The effect of measurement time when evaluating static muscle endurance during sustained static maximal gripping

The purpose of this study was to examine the useful measurement time when evaluating static muscle endurance by comparing various parameters during sustained static gripping for 1, 3 and 6 min. Fifteen males (mean +/- SD age 20.8 +/- 1.3 yr, height 172.9 +/- 4.6 cm, body mass 67.7 +/- 5.7 kg) and fifteen females [mean +/- SD age 20.2 +/- 0.9 yr, height 158.5 +/- 3.2 cm, body mass 55.9 +/- 4.6 kg] volunteered to participate in this study. The subjects performed the sustained static maximal grip test with a sagittal and horizontal arm position for 1, 3 and 6 min on different days. Eleven force-time parameters were selected to evaluate static muscle endurance. The trial-to-trial reliability of each measurement time of sustained static maximal gripping was very high (rxy = 0.887-0.981 (1 min), 0.912-0.993 (3 min), 0.901-0.965 (6 min)). The errors of exertion values between trials were very small (below 10%). A significant correlation was found in the following parameters: the final strength and the exponential function between 1 min and 3 min, all parameters except for the time required to reach 80% of maximal grip, the regression coefficient at post-inflection between 3 min and 6 min, and the decreasing rate between all measurement times (1 min, 3 min, and 6 min). Significant differences between the measurement times were found in all parameters except for the time to 60, 70, and 80% force decreases, and the regression coefficient of pre-inflection. There was a tendency that the longer the measurement time, the larger the decreasing force. It is suggested that for the 6 min measurement, the subjects unconsciously restrained the maximal gripping force, influenced by a psychological factor as the pain became greater. The 1 min measurement may evaluate only the remarkable decreasing phase of the decreasing force, and not evaluate the phase of an almost steady state.     

Rho-kinase induces association of adducin with the cytoskeleton in platelet activation

We examined whether adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA(2) (a stable analog of TXA(2)), Ca(2+) ionophore, phorbol diester, and shear stress induced phosphorylation of alpha-adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of alpha-adducin. STA(2) stimulation led to a redistribution of adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. Phosphoadducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoadducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA(2)-induced alpha-adducin phosphorylation at Thr445 inhibited incorporation of alpha-adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of alpha-adducin at Ser726. These results suggest that Rho-kinase regulates the association of alpha-adducin and spectrin with the actin cytoskeleton in platelet activation.     

Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox), which respond to phorbol ester and arachidonic acid with O()(2) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCdelta, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCdelta in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O()(2) production to rottlerin, a PKCdelta-selective inhibitor. Optimal reconstitution also requires phospholipase C-beta2 and PI3K-gamma. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40(phox) potentiated fMLP-induced O()(2) production and raised the level of O()(2) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O()(2) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.     

Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria provides molecular evidence for a recent radiation event of the Victoria species flock: identity of EST sequences between Haplochromis chilotes and Haplochromis sp. "Redtailsheller"

The Lake Victoria Cichlid fishes have diverged very rapidly. The estimated 500 species inhabiting the lake are believed to have arisen within the last 14,000 years. The fishes' jaws and teeth have diverged markedly to adapt to different feeding behaviors and environments. To examine how the genomes of these fishes differentiated during speciation, we performed comparative analysis of expressed sequenced tag (EST) sequences. We constructed cDNA libraries derived only from the jaw portions of two cichlid species endemic to Lake Victoria. We sequenced 17,280 cDNA clones from Haplochromis chilotes and 9600 cDNA clones from Haplochromis sp. "Redtailsheller" and obtained 543 different genes common to both species. Of these genes, 441 were essentially identical between species and 102 contained base replacements in their open reading frame (ORF) or untranslated (UTR) regions. Comparative analysis of 71 selected sequences has revealed that while the degree of polymorphism is 0.0054/site for H. chilotes and 0.0047/site for H. sp. "Redtailsheller", genetic distance between the two species is 0.0031/site. The genetic distance particularly indicates that the two species diverged about 890,000 years ago.     

