladders and leaves of aquatic utricularia species

In aquatic species of carnivorous utricularia, about 10 - 50 % of the total biomass consists of bladders. Utricularia bladders are physiologically very active organs though their chlorophyll content may greatly be reduced. To specify energetic costs of carnivory, respiration (RD) and net photosynthetic rate (PN) were compared in bladders and leaves or shoot segments of six aquatic utricularia species with differentiated (U. ochroleuca, U. intermedia, U. floridana) or non-differentiated shoots (U. vulgaris, U. australis, U. bremii) under optimum conditions (20 degrees C, [CO (2)] 0.20 mM, 400 micromol m (-2) s (-1) PAR). RD of bladders of six utricularia species (5.1 - 8.6 mmol kg (-1)(FW) h (-1)) was 75 - 200 % greater, than that in leaves in carnivorous or photosynthetic shoots (1.7 - 6.1 mmol kg (-1)(FW) h (-1)). Within individual species, this difference was statistically significant at P < 0.002 - 0.01. However, PN in photosynthetic leaves/shoots (40 - 117 mmol kg (-1)(FW) h (-1)) exceeded that in bladders (5.2 - 14.7 mmol kg (-1)(FW) h (-1)) 7 - 10 times. RD of empty bladders of U. ochroleuca was exactly the same as that in bladders containing prey. Though utricularia bladders are essential for uptake of growth limiting mineral nutrients N and P from prey as the main benefit of carnivory, the current results support previous work showing that bladder function requires greater metabolic (maintenance) cost and very low photosynthetic efficiency (great RD : PN ratio).     

Synthesis of gossypol atropisomers and derivatives and evaluation of their anti-proliferative and anti-oxidant activity

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.     

Synthesis and study of cyclic and linear analogs of neurotensin

New cyclic analogues of neurotensin (NT): [cyclo (13----8), Gly8]NT-(8-13), [cyclo (13----7), Gly7]NT-(7-13), [cyclo (13----5 epsilon), Lys5]NT-(5-13), [cyclo (13----4 epsilon), Lys4]NT-(4-13), and their linear precursors have been synthesized. The latter (protected linear compounds) were prepared by solid-phase peptide synthesis, and cyclization was attained by using diphenylphosphoryl azide. Cyclization of C-terminal hexa- and octapeptide fragments of NT was found to lead to cycloanalogues possessing high depressor activity. As judged by CD spectral data in aqueous solution, the cyclohexapeptide analogue has a relatively rigid conformation different from its linear counter-part and the NT-(9-13) fragment, whereas NT, its cyclohepta- and cyclononapeptides have random structure.     

Comparison of BMI and percentage of body fat of Indian and German children and adolescents

Today, serious health problems as overweight and obesity are not just constricted to the developed world, but also increase in the developing countries (Prentice 2006, Ramachandram et al. 2002). Focusing on this issue, BMI and percentage of body fat were compared in 2094 schoolchildren from two cross-sectional studies from India and Germany investigated in 2008 and 2009. The German children are in all age groups significantly taller, whereas the Indian children show higher values in BMI (e.g. 12 years: Indian: around 22 kg/m2; German: around 19 kg/m2) and in the percentage of body fat (e.g. 12 years: Indian: around 27%; German: around 18-20%) in most of the investigated age groups. The Indian children have significantly higher BMI between 10 and 13 (boys) respectively 14 years (girls). Indian children showed significant higher percentage of body fat between 10 and 15 years (boys) and between 8 and 16 years (girls). The difference in overweight between Indian and German children was strongest at 11 (boys) and 12 (girls) years: 70% of the Indian but 20% of the German children were classified as overweight. In countries such as India that undergo nutritional transition, a rapid increase in obesity and overweight is observed. In contrast to the industrialized countries, the risk of overweight in developing countries is associated with high socioeconomic status. Other reasons of the rapid increase of overweight in the developing countries caused by different environmental or genetic factors are discussed.     

