Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.     

Caspase-dependent apoptotic pathways in CNS injury

Recent studies have suggested a role for neuronal apoptosis in cell loss following acute CNS injury as well as in chronic neurodegeneration. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to contribute significantly to the morphological and biochemical manifestations of apoptotic cell death. Caspases are translated as inactive zymogens and become active after specific cleavage. Of the 14 identified caspases, caspase-3 appears to be the major effector of neuronal apoptosis induced by a variety of stimuli. A role for caspase-3 in injury-induced neuronal cell death has been established using semispecific peptide caspase inhibitors. This article reviews the current literature relating to pathways regulating caspase activation in apoptosis associated with acute and chronic neurodegeneration, and suggests that identification of critical upstream caspase regulatory mechanisms may permit more effective treatment of such disorders.     

Synthesis and Use of New Semispecific Substrates for Trypsin-Catalyzed Peptide Bond Formation

Two series of semispecific acyl donors, hydroxyalkyl esters of Z-Ala-OH and TV-modified carboxamidomethyl (Cam) esters of Z-Xaa-OH (Xaa = Ala, Leu, Phe) were synthesized as substrates for trypsin-catalyzed peptide synthesis. It follows from the specificity constants of these compounds, that the carboxamidomethyl derivatives are well accepted by trypsin due to favourable S₂′ – P₂′ interactions. These new substrates can be successfully used for the trypsin-mediated formation of dipeptide amides. The synthesis outcome depends on the amino acid in the P₁ position, the ability of the leaving group to provide efficient interactions with the enzyme subsite and the hydrophobicity of the nucleophilic amino acid amide. The modified Cam esters give better peptide yields in comparison to the unmodified ones.     

The production of free radicals during the autoxidation of monosaccharides by buffer ions

The production of free radicals during the autoxidation of simple monosaccharides at 37 degrees has been studied by the electron spin resonance (e.s.r.) technique of spin trapping. In the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), monosaccharides undergoing autoxidation produced hydroxyl and 1-hydroxyalkyl radical-derived spin adducts, indicating that hydroxyl and hydroxyalkyl free-radicals are involved in the autoxidation of monosaccharides. The pH profile for the production of free radicals from monosaccharides undergoing autoxidation revealed the formation of both hydroxyl and hydroxyalkyl radicals at relatively high pH, whereas at low pH, only the formation of hydroxyalkyl radicals was observed; the transition between these routes for the production of free radicals occurred at pH 8.0-8.5. Glycolaldehyde, glyceraldehyde, dihydroxyacetone, and erythrose are relatively rapidly enolised (to an ene-diol) and autoxidised with the concomitant production of free radicals. Ribose and glucose enolise and autoxidise very slowly without detectable production of free radicals. A comparison of the pH profiles of the rates of enolisation and the pH dependence of the production of free radicals from glyceraldehyde during autoxidation suggests that a change in reaction mechanism occurs at pH 8.2. Below pH 8.2, the rates of enolisation and autoxidation increase with increasing pH, with a concomitant increase in the formation of hydroxyalkyl spin-adducts. Above pH 8.2, glyceraldehyde undergoing autoxidation shows a much higher rate of enolisation than of autoxidation and, although the formation of hydroxyalkyl radicals is decreased, the production of hydroxyl radicals is also observed. A free-radical mechanism for the autoxidation of monosaccharides is proposed, to account for the pH-dependent characteristics of the production of free radicals and the relationships between the production of free radicals, autoxidation, and enolisation of the monosaccharides.     

Release of Free Fatty Acids and Loss of Hill Activity by Aging Spinach Chloroplasts

The free fatty acid content of spinach chloroplasts, isolated at pH 5.8 to 8.0, has been found to vary between 3.1 and 5.5% of the total chloroplast fatty acids. When chloroplasts were incubated at room temperature for 2 hours, the free fatty acids increased by 42% and the Hill activity decreased by 70%. After 2 hours of incubation at 37 degrees , the free fatty acids increased about 3-fold and the Hill activity decreased to almost 0. The addition of crystalline bovine serum albumin largely prevented the loss of Hill activity at room temperature and at 5 degrees , but had little effect during incubation at 37 degrees . Both the release of free fatty acids and the loss of Hill activity were pH dependent. The losses were the least during incubation at pH 5.8 and the greatest during incubation at pH 8.0. The major free fatty acids released at pH 5.8 were saturated, while those released at pH 7.0 or 8.0 were mainly the unsaturated acids, alpha-linolenic acid and hexadecatrienoic acid.     