Relationships between force curves and muscle oxygenation kinetics during repeated handgrip

The purpose of this study was to clarify the kinetics of muscle oxygenation by near infrared spectroscopy (NIRS) in the phase of the decreasing force, especially the pre- and post-phases of the inflection point, during repeated rhythmic grip (RRG) of 30 grips/min(-1) for 6 minutes. The inflection point was the time at which the decreasing speed of the grip force changed markedly. It was calculated statistically from two regression lines fitted to each decreasing phase by applying a two-phase regression model. Ten healthy males performed the RRG for 6 minutes. Total Hb and Oxy-Hb decreased rapidly about 10 sec (7.0+/-5.9 sec, 9.8+/-5.4 sec, respectively) corresponding to the value decreasing by 90% MVC after the onset of gripping. Deoxy-Hb was maintained at a high value for 76.2+/-27.9 sec, corresponding to the value decreasing by 70-80% MVC. These phases are considered to be the states where oxygen was not satisfactorily supplied to the active muscles because of the obstruction of blood flow caused by an increase in the intra-muscular pressure. Deoxy-Hb decreased for 120+/-21.3 sec after reaching the highest value, and then reached an almost steady state at a higher level than the rest. After this phase, muscle oxygenation kinetics enters the state where oxygen is satisfactorily supplied to active muscles. We considered that the relationship between oxygen supply and demand differs during the initial and the latter phases in RRG. The changing phase in the decreasing speed of the grip force, namely the inflection point of the decreasing force, significantly correlated with the changing phase of the Oxy-Hb and Deoxy-Hb kinetics. The inflection point of the decreasing force seems to correspond to the phase where oxygen supply cannot meet oxygen demand and the increase of Deoxy-Hb. We infer that the pre- and post-phases of the inflection point depend on different physiological factors.     

Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

Skin pigment pattern formation in zebrafish requires pigment‐cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N‐glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.     

Change in DNA Methylation Patterns of SLC6A4 Gene in the Gastric Mucosa in Functional Dyspepsia

Background

The neurochemical serotonin (5-HT) is an important signaling molecule in the gastrointestinal motor and sensory functions. A key regulator of 5-HT levels is the transmembrane serotonin transporter (5-HTT; SLC6A4) that governs the reuptake of 5-HT. Recent studies have indicated 5-HTT expression may be regulated by epigenetic mechanisms. We investigated DNA methylation status of SLC6A4 gene in the gastric mucosa from functional dyspepsia (FD) because of their potential role in dyspeptic symptoms.

Methods

Endoscopic gastric biopsies were obtained from 78 subjects with no upper abdominal symptoms and 79 patients with FD. Bisulfite Pyrosequencing was carried out to determine the methylation status of promoter CpG islands (PCGIs), promoter non-CpG islands (PNCGIs) and gene body non-CpG islands (NPNCGIs) in the SLC6A4 gene. Gene expression was examined by real-time PCR.

Results

In overall, methylation level of PCGIs was significantly lower in FD compared to control subjects (p = 0.04). On the other hand, methylation level of NPNCGIs was significantly higher in FD compared to control subjects (p = 0.03). Lower methylation level in PNCGIs was highlighted in the patients with PDS (p = 0.01), while higher methylation level in NPNCGIs was more prominent in the patients with EPS (p = 0.017). Methylation levels of PCGIs and PNCGIs were inversely correlated, while methylation levels of NPNCGIs was positively correlated with SLC6A4 mRNA levels in FD patients.

Conclusions

Our data suggest that change in DNA methylation pattern of SLC6A4 in the gastric mucosa may have a role for developing FD. A role of epigenetics for developing FD needs to be further evaluated.     