Comparison of running performances and prevalence of overweight and obesity in Hungarian and Ukrainian adolescents

The 20-m shuttle run (20-mSRT) is a widely used field test to estimate peak oxygen consumption (VO2peak) and thus to assess aerobic fitness of adolescents (11). The purpose of this study was to analyse differences in basic anthropometric measurements (stature, body mass, percent body fat, BMI) and in aerobic fitness of Hungarian and Ukrainian adolescent boys and girls. We examined gender differences in maximal speed (km h-1), in peak VO2 (mL kg-1 min-1) and maximal heart rate (HRmax min-1). Two hundred ninety-two Ukrainian (mean age=16.5±0.5) and 374 (mean age=16.5±0.5) Hungarian adolescents volunteered to participate in this study. Differences were analysed using factorial analysis of the variance (ANOVA) and Student's t-test. Statistical significance was set at p<0.05. Hungarian boys and girls were significantly taller, heavier and had higher percent body fat than their Ukrainian counterparts. Altogether 10% of Hungarians and 7% of Ukrainians were classified overweight or obese according to Cole's BMI classification (4). VO2peak of Ukrainians (mean=49.44±5.29 mL kg-1 min-1) were significantly higher than that of Hungarians (mean=41.93±8.40 mL kg-1 min-1). Maximal heart rate also differed significantly (Ukrainians mean=201.12±8.43 min-1 vs. Hungarians mean=185.38±18.38 min-1).In conclusion, aerobic fitness of the Ukrainian adolescents was significantly higher than that of the Hungarians independently of BMI or gender.     

Production and utilization of hydrogen peroxide associated with melanogenesis and tyrosinase-mediated oxidations of DOPA and dopamine

The synthesis and involvement of H(2)O(2) during the early stages of melanogenesis involving the oxidations of DOPA and dopamine (diphenolase activity) were established by two sensitive and specific electrochemical detection systems. Catalase-treated reaction mixtures showed diminished rates of H(2)O(2) production during the autoxidation and tyrosinase-mediated oxidation of both diphenols. Inhibition studies with the radical scavenger resveratrol revealed the involvement in these reactions of additional reactive intermediate of oxygen (ROI), one of which appears to be superoxide anion. There was no evidence to suggest that H(2)O(2) or any other ROI was produced during the tyrosinase-mediated conversion of tyrosine to DOPA (monophenolase activity). Establishing by electrochemical methods the endogenous production H(2)O(2) in real time confirms recent reports, based in large part on the use of exogenous H(2)O(2), that tyrosinase can manifest both catalase and peroxidase activities. The detection of ROI in tyrosinase-mediated in vitro reactions provides evidence for sequential univalent reductions of O(2), most likely occurring at the enzyme active site copper. Collectively, these observations focus attention on the possible involvement of peroxidase-H(2)O(2) systems and related ROI-mediated reactions in promoting melanocytotoxic and melanoprotective processes.     

Total synthesis of globotriaosyl-E and Z-ceramides and isoglobotriaosyl-E-ceramide

Stereoselective, total synthesis of O-alpha-D-galactopyranosyl-(1----4) -O-beta-D-galactopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-N -tetracosanoyl-[2S,3R,4E (and 4Z)]-sphingenine and O-alpha-D -galactopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-O-beta-D -glucopyranosyl-(1----1)-N-tetracosanoyl-(2S,3R,4E)-sphin gen ine was achieved by using O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate, O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha (and beta)-D-glucopyranosyl fluoride, and O-(2,3,4,6-tetra-O-acetyl-alpha-D -galactopyranosyl)-(1----3)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyran osyl)-(1----4)-2,3,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate.     

