Optimal Shift Research Articles

Glycine-assisted enhancement of 1,4-β-d-xylan xylanohydrolase activity at alkaline pH with a pH optimum shift

This is the first report describing the enhancement of xylanase activity by the neutral amino acid glycine. Xylanase activity is increased seven-fold at alkaline pH in the presence of glycine and its pH optimum is shifted from pH 7 to 8 without using any protein engineering techniques. Analysis of the steady-state kinetics revealed that glycine in the reaction mixture increases the K(m) and k(cat) values of the enzyme. Chemoaffinity labeling and studies using glycine esters indicate an involvement of the carboxylate ion of glycine in enhancing xylanase catalytic activity. A novel possible mechanism for the glycine-assisted catalytic action of xylanase is proposed.

Intensity of eccentric exercise, shift of optimum angle, and the magnitude of repeated-bout effect

This study compared the effect of four different intensities of initial eccentric exercise (ECC1) on optimum angle shift and extent of muscle damage induced by subsequent maximal eccentric exercise. Fifty-two male students were placed into 100%, 80%, 60%, or 40% groups (n = 13 per group), performing 30 eccentric actions of the elbow flexors of 100%, 80%, 60%, or 40% of maximal isometric strength [maximal voluntary contraction (MVC)] for ECC1, followed 2-3 wk later by a similar exercise (ECC2) that used 100% MVC load. MVC at six elbow joint angles, range of motion, upper arm circumference, serum creatine kinase activity, myoglobin concentration, and muscle soreness were measured before and for 5 days following ECC1 and ECC2. A rightward shift of optimum angle following ECC1 was significantly (P < 0.05) greater for the 100% and 80% than for the 60% and 40% groups, and it decreased significantly (P < 0.05) from immediately to 5 days postexercise. By the time ECC2 was performed, only the 100% group kept a significant shift (4 degrees). Changes in most of the criterion measures following ECC1 were significantly greater for the 100% and 80% groups compared with the 60% and 40% groups. Changes in the criterion measures following ECC2 were significantly (P < 0.05) greater for the 40% group compared with other groups. Although the magnitude of repeated bout effect following ECC2 was significantly (P < 0.05) smaller for the 40% and 60% groups, all groups showed significantly (P < 0.05) reduced changes in criterion measures following ECC2 compared with the ECC1 100% bout. We conclude that the repeated-bout effect was not dependent on the shift of optimum angle.

CT-guided intensity-modulated radiotherapy for bladder cancer: Isocentre shifts, margins and their impact on target dose

Background and purpose Image-guided radiotherapy (IGRT) has great potential to improve the treatment of tumour sites that exhibit large geometrical uncertainties such as the bladder. This study quantifies the size and direction of the daily 3D isocentre shift resulting from the use of IGRT and its impact on reducing the margins required. The changes to target dose coverage, delivered dose and dose homogeneity appearing when used in conjunction with intensity-modulated radiotherapy (IMRT) are also assessed. Materials and methods The study is performed using a series of 19 bladder cancer patients that underwent weekly repeat conventional CT scanning during their 6–7 weeks treatment course. The isotropic margin required to cover various percentages of the volumes of the repeat scans CTVs is found by growing the planning scan PTV by incrementing the margin in steps of 1 mm until full coverage is obtained. The optimum daily isocentre shift is determined by moving the repeat scan CTV in 3D so that its volume that lies outside the planning CTV is minimised. Reduction in the isotropic margin after applying the optimum isocentre shift is found by repeating the above procedure. Individual optimum sizes of the six margins required in the sup/inf/ant/post/right/left directions are determined using a recently published empirical margin determination method. Finally, five-field IMRT plans are set up (that minimise the dose deviation throughout the target) using different planning CTV margin alternatives, the optimal isocentre shifts are applied and the target dose distributions are assessed after recalculation without changing any of the beam parameters. Results Without use of IGRT (i.e., without shifting to the optimal isocentre), an isotropic margin of 30 mm is required to cover fully all the CTV volumes on the repeat scans for all patients. The direction of the optimum isocentre shift required is random with an average directional shift less than 1 mm and is unlikely to exceed an absolute value of 15 mm (average of 7 mm). Applying the optimum isocentre shift reduces the isotropic margin to 16 mm for full volume coverage. Determining the optimum margin individually in each of six orthogonal directions reduces the target volume by approximately 30% but requires complex daily planning. Applying the optimum isocentre shift to IMRT plans shows little change to the overall mean target dose of 100% (an average increase of 1%), but produces a spread in the daily mean dose ranging from 96% to 106%. The 3D dose variation over the target is within the 95–107% acceptable range in 72% of cases for a 12 mm uniform margin, which increases to 91% if any deviation from the daily prescribed dose is removed. The minimum and maximum doses within the target can show significant changes. Conclusion The use of IGRT in the treatment of bladder cancer leads to a marked reduction in the margins required. When used in conjunction with IMRT, the pre-treatment plan is shown to be acceptable for daily treatment (after shifting the isocentre) in terms of the resulting dose distribution provided the correct daily mean dose is delivered. That can be achieved by rescaling the daily monitor units for each treatment beam.

