The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

Effects of different heat–moisture treatments on the physicochemical properties of brown rice flour

We evaluated the effect of heat–moisture treatment (HMT) on the main chemical components, physical properties, and enzyme activities of two types of brown rice flour: high-amylose Koshinokaori and normal-quality Koshiibuki. Five different HMTs using brown rice (moisture content was 12.0%) were assessed: 0.1 MPa/120 °C for 5 or 10 min, 0.2 MPa/134 °C for 5 or 10 min and 0.3 MPa/144 °C for 10 min. HMT, decreased the α-amylase and lipase activities, and fat acidity, and slightly increased the dietary fiber and resistant starch levels. After 2 months’ storage at 35 °C, rice samples that were treated with 0.2 MPa/134 °C or 0.3 MPa/144 °C for 10 min had a lower fat acidity than untreated samples, which would be useful for long-term storage and export of rice flour. And HMT exhibited inhibition of retrogradation in the pasting and physical properties, which is profitable to promote the qualities of the rice products.     

Effects of freeze–thaw and micro-computed tomography irradiation on structure–property relations of porcine trabecular bone

We study the effects of freeze–thaw and irradiation on structure–property relations of trabecular bone. We measure the porosity, apparent density, mineral content, trabecular orientation, trabecular thickness, fractal dimension, surface area, and connectivity of trabecular bone using micro-computed tomography (micro-CT) and relate them to Young?s modulus and ultimate strength measured by uniaxial compression testing. The analysis is done on six-month porcine trabecular bone from femoral heads. The effects of freeze–thaw are studied by using bones from three different groups: fresh bone and bones frozen for one and five years. We find that the porosity and apparent density have most dominant influence on the elastic modulus and strength of fresh bone. Also, five years of freezing lowers both Young?s modulus and ultimate strength of trabecular bone. Additionally, the effects of radiation are investigated by comparing Young?s modulus before and after micro-CT exposure. We find that the micro-CT irradiation has a negligible effect on the Young?s modulus of trabecular bone. These findings provide insights on the effects of tissue preservation and imaging on properties of trabecular bone.     

The effect of “aging” of fibrinogen molecule on the structure and properties of fibrin gel

The effect of molecular “aging” of fibrinogen stimulated by preincubation in solution on the fibrin three-dimensional architecture, its ability to crosslink fibrin-stabilizing factor, and the sensitivity of fibringel to plasmin hydrolysis have been studied. The method of elastic light scattering was used to demonstrate that fibrin generated from “defective” fibrinogen had a coarser structure with a higher mean mass-length ratio of polymeric fibers compared to native fibrinogen (2.24 × 10⁹ and 1.46 × 10⁹ g/(mol cm), respectively). Crosslinking had no effect on the architecture of both control and experimental fibrin samples. Spectrophotometric and electrophoretic analysis has shown a higher sensitivity of coarse fibrin gels to plasmin. A close correlation between spontaneous local conformational reconstructions in fibrinogen molecule and its functional activity is concluded.     

Effects of in combination antioxidant supplementation on microscopic and oxidative parameters of freeze–thaw bull sperm

The present study was conducted to determine the effect of reduced glutathione (GSH) and superoxide dismutase (SOD) supplementation in the bull semen freezing extender on post-thaw parameters of Holstein and Simmental bull sperm. Semen were collected from seven bulls (four Holstein and three Simmental) and cryopreserved in the Tris-egg-yolk based extender as control group and supplemented with various concentrations of GSH × SOD (5 × 100, 7.5 × 100, 5 × 150, and 7.5 × 150 mM × IU mL^?1) in treatment groups. Microscopic parameters were evaluated in terms of total motility parameters using computer assisted semen analysis and viability and membrane integrity were assessed using Eosin–Nigrosin stains and hypo-osmotic swelling test (HOST), respectively after thawing the semen. Malonaldialdehyde (MDA) level, SOD and glutathione peroxides (GPx) activities were assessed immediately after thawing. Results showed that supplementation of the cryopreservation medium with various concentrations of GSH × SOD improved total motility (TM) and progressive motility parameters for Holstein (P < 0.05) semen, and values of TM and HOST for Simmental semen compared to the control group (P < 0.01) after semen thawing. Addition of antioxidant to Holstein semen samples decreased the level of MDA and increased GPx activities compared to control groups (P < 0.05). SOD activities increased in Simmental bull samples compare to the control group (P < 0.01), but not differ in Holstein, while these activities. In conclusion, supplementation of antioxidant to the semen extender as combination (GSH × SOD) improved the semen post-thaw qualities which may be associated with a reduction in lipid peroxidation as well as an increase in the antioxidant enzyme activities.     