Metabolic and thermal physiology of pigeons and doves

Pigeons and doves (Columbidae) are an interesting group to examine for physiological adaptations to climate and diet because this cosmopolitan family comprises more than 300 species that are mostly granivores, although some are specialized frugivores. We determined allometric and phylogenetic effects on body temperature (T(b)), basal metabolic rate (BMR; J h(-1)), and wet thermal conductance (C(wet); J h(-1) C(-1)), and we examined mass (M) and phylogenetically corrected residuals for further effects of climate, diet, and landmass size (mainland or island). Independent contrasts, correlograms, autoregression, and phylogenetic eigenvector regression (PVR) were used to examine phylogenetically related effects. We found a small but significant phylogenetic pattern for body mass of columbids. For T(b), there was no significant effect of mass or phylogeny. There was a significant effect of climate on T(b) and no significant effects of diet or landmass without mass or phylogenetic correction, but after mass and phylogenetic correction, there were no effects of climate, diet, or landmass. For BMR, there was a strong allometric effect, and residuals were significantly lower for arid and tropical species but not for temperate species, compared to predictions for nonpasserine birds. There was a nearly significant autoregressive phylogenetic relationship for BMR parl0;r=0.44), and the strong allometry of BMR remained for independent contrasts (slope=0.731), autoregressive residuals (0.698), and PVR (0.705). Residuals, from regression of autoregression and PVR residuals of M and BMR, were significantly associated with climate: arid pigeons had a lower BMR residual than tropical and temperate pigeons. PVR residuals were significantly affected by landmass (island columbids had a smaller residual than mainland columbids), but autoregression residuals were not. There was no association of autoregression or PVR residuals with diet. For C(wet), there was a strong allometric effect, and residuals for columbids were significantly higher compared to other birds. There was no significant relationship for C(wet) of columbids to climate, diet, or landmass. There was no significant autoregressive or PVR relationship for C(wet), and the strong allometry remained after phylogenetic analysis by independent contrasts (slope=0.501), autoregression (0.509), and PVR (0.514). Residuals from autoregression and PVR were not significantly correlated with climate, diet, or landmass (mainland/island).     

Syntheses and Herbicidai Activities of Phenoxypyrimidines and Phenoxytriazines

Series of phenoxypyrimidines and phenoxytriazines were prepared to be evaluated as herbicides. Among them, 2-(2,6-dichlorophenoxy)-pyrimidine (XV), 2-phenoxy-4,6-dimethyl- pyrimidine (XVII), 2-(3-methyl-4-chlorophenoxy)-4,6-bis(ethylamino)-5-triazine (LIV), 2-(2,4-dichlorophenoxy)-4,6-bis(ethylamino)-s-triazine (LVIII), and 2-(2,6-dichlorophenoxy)-4,6-bis(ethylamino)-s-triazine (LX) showed high pre-emergent herbicidai activity to radish. On the other hand, 2-chloro-4-(2,6-dichlorophenoxy)-6-methylpyrimidine (XXX) revealed high efficiency to millet. Some structure-activity relationship is discussed.     

Comparison of Glycolipids and Plastids in Callus Cells and Leaves of Alnus and Betula

The fine structure of plastids and fatty acid composition ofglycolipids (e.g. monogalactosyl diacylglycerides, MGDG; digalactosyldiacylglycerides, DGDG) in callus cells of Alnus glutinosa,A. incana and Betula pendula cultured in light was comparedwith that in intact leaves. The tissues were qualitatively verysimilar but a rather high amount oflignoceric acid (24:0) wascharacteristic for the callus of A. incana. This fatty acidwas found only in trace amount in other tissues. Linolenic (18:3)and palmitic (16:0) acids are the most abundant (25–65%and 17–27% respectively) fatty acids in all tissues studied.The proportion of 18:1 and 18:2 was much higher in the calluscompared with corresponding intact leaves, which are especiallyrich (48–65%) in 18:3. In callus cultures a higher proportion(17–19%) of linoleic acid (18:2) is found in both Alnusspecies than in the two callus strains of Betula (9–12%). All leaf and callus samples contained esterified steryl glycosidesand two cerebrosidelike spots in thin-layer chromatography,but they were more prominent in callus cultures than in leaves.The callus cells have plastids with rather well developed thylakoidswhich explains the similarity of the main glycolipid components(MGDG and DGDG) to that of leaves. (Received April 23, 1984; Accepted August 17, 1984)     

Plant biochemistry of xenobiotics: isolation and properties of soybean O- and N-glucosyl and O- and N-malonyltransferases for chlorinated phenols and anilines.

