A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.     

Application of Overall Dynamic Body Acceleration as a Proxy for Estimating the Energy Expenditure of Grazing Farm Animals: Relationship with Heart Rate

Estimating the energy expenditure of farm animals at pasture is important for efficient animal management. In recent years, an alternative technique for estimating energy expenditure by measuring body acceleration has been widely performed in wildlife and human studies, but the availability of the technique in farm animals has not yet been examined. In the present study, we tested the potential use of an acceleration index, overall dynamic body acceleration (ODBA), as a new proxy for estimating the energy expenditure of grazing farm animals (cattle, goats and sheep) at pasture with the simultaneous evaluation of a conventional proxy, heart rate. Body accelerations in three axes and heart rate for cows (n = 8, two breeds), goats (n = 6) and sheep (n = 5) were recorded, and the effect of ODBA calculated from the body accelerations on heart rate was analyzed. In addition, the effects of the two other activity indices, the number of steps and vectorial dynamic body acceleration (VeDBA), on heart rate were also investigated. The results of the comparison among three activity indices indicated that ODBA was the best predictor for heart rate. Although the relationship between ODBA and heart rate was different between the groups of species and breeds and between individuals (P<0.01), the difference could be explained by different body weights; a common equation could be established by correcting the body weights (M: kg): heart rate (beats/min) = 147.263∙M ^-0.141 + 889.640∙M ^-0.179∙ODBA (g). Combining this equation with the previously reported energy expenditure per heartbeat, we estimated the energy expenditure of the tested animals, and the results indicated that ODBA is a good proxy for estimating the energy expenditure of grazing farm animals across species and breeds. The utility and simplicity of the procedure with acceleration loggers could make the accelerometry technique a worthwhile option in field research and commercial farm use.     

Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.     

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

The potential of the active ingredient of a commercial bioinsecticide, XenTari^® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari^® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.     

Effects of coral colony morphologies on mass transfer and susceptibility to thermal stress

This study tested the hypothesis that corals of the same species, but of varying size and shape, may respond differently to thermal stress because of different mass transfer capacities. High mass transfer rates are an advantage under thermal stress, and mass transfer rates are assumed to scale with size. Yet large, corymbose Acropora colonies are more vulnerable to thermal stress than small corymbose Acropora colonies. We took a two-tiered approach to examine the differences in the susceptibility of different coral morphologies to thermal stress. Firstly, the response of several coral species of different sizes and shapes were measured in the field through a thermal stress event. Secondly, diffusion experiments were conducted using gypsum-coral models of different morphologies to estimate mass transfer rates, to test whether dissolution rates differed in accordance with colony morphology and colony size. Coral colonies with a high height to diameter ratio were subjected to more partial mortality than flat colonies. These results agree with mass transfer theory. The diffusion experiments showed that in a low-flow environment, small encrusting colonies had higher rates of dissolution than large flat or small branched colonies. These results, however, disagree with mass transfer theory. We show that the volume of space between colony branches predicts the response to thermal stress in the field. Small encrusting colonies were most likely to maintain mass transfer and were therefore more likely to survive thermal stress than large branched colonies. We predict that an increase in the frequency and intensity of thermal stresses may see a shift from large branched coral colonies to both small colonies, and flat-massive colonies with low aspect ratios.     

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.     

Comparison of stable nitroxide, 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls, with respect to protection from radiation, prevention of DNA damage, and distribution in mice

We compared three 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls (PROXYLs): carbamoyl-, methoxycarbonyl-, and hydroxymethyl-PROXYL (CM-, MC-, and HM-PROXYL, respectively) with respect to radioprotection, prevention of DNA damage, and in vivo distribution in mice. The PROXYLs provided protection to C3H mice against lethal X-irradiation (8 Gy) with the following order of magnitude, HM- > CM- approximately MC-PROXYL. In contrast, radioprotection at the cellular level assessed by the colony formation of leukemia cell line L5178Y showed no difference among them. The degree of protection from X ray-induced oxidation of DNA bases measured by the formation of 8-hydroxydeoxyguanosine in salmon DNA and the cleavage of DNA measured by electrophoresis of plasmid pBR322 DNA did not differ among the PROXYLs. Redox potentials were also similar for each. However, the blood concentration of the PROXYLs injected ip into the mice showed different maximum concentrations (HM- > CM- approximately MC-PROXYL), although all reached a maximum at around 5-10 min and gradually decreased thereafter. Their concentration in bone marrow showed a similar pattern, suggesting that the difference in in vivo radioprotection among the three PROXYLs is due to the difference in their distribution to bone marrow. In general, the radioprotection provided by stable nitroxides is affected not only by redox potential and reactivity in vitro but also by pharmacokinetics.     

Oat5 and NaDC1 Protein Abundance in Kidney and Urine After Renal Ischemic Reperfusion Injury

The aim of this study was to evaluate the abundance of the organic anion transporter 5 (Oat5) and the sodium-dicarboxylate cotransporter 1 (NaDC1) in kidney and urine after renal ischemic reperfusion injury. Renal injury was induced in male Wistar rats by occlusion of both renal pedicles for 0 (Group Sham), 5 (Group I5R60), or 60 (Group I60R60) min. The studies were performed after 60 min of reperfusion. The expression of Oat5 and NaDC1 was evaluated by IHC and Western blotting. Oat5 and NaDC1 abundance and alkaline phosphatase activity (AP) were assayed in urine. A decreased expression in renal homogenates and apical membranes and an increase in urinary excretion of Oat5 and NaDC1 were observed in I60R60 rats, as well as alterations of other widely used parameters for renal dysfunction and injury (plasma creatinine, urinary AP activity, kidney weight, histological lesions). In contrast, in the I5R60 group, only an increase in urinary excretion of Oat5 and mild histopathological damage was detected. This is the first study on Oat5 and NaDC1 detection in urine. These results suggest that urinary excretion of Oat5 might be an early indicator of renal dysfunction, which is useful for detection of even minor alterations in renal structural and functional integrity. (J Histochem Cytochem 57:17–27, 2009)