Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.     

Improvement in spermatogenic function after subcutaneous implantation of a capsule containing an aromatase inhibitor in four oligozoospermic dogs and one azoospermic dog with high plasma estradiol-17beta concentrations

A capsule containing an aromatase inhibitor (4-androsten-4-ol-3,17-dione) was subcutaneously implanted in four oligozoospermic beagle dogs and one azoospermic beagle dog with high plasma estradiol-17beta (E2) concentrations (15-19 pg/ml) and low plasma testosterone (T) concentrations (0.6-0.8 ng/ml) for 8 weeks and the effect of the aromatase inhibitor on spermatogenic dysfunction was assessed. Plasma E2 and T concentrations and semen quality were examined at 1 week intervals from 3 weeks before to 12 weeks after the start of treatment. Testicular biopsies were done twice (capsule implantation and removal). Plasma E2 concentrations of all dogs decreased (9-14 pg/ml) and plasma T concentrations increased (2.0-2.6 ng/ml) from 3 weeks after capsule implantation to capsule removal. The mean number of spermatozoa ejaculated by all four oligozoospermic dogs between 4 and 9 weeks after implantation was higher (127 x 10(6) to 205 x 10(6)) than before implantation (20 x 10(6) to 38 x 10(6)) (P < 0.05 and 0.01). Very low numbers (2 x 10(4) to 4 x 10(4)) of immotile spermatozoa were observed between 7 and 8 weeks after implantation in the semen collected from the dog with azoospermia. Before implantation, a few spermatozoa were seen in only one-fifth of the seminiferous tubules in this dog; 8 weeks after implantation, the mean diameter and mean number of round spermatids in the seminiferous tubules in all five dogs were higher than before implantation (P < 0.05). Implantation of the capsule containing the aromatase inhibitor in infertile dogs with abnormally high plasma E2 concentrations improved their spermatogenic function, concurrent with decreased plasma E2 and increased plasma T.     

A new D,L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis

A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. beta-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.     

Acute effect of static stretching on power output during concentric dynamic constant external resistance leg extension

The purpose of the present study was to clarify the effect of static stretching on muscular performance during concentric isotonic (dynamic constant external resistance [DCER]) muscle actions under various loads. Concentric DCER leg extension power outputs were assessed in 12 healthy male subjects after 2 types of pretreatment. The pretreatments included (a) static stretching treatment performing 6 types of static stretching on leg extensors (4 sets of 30 seconds each with 20-second rest periods; total duration 20 minutes) and (b) nonstretching treatment by resting for 20 minutes in a sitting position. Loads during assessment of the power output were set to 5, 30, and 60% of the maximum voluntary contractile (MVC) torque with isometric leg extension in each subject. The peak power output following the static stretching treatment was significantly (p < 0.05) lower than that following the nonstretching treatment under each load (5% MVC, 418.0 +/- 82.2 W vs. 466.2 +/- 89.5 W; 30% MVC, 506.4 +/- 82.8 W vs. 536.4 +/- 97.0 W; 60% MVC, 478.6 +/- 77.5 W vs. 523.8 +/- 97.8 W). The present study demonstrated that relatively extensive static stretching significantly reduces power output with concentric DCER muscle actions under various loads. Common power activities are carried out by DCER muscle actions under various loads. Therefore, the result of the present study suggests that relatively extensive static stretching decreases power performance.     

Growth and grazing mortality rates of phylogenetic groups of bacterioplankton in coastal marine environments

Dilution culture experiments were conducted in western North Pacific coastal regions to determine growth and grazing mortality rates of bacterial phylogenetic groups (alpha-, beta-, and gamma-proteobacteria and the Cytophaga-Flavobacter cluster) detected by fluorescent in situ hybridization. Growth rates varied greatly (1.2- to 4.0-fold) among different groups, and they were related to environmental variables (chlorophyll a concentrations and temperature) in a group-specific fashion. Growth rates of alpha-proteobacteria, the most abundant group in all the samples examined, were generally lower than those of less abundant groups, including the Cytophaga-Flavobacter cluster and gamma-proteobacteria. Grazing mortality rates and mean cell volumes varied little among different groups. These results provide insights into factors that affect distributions of different groups, but growth and grazing mortality alone did not fully explain bacterial community compositions at a broad phylogenetic level.     

Speciation and reduced hybrid female fertility in house mice

In mammals, intrinsic postzygotic isolation has been well studied in males but has been less studied in females, despite the fact that female gametogenesis and pregnancy provide arenas for hybrid sterility or inviability that are absent in males. Here, we asked whether inviability or sterility is observed in female hybrids of Mus musculus domesticus and M. m. musculus, taxa which hybridize in nature and for which male sterility has been well characterized. We looked for parent‐of‐origin growth phenotypes by measuring adult body weights in F1 hybrids. We evaluated hybrid female fertility by crossing F1 females to a tester male and comparing multiple reproductive parameters between intrasubspecific controls and intersubspecific hybrids. Hybrid females showed no evidence of parent‐of‐origin overgrowth or undergrowth, providing no evidence for reduced viability. However, hybrid females had smaller litter sizes, reduced embryo survival, fewer ovulations, and fewer small follicles relative to controls. Significant variation in reproductive parameters was seen among different hybrid genotypes, suggesting that hybrid incompatibilities are polymorphic within subspecies. Differences in reproductive phenotypes in reciprocal genotypes were observed and are consistent with cyto‐nuclear incompatibilities or incompatibilities involving genomic imprinting. These findings highlight the potential importance of reduced hybrid female fertility in the early stages of speciation.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.     

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.