O-Glucosyltransferase (O-GT), O-malonyltransferase (O-MAT), N-glucosyltransferase (N-GT), and N-malonyltransferase (N-MAT) activities have been detected in cultured soybean cells, using pentachlorophenol and 3,4-dichloroaniline as xenobiotic standard substrates. The O-GT was purified approximately 1000-fold, and the N-MAT approximately 70-fold. There was an extensive copurification of O-GT and O-MAT. The following functional molecular weight values were obtained, 47 kDA (O-GT), 48 kDA (O-MAT) 43 kDa (N-GT), and 48 kDa (N-MAT). O-GT and N-MAT appeared to be monomeric polypeptides with isoelectric points of approximately 4.8 and approximately 6.1, respectively. The O-GT, N-GT, and N-MAT activities had marked substrate specificities for chlorinated aromatic xenobiotics and thus illustrate the existence of plant isoenzymes with specificity for xenobiotics.     

Isolation and identification of prostaglandins of the E and F series in testes and semen of lowland-black-white breed of bulls

The prostaglandins of E and F series were obtained from testes and semen of sexually mature bulls of lowland-black-white breed. From 1 g of fresh testes tissue we obtained 7.01 X 10(-9) M prostaglandin of the F series (PGF) and 20.65 X 10(-9) M prostaglandin of the E series (PGE): from 11 of semen 3.28 X 10(-6) M of PGF and 10.58 X 10(-6) M of PGE were obtained as well. The prostaglandins thus obtained displayed biological activity in experiments on the isolated small intestine of the rabbit.     

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.     

Unfolding of iron and copper complexes of human lactoferrin and transferrin

1. Human lactoferrin and transferrin are capable of binding two iron or copper ions into specific binding sites in the presence of bicarbonate. 2. Urea and several alkyl ureas have been effective in unfolding these metal-protein complexes. 3.Biphasic transitions are observed for the unfolding of each of the metal complexes of these proteins as determined by direct visible spectroscopy suggesting the release of iron(III) and Cu(II) ions from both of these metal-binding proteins during the unfolding process. 4. Greater stabilization and increased resistance to protein unfolding is observed for all iron(III) complexes compared to Cu(II) complexes of lactoferrin and transferrin as determined by isothermal unfolding and thermal denaturation. 5. Relative stabilization of the different metal-protein complexes investigated within this study were determined to be as follows: Lf-Fe(III) greater than Lf-Cu(II); Tf-Fe(III) greater than Tf-Cu(II), and Lf-Fe(III) greater than Tf-Fe(III); Lf-Cu(II) greater than Tf-Cu(II).     

Genetic and environmental factors that influence production and quality of milk of Alpine and Saanen goats

Data from 1279 lactations of 783 Alpine and Saanen goats of the herd of our university in Minas Gerais, Brazil, were used to study environmental effects on and to estimate genetic parameters for milk production until 270 days of lactation (MP270) and for production and percentages of fat (PFAT and %FAT), protein (PPROT and %PROT), lactose (PLACT and %LACT), and total dry extract (PEXTR and %EXTR). Environmental effects were estimated by a statistical model that included contemporary group effect, type of kidding, genetic grouping, and kidding order. A multi-trait animal model with animal and permanent environment random effects was used to estimate genetic parameters and the significant environmental effects (fixed). Contemporary group influenced all traits; genetic grouping did not influence %LACT; type of kidding did not influence PFAT, %PROT or %LACT, and kidding order did not influence %FAT or %EXTR. Heritability and repeatability estimates were, respectively, 0.19 and 0.37 (MP270); 0.10 and 0.20 (PFAT); 0.12 and 0.24 (PPROT); 0.15 and 0.27 (PLACT); 0.13 and 0.24 (PEXTR); 0.21 and 0.34 (%FAT); 0.39 and 0.44 (%PROT); 0.17 and 0.29 (%LACT); 0.31 and 0.47 (%EXTR). Estimates of genetic correlations among MP270 and production of milk constituents were positive and high, but correlations between MP270 and %FAT, MP270 and %PROT, MP270 and %ESTR were moderate and negative. These heritability estimates show that satisfactory genetic gains can be obtained by selection, especially for milk constituents.     