The effect of vitamin A deficiency and dietary α-tocopherol on the stability of rat-liver lysosomes

1. Vitamin A-deficient rats and pair-fed controls were maintained on either normal or raised amounts of dietary alpha-tocopherol. 2. Their livers were fractionated and ;free' and ;total' lysosomal phosphatase were determined in the various fractions. The rate of release of this enzyme was determined in the mitochondria-lysosome-rich fraction during incubation at pH5 and 37 degrees . 3. The deficient livers showed increased enzymic activity. 4. Prolonged incubation caused more rapid enzyme release from the mitochondria-lysosome-rich fraction of the vitamin A-deficient rats receiving the normal amount of dietary alpha-tocopherol than from the equivalent fraction of their pair-fed controls receiving vitamin A. Raised dietary alpha-tocopherol reversed this phenomenon.     

Effects of soil freeze–thaw cycles differ between experimental plant communities

Soil freeze–thaw cycles (FTC) influence nutrient cycling, but their consequences for productivity and composition of vegetation are not well investigated. Ongoing global warming will increase the recurrence of FTC in cool-temperate and high-latitude regions.Here, we report on the above- and belowground biomass production as well as the nitrogen nutrition of two common vegetation types, grassland and heath, after more frequent FTC in a controlled field experiment in Central Europe. Furthermore, we analyze the duration of the observed effects. Five FTC were induced by buried heating wires in addition to three naturally occurring FTC during winter 2005/06. More frequent FTC significantly increased aboveground production of experimental grassland early in the following growing season. However, no reaction was found for experimental heath within the first growing season. Biomass production of heath communities dropped significantly and C/N ratio increased in the freeze–thaw treated plots in the second year after the manipulation, whereas production in the grassland communities was no longer affected significantly, except for an increase in C/N ratio. This response can at least partly be explained by changes in nutrient availability, as plant available nitrate increased in the manipulated grassland plots and decreased in the manipulated heath plots.The results show the high ecological importance of climate changes during winter, with the outcomes differing strongly between contrasting vegetation types. Furthermore, we show that short term climatic events can cause long-lasting effects, sometimes emerging in the vegetation only after considerable time lags (here: one growing season).     

The effect of aging on brain barriers and the consequences for Alzheimer’s disease development

Life expectancy has increased in most developed countries, which has led to an increase in the proportion of elderly people in the world’s population. However, this increase in life expectancy is not accompanied by a lengthening of the health span since aging is characterized with progressive deterioration in cellular and organ functions. The brain is particularly vulnerable to disease, and this is reflected in the onset of age-related neurodegenerative diseases such as Alzheimer’s disease. Research shows that dysfunction of two barriers in the central nervous system (CNS), the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier (BCSFB), plays an important role in the progression of these neurodegenerative diseases. The BBB is formed by the endothelial cells of the blood capillaries, whereas the BCSFB is formed by the epithelial cells of the choroid plexus (CP), both of which are affected during aging. Here, we give an overview of how these barriers undergo changes during aging and in Alzheimer’s disease, thereby disturbing brain homeostasis. Studying these changes is needed in order to gain a better understanding of the mechanisms of aging at the brain barriers, which might lead to the development of new therapies to lengthen the health span (including mental health) and reduce the chances of developing Alzheimer’s disease.     

Studies of the effect of organic solvents on the stability of β-glucosidase fromFusarium oxysporum

Summary The effect of organic solvents on the stability of -glucosidase in a powder form, isolated fromFusarium oxysporum, has been studied using several organic solvents of different degree of hydrophobicity. It was found that -glucosidase remains quite stable after a prolonged incubation in the presence of most of the organic solvents used, even at temperatures as high as 70°C. Only dimethyformamide (DMF) and tetrahydrofuran (THF) reduce considerably the enzyme activity in a short preincubation period. Studies on the effect of the pH of the buffer used prior to lyophilization, as well as of exogenous added water to the incubation mixture, on enzyme stability show that it is more stable in pH 5.0 and in the lowest water content. In addition it was found that the presence of glucose in the lyophilization procedure gives a significant protection to the enzyme when it is incubated for 30 h in pentanol and n-hexane.     