The iminosugar isofagomine increases the activity of N370S mutant acid β-glucosidase in Gaucher fibroblasts by several mechanisms

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid beta-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 +/- 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Response Times and Their Temperature Dependence Measured for a Series of Liquid Crystals Using Gain Spectra of Stimulated Light Scattering

In the present work we have shown the opportunity of using the process of stimulated light scattering due to recording of dynamic orientation gratings for directly measuring the characteristic orientation relaxation time of liquid crystals (LCs) and related material parameters, the elastic constants and the coefficients of orientational viscosity. The measurement consists in obtaining the gain spectra of LCs and determining the magnitude of the gain coefficient and the optimum frequency shift between interfering laser beams. We have designed a setup where the gain spectra of stimulated light scattering process in LCs are obtained within seconds. These spectra are highly specific to LCs serving as a precise and fast tool for studying LC materials and their mixtures with the purpose of optimizing their linear and nonlinear optical properties. Using this technique we have found that certain azo LC materials are faster compared to several well-known Merck's formulations for LC displays.

The Genetic Basis of Thermal Reaction Norm Evolution in Lab and Natural Phage Populations

Two major goals of laboratory evolution experiments are to integrate from genotype to phenotype to fitness, and to understand the genetic basis of adaptation in natural populations. Here we demonstrate that both goals are possible by re-examining the outcome of a previous laboratory evolution experiment in which the bacteriophage G4 was adapted to high temperatures. We quantified the evolutionary changes in the thermal reaction norms—the curves that describe the effect of temperature on the growth rate of the phages—and decomposed the changes into modes of biological interest. Our analysis indicated that changes in optimal temperature accounted for almost half of the evolutionary changes in thermal reaction norm shape, and made the largest contribution toward adaptation at high temperatures. Genome sequencing allowed us to associate reaction norm shape changes with particular nucleotide mutations, and several of the identified mutations were found to be polymorphic in natural populations. Growth rate measures of natural phage that differed at a site that contributed substantially to adaptation in the lab indicated that this mutation also underlies thermal reaction norm shape variation in nature. In combination, our results suggest that laboratory evolution experiments may successfully predict the genetic bases of evolutionary responses to temperature in nature. The implications of this work for viral evolution arise from the fact that shifts in the thermal optimum are characterized by tradeoffs in performance between high and low temperatures. Optimum shifts, if characteristic of viral adaptation to novel temperatures, would ensure the success of vaccine development strategies that adapt viruses to low temperatures in an attempt to reduce virulence at higher (body) temperatures.

Microwave properties of Ba0.5Sr0.5TiO3 thin film coplanar phase shifters

Coplanar waveguide transmission lines have been used to show that the temperature dependent properties of Ba0.5Sr0.5TiO3 thin films used for microwave phase shifters in the frequency range 45MHz–50GHz are correlated strongly with the microstructure of the films. The highest tunability and figure of merit of the phase shifters were obtained for films with the narrowest ferroelectric-paraelectric phase transition range, lowest mosaic spread, and widest columnar microstructure. The study also showed that the operating temperature plays an important role in achieving the optimum phase shift for microwave applications.

Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.

An extracellular alkaline protease from a novel haloalkaliphilic bacterium (Ve1) was purified to the homogeneity, with a molecular weight of 30–32 kDa. The bacterium was related to Bacillus pseudofirmus on the basis of 16S rRNA gene sequencing. The enzyme was active in the range of pH 8.5–12 with the optimum at 10–11. The requirement of the salt for enzyme catalysis was increased on increasing temperature and a shift in temperature optima from 37 to 55 °C was evident in the presence of 2% salt. The enzyme was highly stable at 37 °C and retained 50% activity at 45 °C. However, the enzyme was quite unstable at temperatures beyond 55 °C. However, the enzyme was quite stable at higher temperature in the presence of NaCl and CaCl 2. The enzyme was stable with different surfactants; in-fact, SDS and Triton X-100 were slightly stimulatory. The activity was affected by mono and divalent cations to varying extent. The protease was sensitive to urea denaturation and the renaturation of denatured protein under in vitro conditions was affected by different factors. While protein concentration played pronounced role in the renaturation by dialysis (89% renaturation); pH, salt and redox conditions did not affect the renaturation significantly. The study on this enzyme assumes significance in the light of dual extremities of pH and salt coupled with moderate temperature stability.

The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.

Catalytic potential of a poly(AAc‐co‐HPMA‐cl MBAm)‐matrix‐immobilized lipase from a thermotolerant Pseudomonas aeruginosa MTCC‐4713

AbstractA purified alkaline thermo‐tolerant lipase from Pseudomonas aeruginosa MTCC‐4713 was immobilized on a series of five noble weakly hydrophilic poly(AAc‐co‐HPMA‐cl MBAm) hydrogels. The hydrogel synthesized by copolymerizing acrylic acid and 2‐hydroxy propyl methacrylate in a ratio of 5 : 1 (HG5:1 matrix) showed maximum binding efficiency for lipase (95.3%, specific activity 1.96 IU mg−1 of protein). The HG5:1 immobilized lipase was evaluated for its hydrolytic potential towards p‐NPP by studying the effect of various physical parameters and salt‐ions. The immobilized lipase was highly stable and retained ∼92% of its original hydrolytic activity after fifth cycle of reuse for hydrolysis of p‐nitrophenyl palmitate at pH 7.5 and temperature 55°C. However, when the effect of pH and temperature was studied on free and bound lipase, the HG5:1 immobilized lipase exhibited a shift in optima for pH and temperature from pH 7.5 and 55°C to 8.5 and 65°C in free and immobilized lipase, respectively. At 1 mM concentration, Fe3+, Hg2+, NH4+, and Al3+ ions promoted and Co2+ ions inhibited the hydrolytic activities of free as well as immobilized lipase. However, exposure of either free or immobilized lipase to any of these ions at 5 mM concentration strongly increased the hydrolysis of p‐NPP (by ∼3–4 times) in comparison to the biocatalysts not exposed to any of the salt ions. The study concluded that HG5:1 matrix efficiently immobilized lipase of P. aeruginosa MTCC‐4713, improved the stability of the immobilized biocatalyst towards a higher pH and temperature than the free enzyme and interacted with Fe3+, Hg2+, NH4+, and Al3+ ions to promote rapid hydrolysis of the substrate (p‐NPP). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4252–4259, 2006

Kinetics of the DNA polymerase pyrococcus kodakaraensis

The polymerase chain reaction is one of the most important reactions in molecular biology. Single stranded DNA is copied in a complex series of steps, at the core of which lies the action of the DNA polymerase. At each nucleotide along the template, the polymerase screens the dNTP pool until it finds the complementary dNTP. The insertion of each dNMP is a balance between high fidelity and rapid elongation. In this study the kinetics of the β type polymerase pyrococcus kodakaraensis (KOD) is analyzed. The kinetics is influenced by reaction conditions such as the dNTP pool composition and temperature. In a previous study by Viljoen et al. [2005, A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection. Computational Biology and Chemistry 29, 101–110], a macroscopic kinetics expression of the polymerase chain reaction has been derived. The model contains four parameters that are intrinsic to a specific polymerase. The experiments to measure the temperature-dependence of the parameters for KOD DNA polymerase are reported. The results indicate that the optimal temperature for an equimolar dNTP pool is 72.5 ∘ C and the optimum temperature shifts to lower temperatures when the dNTP pool composition is biased.