Folding and unfolding of gammaTIM monomers and dimers

Kinetic simulations of the folding and unfolding of triosephosphate isomerase (TIM) from yeast were conducted using a single monomer gammaTIM polypeptide chain that folds as a monomer and two gammaTIM chains that fold to the native dimer structure. The basic protein model used was a minimalist Gō model using the native structure to determine attractive energies in the protein chain. For each simulation type--monomer unfolding, monomer refolding, dimer unfolding, and dimer refolding--thirty simulations were conducted, successfully capturing each reaction in full. Analysis of the simulations demonstrates four main conclusions. First, all four simulation types have a similar "folding order", i.e., they have similar structures in intermediate stages of folding between the unfolded and folded state. Second, despite this similarity, different intermediate stages are more or less populated in the four different simulations, with 1), no intermediates populated in monomer unfolding; 2), two intermediates populated with beta(2)-beta(4) and beta(1)-beta(5) regions folded in monomer refolding; 3), two intermediates populated with beta(2)-beta(3) and beta(2)-beta(4) regions folded in dimer unfolding; and 4), two intermediates populated with beta(1)-beta(5) and beta(1)-beta(5) + beta(6) + beta(7) + beta(8) regions folded in dimer refolding. Third, simulations demonstrate that dimer binding and unbinding can occur early in the folding process before complete monomer-chain folding. Fourth, excellent agreement is found between the simulations and MPAX (misincorporation proton alkyl exchange) experiments. In total, this agreement demonstrates that the computational Gō model is accurate for gammaTIM and that the energy landscape of gammaTIM appears funneled to the native state.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

Formulation and Evaluation of Bi-layer Tablet of Metoclopramide Hydrochloride and Ibuprofen

The aim of this study was to prepare bi-layer tablet of Metoclopramide Hydrochloride (MTH) and Ibuprofen (IB) for the effective treatment of migraine. MTH and IB were formulated as immediate and sustained release layer respectively. MTH was formulated as immediate release layer by using various disintegrants like Ac-Di-Sol, Polyplasdone XL, Explotab, Agar and Gellan Gum. Treated form of gellan gum and agar was prepared and compared for their disintegrant efficiency with other disintegrants. IB was formulated as sustained release layer using hydrophilic matrix (hydroxypropylmethylcellulose [HPMC K₄M]). The effect of concentration of hydrophilic matrix (HPMC K₄M), binder (polyvinylpyrollidone [PVP K₃₀]) and buffer (sodium bicarbonate) on IB release was studied. The dissolution study of sustained release layer showed that an increasing amount of HPMC or PVP K₃₀ results in reduced IB release. The inclusion of buffer (sodium bicarbonate) enhanced the release of IB from sustained release layer. The rational for formulation of bi-layer tablet of these two drugs in combination was (1) MTH increases the absorption of acidic non-steroidal anti-inflammatory drug (NSAID) by increasing gastric motility. So sequential release of MTH (as immediate release) and IB (as sustained release) was suitable for treatment of migraine. (2) MTH was degraded when prolonged contact with acidic NSAID. Bi-layer tablet was suitable for preventing direct contact of these two drugs and thus to maximize the efficacy of combination of two drugs for migraine.