Ruthenium Red and the Bacterial Glycocaly

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.     

介孔分子筛MCM-41固定中性脂肪酶条件的研究

以介孔分子筛MCM-41材料为载体,采用物理吸附法对中性脂肪酶进行了固定化处理,并研究不同条件对固定化脂肪酶催化活性的影响,从而得到该种材料对脂肪酶的最佳固定化条件。给酶量为45960 U/g,固定化温度为45℃,pH值为7.5,时间为3 h,此时固定化酶的活力约为4666 U/g。固定化酶和游离酶的最适反应温度都为40℃,最适pH值为7.5,比游离酶低。固定化酶温度稳定性和pH稳定性较游离酶有所提高。     

Flow kinetics of immobilized beta-glucosidase

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.     

Sexual isolation in Drosophila. II. Intraspecific variation in mate recognition systems

A simple behavioral model is used to investigate whether differences in the specific-mate-recognition system (SMRS), occur within species of the Drosophila genus. This model takes into account, and overcomes, the distorting effect of vigor differences on experimental results. Analysis shows significant deviations from the expected values under the assumption of identical SMRSs in around one fifth of the multiple-choice experiments performed with natural strains of twelve different Drosophila species. Different selection procedures raise the number of significant assortative mating results between strains of D. melanogaster and D. pseudoobscura from 3.0% to 32.8%. Finally, sub- or semispecific taxa show variations in their SMRS even more frequently (74.5%). Differences in male vigor and female receptivity are also found. These results show that a classification of Drosophila species based on SMRS stability, as proposed by the “Recognition concept of species”, is virtually impossible.     

Characterization of free radical-mediated damage of canine cardiac sarcoplasmic reticulum

In vitro generation of free radicals by xanthine oxidase acting on xanthine as substrate depressed steady-state calcium uptake by canine cardiac sarcoplasmic reticulum vesicles. The effect of free radicals on the calcium-dependent ATPase activity of the SR vesicles was pH dependent. At pH 7.0, ATPase activity was decreased, but at pH 6.4 it was unchanged. Exposure to free radicals increased the passive permeability of the vesicles to calcium. This increase was inhibited by superoxide dismutase (SOD) at pH 7.0, and SOD plus mannitol at pH 6.4. The increased permeability per se was insufficient to explain the effects of free radicals on ATPase activity, since the calcium ionophore A23187 was unable to mimick these effects. Direct measurement of the number and turnover of the pump units indicated that the number of units was unchanged but turnover was decreased by free radicals at pH 7.0. The overall data suggest at least two mechanisms of free radical damage, one associated with an increase in passive permeability and another associated with an as yet undefined change in some specific steps of the ATPase reaction.     

pH dependence of lipid peroxidation and albumin oxidative modification: possible implications to the pH paradox

The hydrogen ion concentration may have an important influence in biological free radical reactions. We studied the effect of an acidic pH on two models of free radical-mediated damage: copper-induced lipid peroxidation in plasma and copper/hydrogen peroxide-induced oxidative modification of albumin. A reduction of pH from 7·4 to 6·6 decreased diene conjugation by 32%, thiobarbituric acid-reactive substances formation by 25% and fluorescence generation by 53% in plasma exposed to cupric chloride. At pH values lower than 6·6 an even greater inhibition of lipid peroxidation in plasma was obtained. Visible fluorescence development in albumin by exposure to site-specific generation of free radicals was also increasingly reduced by decreasing pH values. From pH 7·4 to 6·6 there was a 50% fluorescence generation inhibition. The observed partial protection of lipids and proteins against oxidative damage by an acidic pH alerts to the need for rigorously controlling the pH values when assaying compounds for antioxidant properties in vitro. It may also contribute to the explanation for the protective effect of an acidic pH against anoxic cell injury and for cell death that is precipitated by a rapid return to a normal pH following reperfusion (the ‘pH paradox’).     

Lifetimes and NADH quenching of tryptophan fluorescence in pig heart cytoplasmic malate dehydrogenase.

The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.     

Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system

The production of agar-oligosaccharides from agarose by free and immobilized agarase, obtained from a Pseudomonas aeruginosa AG LSL-11 was investigated and the activity, longevity and the operational stability of immobilized enzyme was compared with that of the free enzyme. The agar hydrolyzed products of free enzyme and immobilized enzyme were neoagarobiose, neoagarotetraose and neoagarohexaose as evidenced by LC-MS analysis. The immobilization of agarase was confirmed by SEM and also by the enzymatic transformation of agarose into agaroligosaccharides. The free agarase showed maximum activity at 40°C, whereas it’s immobilized counterpart showed maximum activity at 45oC, however, the optimum pH for both systems remained unchanged (pH 8.0). The relative activities of free agarase at pH 9.0 and 10.0 were 90 and 74%, respectively, whereas, the corresponding activities of the immobilized system were determined to be 97 and 90%. The stabilities of free agarase at pH 9.0 and 10.0 were 80 and 60% respectively, but for the immobilized system the respective residual activities were estimated to be 97 and 85%. Immobilized agarase appears to be more tolerant to high temperatures in terms of its activity and stability as it is compared to that of the free enzyme which retained 74 and 50.84% of relative activity at 55 and 60°C while, free agarase retained only 40 and 16.79% of its original activity. Furthermore, the immobilized agarase could be reused in batches efficiently for eight cycles, and could be stored for 3 months at 4°C as wet beads and for more than 6 months as dry beads.     

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.     

The influence of pH on substrate form specificity of phosphoenolpyruvate carboxylase purified from Crassula argentea

Purified phosphoenolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free mg2+ to selectively use the metal-complexed form of phosphoenolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phosphoenolpyruvate concentrations was obtained for both enzymes, consistent with the use of the complex as the substrate. Kinetic studies at pH 7.0 indicated that at this pH the total concentration of phosphoenolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C.argentea while the C4 enzyme still utilized the complex. The loss of specificity induced by the decrease in the pH of the assay medium was accompanied by a decrease in the Km of this enzyme for phosphoenolpyruvate whatever the form considered and an increase in Vmax/Km. In contrast, a similar decrease of pH led to an increased Km of the C4 enzyme for phosphoenolpyruvate and a decrease of Vmax/Km. For the enzyme from C. argentea (previously shown to contain an essential arginine at the active site), protection of activity by the different forms of substrate against inactivation by the specific arginyl reagent 2,3-butanedione changes markedly with pH. At pH 8.1, the metal complex is the better protector while at pH 7.0 free phosphoenolpyruvate gives the best protection consistent with the observed kinetic changes in substrate form utilization. The relationship between the enzyme affinity for substrate, substrate specificity, and the requirement for magnesium for substrate turnover is discussed.     

Binding to cellular receptors results in increased iron release from transferrin at mildly acidic pH

In order to better understand the cellular delivery of iron from serum transferrin (Tf), we compared iron release from receptor-bound and free Tf. While free Tf did not release all iron until below pH 4.6, receptor-bound Tf released significantly more iron at mildly acidic pH, with essentially all iron released between pH 5.6 and 6.0. Since Tf is acidified to a minimum pH of 5.4 in K562 cells, this result accounts for the nearly complete extraction of iron from Tf by these cells. Comparison of fluorescence from Tf conjugated with lissamine rhodamine sulfonyl chloride (LRSC-Tf) free in solution and bound to receptor provides further evidence that the Tf receptor modulates low pH-mediated conformational changes in Tf. As pH was decreased from neutrality, the fluorescence of free LRSC-Tf began to increase below pH 6.2; the fluorescence of LRSC-Tf bound to human receptors did not increase until below pH 5.6. Binding to the Tf receptor, while facilitating iron release from Tf, appears to partially inhibit a conformational change that causes the increase in LRSC-Tf fluorescence at low pH. The fluorescence of human LRSC-Tf bound to murine receptors increases at a higher pH, 6.0, indicating that there are differences in conformational stabilization of Tf by receptors of different species. The results suggest that the Tf receptor, in addition to providing a means by which cells may internalize Tf, functions to increase the release of iron from Tf in the endosome.     