Is there an optimal ribosome concentration for maximal protein production?

A core model is presented for protein production in Escherichia coli to address the question whether there is an optimal ribosomal concentration for non-ribosome protein production. Analysing the steady-state solution of the model over a range of mRNA concentrations, indicates that such an optimum ribosomal content exists, and that the optimum shifts to higher ribosomal contents at higher specific growth rates.

A novel reactive perstraction system based on liquid‐core microcapsules applied to lipase‐catalyzed biotransformations

A novel reactive perstraction system has been developed based on liquid-core capsules, involving an enzyme-catalyzed reaction coupled with simultaneous in situ product recovery. Lipase-catalyzed reactions, hydrolysis of triprionin and nitrophenyl laurate, were selected to test the system and demonstrate the feasibility of immobilization of enzymes to the membranes of liquid-core capsules and the ability to extract hydrophobic products of the reaction within the capsule core. The lipase from Candida rugosa was immobilized to the microcapsules by adsorption and by covalent binding through activation with glutaraldehyde. In both cases improved temperature and operational stability were achieved. Both types of immobilization resulted in a basic shift of the pH optimum for activity, from 7.5 to 9.0. The presence of an organic phase within the capsule core allowed direct product separation and lead to a decrease in product inhibition of the lipase-catalyzed reaction.

Properties of immobilized pepsin on Modified PMMA microspheres

In this work we use micro-size poly(methyl methacrylate)/acrylaldehyde microspheres as a support for pepsin immobilization. The aldehyde groups on the microspheres offer a very simple, mild and firm combination for enzyme immobilization. The amount of enzyme we can bind to this support reaches 82 mg/g, which is much higher than for other supports (mostly less than 10 mg/g). Compared to free enzyme, the Km of immobilized enzyme is increased, whereas the Vmax is decreased. Further, the Vmax/Km value for immobilized pepsin is about 50% of the value for free enzyme. This is better than values reported previously, generally lower than 35%. The optimum temperature shifts from 43 degrees C for free pepsin to 47 degrees C. However, the optimum pH does not change between free and immobilized enzyme. This improved resistance of the immobilized enzyme towards changes in temperature and pH also shows that the aldehyde modified poly(methyl methacrylate)/acrylaldehyde microspheres can be a valuable support for pepsin immobilization.

Thermal reaction norms in two Coenagrion damselfly species: contrasting embryonic and larval life‐history traits

Summary1. We studied the temperature‐dependence of important life‐history traits both at the embryonic (egg hatching success, embryonic development time and hatchling size) and the larval stage (larval growth rate, larval survival and larval size after 100 days) using full‐sib families of two congeneric damselflies, Coenagrion hastulatum and Coenagrion puella, that differ in latitudinal distribution. Larvae were reared in the laboratory from the egg stage at four temperatures (12, 17, 22 and 27 °C).2. The observed patterns of thermal plasticity in embryonic traits showed that the northern species was more successful than the southern species at lower temperatures, in line with the pattern of temperature adaptation in thermal reaction norms.3. At the larval stage, we found no consistent pattern of latitudinal compensation. The thermal family reaction norms indicate, however, the potential for latitudinal compensation to evolve. We observed an ontogenetic shift in thermal optima for larval growth rate, with a higher optimal temperature for growth rate during the first 2 weeks of the larval stage.4. This is the first indication of the existence of latitudinal compensation at the interspecific level in an invertebrate; it is stage‐specific, being present only in the embryonic stage. We argue that compensation in the embryonic stage may be much more likely than in the larvae and stress the importance of including more then one life‐history stage when drawing conclusions about the adaptiveness of patterns in thermal reaction norms.

Mutations of the PC2 Substrate Binding Pocket Alter Enzyme Specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.