The sarcosine effect on protein stability: A case of nonadditivity?

We have used differential scanning calorimetry to determine the effect of low concentrations (C = 0-2 M) of the osmolyte sarcosine on the Gibbs energy changes (deltaG) for the unfolding of hen-egg-white lysozyme, ribonuclease A, and ubiquitin, under the same buffer and pH conditions. We have also computed this effect on the basis of the additivity assumption and using published values of the transfer Gibbs energies for the amino acid side chains and the peptide backbone unit. The values thus predicted for the slope delta deltaG/deltaC agree with the experimental ones, but only if the unfolded state is assumed to be compact (that is, if the accessibility to solvent of the unfolded state is modeled using segments excised from native structures). The additivity-based calculations predict similar delta deltaG/deltaC values for the three proteins studied. We point out that, to the extent that this approximate constancy of delta deltaG/deltaC holds, osmolyte-induced increases in denaturation temperature will be larger for proteins with low unfolding enthalpy (small proteins that bury a large proportion of apolar surface). The experimental results reported here are consistent with this hypothesis.     

The effect of context on the folding of β-hairpins

Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.     

The effect of chondroitin sulphate–protein on the formation of collagen fibrils in vitro

1. It was found that the precipitation of collagen fibrils at 37 degrees from mixtures of chondroitin sulphate-protein and tropocollagen at physiological ionic strength and pH takes place in two distinct phases. The first occurs immediately on mixing either at 4 degrees or at 37 degrees , and the second occurs only at 37 degrees and after a lag phase whose magnitude depends on the proportions of components. 2. When the second stage of precipitation was inhibited by mixing the reactants at 4 degrees , the initial precipitate was found to contain ;native-type' collagen fibrils and chondroitin sulphate-protein. 3. On the basis of kinetic experiments it was concluded that aggregates of chondroitin sulphate-protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils. 4. The gels that result after continued incubation at 37 degrees are fibrous in appearance if formed in the presence of the initial precipitate of chondroitin sulphate-protein and tropocollagen. 5. On the basis of these experiments in vitro the authors propose a sequence of events for collagen fibrogenesis in vivo.     

The effect of recrystallization of aqueous solutions of metal sulfates on the acid–base balance

A comparative study of pH values in recrystallized aqueous solutions of manganese, copper, and iron sulfates and the calculated values for the pH levels of similar solutions obtained directly from solid salts has been performed for the first time. The trend for the positive deviation of the pH_rec values from the pH_calc values has been established. The positive deviation was the strongest in the case of iron sulfate solutions. The sample of the recrystallized aqueous solution (recrystallized water) consisted of water obtained by melting of the initial aqueous solution after freezing of 60% of the solution (by volume). Four hypotheses were proposed to explain the positive deviation of pH_rec from pH_calc: (1) low СО₂ concentration; (2) more efficient adsorption of anions on the freezing front compared to that of cations; (3) mechanochemical and radiation chemical processes that occur upon the melting of ice; and (4) Fe²⁺-catalyzed decomposition of hydrogen peroxide formed in the recrystallized solution. Analysis of the putative causes showed that the fourth hypothesis provided the most adequate explanation of the difference between pH_rec and pH_calc. The formation of hydrogen peroxide in recrystallized water is proposed to occur due to the recombination of OH radicals formed during the radiation chemical processes.     