UPTAKE OF CADMIUM BY FRESHWATER GREEN ALGAE: EFFECTS OF PH AND AQUATIC HUMIC SUBSTANCES1

The effects of humic substances and low pH on short‐term Cd uptake by Pseudokirchneriella subcapitata (Korshikov) Hindak and Chlamydomonas reinhardtii Dang were investigated under defined exposure conditions. The uptake experiments were run in the presence of either a synthetic organic ligand (nitrilotriacetate) or natural organic ligands (Suwannee River fulvic or humic acid). An ion‐exchange method was used to measure the free Cd²⁺ concentrations in the exposure solutions. At pH 5, measured free Cd²⁺ concentrations agreed with estimations made using the geochemical equilibrium model WHAM, but at pH 7 the model overestimated complexation by both Suwannee River fulvic and humic acids compared with the ion‐exchange measurements. Consistent with the metal internalization step being rate limiting for overall short‐term uptake, intracellular Cd uptake was linear for exposure times less than 20 min at pH 5 or pH 7 for both algal species. After taking into account complexation of Cd in solution, Suwannee River humic substances had no additional effects on cadmium uptake at pH 7, as would be predicted by the free ion model. This absence of effects other than complexation persisted at pH 5, where the tendency of humic substances to adsorb to the algal cell surface is favored. Changes in pH strongly influenced Cd uptake, with the intracellular flux of Cd being at least 20 times lower at pH 5 than at pH 7 for P. subcapitata. Our results support models such as the free ion model or the biotic ligand model, in which humic substances act indirectly on Cd uptake by reducing the bioavailability of Cd by complexation in solution.     

Studies on the Water-Insoluble Enzyme

Water-insoluble yeast invertase was prepared by binding native invertase to DEAE-cellulose. Some characteristics of the bound invertase and the continuous hydrolysis of sucrose by use of it are described. The activity of bound invertase corresponded to about 1/2 at pH 3.4 when compared with the maximum activity of free form and it could hydrolyze sucrose into invert sugar perfectly. The apparent optimum pH of bound invertase was shifted toward acid pH by about 2 pH units in comparison with free invertase. Stability of bound invertase to temperature was slightly less in comparison with free invertase at pH 5.2. The continuous sucrose hydrolysis was carried out using bound invertase at pH 3.6 and it could be used about ten times until the hydrolysis ratio decreased to the half of the initial.     

Stabilization of retinol through incorporation into liposomes

Chemical and photochemical processes during storage and preparation rapidly degrade retinol, the most active form of vitamin A. Therefore, the efficacy of incorporation into liposomes in order to modulate the kinetics of retinol degradation was investigated. Retinol was readily incorporated into multilamellar liposomes that were prepared from soybean phosphatidylcholine; the extent of the incorporation was 98.14 +/- 0.93% at pH 9.0 at a ratio of 0.01 : 1 (wt : wt) retinol : phospholipid. It was only marginally lower at higher retinol concentrations. The pH of the hydration buffer had a small effect. The incorporation efficiency ranged from 99.25 +/- 0.47% at pH 3 to 97.45 +/- 1.13% at pH 11. The time course of the retinol degradation in the aqueous solution in liposomes was compared to that of free retinol and free retinol with alpha-tocopherol under a variety of conditions of pH (3, 7, and 11), temperature (4, 25, 37, and 50 degrees ), and light exposure (dark, visible, and UV). The retinol that was incorporated into the liposomes degraded significantly slower than the free retinol or retinol with alpha-tocopherol at pH 7 and 11. At pH 3, where the free retinol degrades rapidly, the degradation kinetics were similar in liposomes and the presence of alpha-tocopherol. At pH 7.0 and 4 degrees in the light, for example, free aqueous retinol was completely degraded within 2 days, while only 20% of the retinol in the liposomes were degraded after 8 days. In general, the protective effect of the liposome incorporation was greater at low temperatures, at neutral and high pH, and in the dark. The results suggest that protection is greater in the solid, gel phase than in the fluid liquid crystalline phase lipids. These results indicate that the incorporation into liposomes can extend the shelf-life of retinol under a variety of conditions of temperature, pH, and ambient light conditions.     

Immobilization of urease on gelatin — poly (HEMA) copolymer preparation and characterization

Urease was immobilized onto gelatin-poly (HEMA) copolymer by covalent linkage. Maximum amount of urease was immobilized onto the support at a pH of 8.5. The optimal pH of the immobilized urease was similar to that of free urease; the optimal temperature showed an increase of 10 °C over the free enzyme. The stability of the immobilized urease for a range of pH, temperature and shelf life was greater than the corresponding values for the free enzyme. The same result was obtained for k _m also.Grateful acknowledgement is made to CSIR, Govt. of India for the research associateship conferred on Dr. M. Chellapandian which helped the progress of this piece of research investigation.