Advances in array detectors for X-ray diffraction techniques

Improved focal plane array detector systems are described which can provide improved readout speeds, random addressing and even be employed to simultaneously measure position, intensity and energy. This latter capability promises to rekindle interests in Laue techniques. Simulations of three varieties of foil mask spectrometer in both on- and off-axis configurations indicate that systems of stacked silicon detectors can provide energy measurements within 1% of the true value based on the use of single 'foils' and approximately 10000 photons. An eight-detector hybrid design can provide energy coverage from 4 to 60 keV. Energy resolution can be improved by increased integration time or higher flux experiments. An off-axis spectrometer design in which the angle between the incident beam and the detector system is 45 degrees results in a shift in the optimum energy response of the spectrometer system. In the case of a 200 microm-thick silicon absorber, the energy optimum shifts from 8.7 keV to 10.3 keV as the angle of incidence goes from 0 to 45 degrees. These new designs make better use of incident photons, lower the impact of source flicker through simultaneous rather than sequential collection of intensities, and improve the energy range relative to previously reported systems.

Quantitative assay of senescence-associated β-galactosidase activity in mammalian cell extracts

Senescence-associated β-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal β-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained. This article describes a quantitative method for measuring this activity and characterizes the method using extracts from senescent, quiescent, and presenescent human fibroblasts. The assay is capable of detecting relatively subtle changes in activity and confirms previous indications based on staining that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. Investigation of the pH dependence of the activity in the cell extracts suggests that the senescent phenotype is correlated with an increase in total β-galactosidase rather than with a shift in the pH optimum of the enzyme. This assay for measuring senescence-associated changes in β-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated.

Production of amino acids from bovine serum albumin by continuous sub-critical water hydrolysis

The aim of this work was the determination of the best experimental conditions for the production of amino acids from bovine serum albumin (BSA) by continuous sub-critical water hydrolysis. The results were compared with conventional acid hydrolysis in HCl. At a residence time of 30 s and a pressure of 25 MPa, the highest amount of amino acids was obtained at 583 K. An increase in residence time to 90 s led to a shift of the temperature optimum to 543 K. The highest amino acid yield in sub-critical water was obtained at 563 K and 65 s. No significant influence of operating pressure (15–27 MPa) could be observed at the tested temperature (523 K) and residence time (30 s). The addition of carbon dioxide led to an increase in amino acid yield due to the acceleration of acid hydrolyzed catalysis steps. This protein treatment may provide a practical and economical solution for the disposal of protein-rich sources like marine wastes, hairs, and feathers, which are considered as waste so far.

Temperature Controls a Dependence of Barley Plant Growth on Mineral Nutrition Level

The effect of temperature regime on growth and other morphological characteristics of barley plants (Hordeum distichum L., cv. Andrei) as dependent on the level of mineral nutrition was investigated in a controlled experiment. Plants were raised hydroponically at a high (0.22 g/(g day)) and low (0.05 g/(g day)) relative rates of the addition of mineral nutrients (R A). Mineral nutrients were daily added to the nutrient solutions in exponentially increased amounts to provide steady-state plant growth. At the optimum temperature regime (21/17°C, day/night), the plant relative growth rate (RGR) was proportional to the preset R A during the entire exponential period. Low R A led to a decrease in the nitrogen content in plants, plant weight, and respiratory activity, as well as to the increase in the relative root weight. Biomass accumulation at lowered temperature regime (13/8°C) and a high R A was 1.8-fold lower than at optimum temperature regime. Although under these conditions, the ratio of respiration to gross photosynthesis reduced threefold due to the decrease in the respiration rate, RGR of plants was equal to 0.11 ± 0.02 g/(g day), which was twice lower than the preset R A. These pointed to the decrease in plant ability to maintain a certain ratio of photosynthesis to respiration within a day. At a deficiency of mineral nutrition and low temperature, RGR reached the preset R A. Plants adapted to lowered temperature by a shift of the temperature optimum of their metabolism (heat production) to lower values. As a whole, a low variability of such growth parameters as RGR, C/N, and root to shoot weight ratio at different R A and lowered temperatures testified to the lessening of growth limitation by the mineral nutrition.