Association between common genetic variants in pre-microRNAs and gastric cancer risk in Japanese population

Background: Common single‐nucleotide polymorphisms (SNPs) in microRNAs (miRNA) have been shown to be associated with susceptibility to several human cancers. We evaluated the associations of three SNPs (rs11614913, rs2910164, and rs3746444) in pre‐miRNAs (miR‐196a2, miR‐146a, and miR‐499) with the risk of gastric cancer (GC) and peptic ulcer diseases, and with the severity of Helicobacter pylori‐induced gastritis in Japanese population. Methods: The rs11614913 (C>T), rs2910164 (G>C), and rs3746444 (A>G) SNPs were genotyped in 552 GC, and 697 non‐cancer subjects, including 141 gastric and 73 duodenal ulcer, and 483 non‐ulcer subjects. The degree of histologic gastritis was classified according to the updated Sydney System, and the serum pepsinogen levels were measured in selected 579 and 204 cases. Results: The rs2910164 CC genotype held a significantly higher risk of GC when compared to non‐cancer subjects (adjusted odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.02–1.66, p =.03). Similarly, the rs2910164 C carrier was associated with higher risk of GC when compared to both non‐cancer and non‐ulcer subjects (OR = 1.39, 95%CI = 1.00–1.93, p =.05, adjusted OR = 1.57, 95%CI = 1.09–2.27, p =.016, respectively). The rs2910164 CC genotype was associated with non‐cardia and upper third, diffuse type and advanced stage GC. The rs11614913 TT genotype was associated with higher degree of mononuclear cell infiltration (score 0–1 vs 2～, adjusted OR = 1.62, 95%CI = 1.05–2.49, p =.03). Conclusions: The rs2910164 (G>C) SNP in the miR‐146a is associated with susceptibility to GC. In addition, the rs11614913 (C>T) SNP in the miR‐196a2 is associated with the degree of H. pylori‐induced mononuclear cell infiltration.     

Multiple pathways of excitation energy flow in the photosynthetic pigment system of a cryptophyte,Cryptomonas sp. (CR-1)*

Energy transfer pathways in a cryptophyte, Cryptomonas sp. (CR-1 strain) were investigated mainly by the steady state fluorescence spectroscopy and the time-resolved spectrum. Cryptomonas sp. (CR-1) contains chlorophyll (Chi) a, Chi c₂, carotenoids and cryptomonad phycoer-ythrin (Cr-PE₅₆₅), the last of which is known to be located in the lumenal side of the thyiakoid membranes. The spectral heterogeneity cf pigments was resolved by fluorescence spectra; there were at least five emission bands of Chi a at -196°C. Chlorophyll C₂ and carotenoids transferred independently to the common Chi a form (Chi a₆₆₃), and Cr-PE₅₆₅, to the different form (Chi a₆₈₂). Chlorophyll c₂ was not an intermediary component of energy transfer from carotenoid to Ch a; this is a common phenomenon to green algae and brown algae. The Chi a₆₆₃ and Chi a₆₈₂ are postulated to be located in the light-harvesting chlorophyll protein (LHC) II; thus, the energy is accumulated on Chi a₆₈₂‘n LHC II. The energy transfer step in Cr-PE_;565, was short, which was shown by a small, time-dependent red-shift of the emission. In the photosystem (PS) II core, two fluorescence components were resolved at 688 and 696 nm. The former was the trap at cryogenic temperatures. A large red-shift induced by the low temperature was explained by an equilibrium between Chi a₆₈₂ in LHC II and Chi a₆₈₈ in PS II core. The presence of Chi a₆₈₂ emission at physiological temperature is a unique feature of this alga. This was also reported in dinophyceae, which contain peridinin-ChI a-protein in the lumenal side of the thyiakoid membrane. Thus, this modification might be common in systems where the antenna complexes bind to the LHC II on the lumenal side. Based on the spectral data, we proposed a model for the molecular organization of PS II and the energy transfer pathways in cryptophyceae.