Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.     

Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus

We prepared the specific antibodies for EXT1 and EXT2, hereditary multiple exostoses (HME) gene products, and characterized their expression, subcellular localization, and protein association among EXT members. Biochemical analyses indicate that EXT1 and EXT2 can associate and form homo/hetero-oligomers in vivo with or without HME-linked mutations, EXT1 (R340C) and EXT2 (D227N), when exogenously expressed in COS-7 cells. An immunocytochemical analysis showed that both EXT1 and EXT2 localized in Golgi apparatus, irrespective of HME mutations. An immunohistochemical analysis on developing bones further showed that both EXT1 and EXT2 were concomitantly expressed in hypertrophic chondrocytes of forelimb bones from 1-day-old neonatal mouse, but down-regulated in maturing chondrocytes of developing cartilage from 21-day-old mouse. Taken together with the recent finding that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate [Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lindholt, K. (1998) J. Biol. Chem. 273, 26265-26268], our results implied a molecular basis that a HME-linked mutation found in EXT genes could interfere the physiological function(s) of EXT homo/hetero-oligomers as glycosyltransferases in the developing bones of HME patients.     

Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms

Objective

Intra-arterial stem cell transplantation exerts neuroprotective effects for ischemic stroke. However, the optimal therapeutic time window and mechanisms have not been completely understood. In this study, we investigated the relationship between the timing of intra-arterial transplantation of allogeneic mesenchymal stem cells (MSCs) in ischemic stroke model in rats and its efficacy in acute phase.

Methods

Adult male Wistar rats weighing 200 to 250g received right middle cerebral artery occlusion (MCAO) for 90 minutes. MSCs (1×10⁶cells/ 1ml PBS) were intra-arterially injected at either 1, 6, 24, or 48 hours (1, 6, 24, 48h group) after MCAO. PBS (1ml) was intra-arterially injected to control rats at 1 hour after MCAO. Behavioral test was performed immediately after reperfusion, and at 3, 7 days after MCAO using the Modified Neurological Severity Score (mNSS). Rats were euthanized at 7 days after MCAO for evaluation of infarct volumes and the migration of MSCs. In order to explore potential mechanisms of action, the upregulation of neurotrophic factor and chemotactic cytokine (bFGF, SDF-1α) induced by cell transplantation was examined in another cohort of rats that received intra-arterial transplantation at 24 hours after recanalization then euthanized at 7 days after MCAO for protein assays.

Results

Behavioral test at 3 and 7 days after transplantation revealed that stroke rats in 24h group displayed the most robust significant improvements in mNSS compared to stroke rats in all other groups (p’s<0.05). Similarly, the infarct volumes of stroke rats in 24h group were much significantly decreased compared to those in all other groups (p’s<0.05). These observed behavioral and histological effects were accompanied by MSC survival and migration, with the highest number of integrated MSCs detected in the 24h group. Moreover, bFGF and SDF-1α levels of the infarcted cortex were highly elevated in the 24h group compared to control group (p’s<0.05).

Conclusions

These results suggest that intra-arterial allogeneic transplantation of MSCs provides post-stroke functional recovery and reduction of infarct volumes in ischemic stroke model of rats. The upregulation of bFGF and SDF-1α likely played a key mechanistic role in enabling MSC to afford functional effects in stroke. MSC transplantation at 24 hours after recanalization appears to be the optimal timing for ischemic stroke model, which should guide the design of clinical trials of cell transplantation for stroke patients.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Male Isaza (Gymnogobius isaza, Gobiidae) prefer large mates: a counterstrategy against brood parasitism by conspecific females

Like many other gobies, male Isaza (Gymnogobius isaza) which are endemic to Lake Biwa, Japan, exclusively care for broods in nests. This goby may have an optimal range of brood size (i.e., an average clutch size of about 2000–3000 eggs) within which they may produce larger numbers of hatching young because much larger broods may be destroyed by fungal infection before hatching. This optimal brood size hypothesis (Takahashi et al. in J Ethol 22:153–159, 2004) predicts that (1) after spawning, both males and females will refuse additional spawning by other gravid females (second females) to keep brood sizes within optimal ranges, (2) larger fish will repel second females more successfully than will smaller fish, and thus, (3) both sexes prefer larger mates. To examine these predictions, we first observed Isaza’s aggressive behaviors in aquaria and investigated whether fish attacked and repelled second females that were introduced after spawning, and, if so, what were the sizes of fish that did so. Large fish, regardless of sex, aggressively prevented second females from entering the nest, but second females larger than the pairs displaced the pair females forcibly and spawned eggs into their clutches. Mate choice experiments showed that males preferred large females. Although females’ choice of large mates was not confirmed, many results may largely coincide with the predictions of the optimal brood size hypothesis. Thus, Isaza males’ choice of large mates will be advantageous for defending against brood parasitism by conspecific females and for achieving optimal clutch size.     

Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling

To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2^−/− cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE₂-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.     

The obligate alkaliphile <Emphasis Type="Italic">Bacillus clarkii</Emphasis> K24-1U retains extruded protons at the beginning of respiration

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (ΔpH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Δψ), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the ΔpH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na⁺ channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Δψ-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient ΔpH under alkaline conditions.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.