The effect of physical training on the N-methyl-N-nitrosourea-induced mammary carcinogenesis of Sprague–Dawley rats

The impact of physical activity on carcinogenesis has been demonstrated in many studies. Taking into account the discrepant results of physical exercise on the cell proliferation and apoptosis of breast cancer, we aimed to examine the impact of physical training on N-methyl-N-nitrosourea-(MNU)-induced mammary carcinogenesis. Fifty female rats were divided into four groups according to the intensity of physical activity they undertook. The number of developed tumors, tumor volume, and histopathological diagnoses were noted. Apoptosis and cell proliferation were studied by the number of TUNEL-positive and Ki-67-expressing cells. We demonstrated a statistically significant decrease in the tumor number between all trained groups and the control group. The results were most pronounced in the group with a moderate intensity of training. Moreover, we showed a decrease in tumor volume as training intensity increased, though the differences were not statistically significant. The mean number of TUNEL-positive cancer cells was significantly higher in the training groups than in the control group. These data suggest that physical training, especially of moderate intensity, may alleviate MNU-induced mammary carcinogenesis. The results could suggest that physical exercise-induced apoptosis may be a protective mechanism.     

The effect of genetic complexity on the time-course of ribonucleic acid–deoxyribonucleic acid hybridization

1. The rate of RNA-DNA hybridization was studied under conditions of RNA excess, with RNA synthesized in vitro. The initial rate of the reaction was proportional to the initial RNA concentration. Throughout the observed course of the reaction there was a linear relationship between the reciprocal of the amount of RNA hybridized/mug. of DNA and the reciprocal of time. The slope of the reciprocal plot was inversely proportional to the initial RNA concentration. 2. A comparison was made of the hybridization of DNA from Escherichia coli and from bacteriophages T4 and lambda with homologous RNA. The initial rate of hybridization was inversely proportional to the genetic complexity of the hybridizing system. The slope of the reciprocal-time plot was directly proportional to genetic complexity. These results are interpreted to indicate that the rate of hybridization reflects the mean concentration of the various unique RNA species in a preparation.     

Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.     

The effect of disturbance on an ant–plant mutualism

Protective ant–plant mutualisms—where plants provide food or shelter to ants and ants protect the plants from herbivores—are a common feature in many ecological communities, but few studies have examined the effect of disturbance on these interactions. Disturbance may affect the relationship between plants and their associated ant mutualists by increasing the plants’ susceptibility to herbivores, changing the amount of reward provided for the ants, and altering the abundance of ants and other predators. Pruning was used to simulate the damage to buttonwood mangrove (Conocarpus erectus) caused by hurricanes. Pruned plants grew faster than unpruned plants, produced lower levels of physical anti-herbivore defenses (trichomes, toughness), and higher levels of chemical defenses (tannins) and extrafloral nectaries. Thus, simulated hurricane damage increased plant growth and the amount of reward provided to ant mutualists, but did not have consistent effects on other anti-herbivore defenses. Both herbivores and ants increased in abundance on pruned plants, indicating that the effects of simulated hurricane damage on plant traits were propagated to higher trophic levels. Ant-exclusion led to higher leaf damage on both pruned and upruned plants. The effect of ant-exclusion did not differ between pruned and unpruned plants, despite the fact that pruned plants had higher ant and herbivore densities, produced more extrafloral nectaries, and had fewer physical defenses. Another common predator, clubionid spiders, increased in abundance on pruned plants from which ants had been excluded. I suggest that compensatory predation by these spiders diminished the effect of ant-exclusion on pruned plants.     

The effect of landscape heterogeneity on host–parasite dynamics

Environmental heterogeneity has been shown to have a profound effect on population dynamics and biological invasions, yet the effect of its spatial structure on the dynamics of disease invasion in a spatial host–parasite system has received little attention. Here we explore the effect of environment heterogeneity using the pair approximation and the stochastic spatially explicit simulation in which the lost patches are clustered in a fragmented landscape. The intensity of fragmentation is defined by the amount and spatial autocorrelation of the lost habitat. More fragmented landscape (high amount of habitat loss, low clustering of lost patches) was shown to be detrimental to the parasitic disease invasion and transmission, which implies that the potential of using artificial disturbances as a disease-control agency in biological conservation and management. Two components of the spatial heterogeneity (the amount and spatial autocorrelation of the lost habitat) formed a trade-off in determining the host–parasite dynamics. An extremely high degree of habitat loss was, counter-intuitively, harmful to the host. These results enrich our understanding of eco-epidemiological, host–parasite systems, and suggest the possibility of using the spatial arrangement of habitat patches as a conservation tool for guarding focal species against parasitic infection and transmission.