Skewed reproductive success among male white-spotted charr land-locked by an erosion control dam: Implications for effective population size

In 1996 and 1997, the spawning behavior of fluvial white-spotted charr Salvelinus leucomaenis, was observed in the upstream area of an erosion control dam. A small number of males with relatively large body size mated successfully with females as a pair, while almost all satellite males did not sneak successfully, resulting in a non-random mating system. The low sneaking success of subordinate males, in addition to the monopolization of spawning opportunities by a few dominant males, is one of the most important causes of skewed reproductive success among males. The total number of adult fishes in the study area (N: approximately half of the whole tributary above a dam) was estimated as 148 and 102 in 1996 and 1997, respectively. Based on these findings and some further assumptions, the estimated effective population size (N_e) was low in both years. The N_e/N ratio ranged from 0.33 to 0.36 in both years. In addition to reduced population size by construction of an impassable dam, the above-dam population suffered low N_e due to skewed reproductive success among males. The low N_e may be one cause of extinction in above-dam populations of fluvial charr, especially just after the construction of impassable barriers.     

Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution

Human induced pluripotent stem (hiPS) cells have great potential for regenerative medicine and drug discovery. It is essential to establish highly efficient and reliable methods for hiPS cell cryopreservation. We examined cryopreservation of hiPS cells by the vitrification method using a dimethyl sulfoxide Me2SO-free and serum-free medium, VS2E, that uses Euro-Collins solution as a base with 40% (v/v) ethylene glycol and 10% (w/v) polyethylene glycol as cryoprotectants. This combination of vitrification and cryoprotectants resulted in a higher recovery rate of hiPS cells than with a commercially-available vitrification solution, DAP213, which contained Me2SO and serum components. After vitrification and warming, hiPS cells were cultured easily. Even after several subculturing steps, cells expressed undifferentiated cell markers, such as Oct-3/4 and SSEA-4, and also exhibited alkaline phosphatase activity. The pluripotency of hiPS cells was maintained, as demonstrated by teratoma formation upon hiPS cell transplantation into severe combined immunodeficient mice. Thus, we successfully preserved hiPS cells under liquid nitrogen with high efficiency using Me2SO-free vitrification solution and rapid cooling.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

Protein N-Myristoylation Plays a Critical Role in the Endoplasmic Reticulum Morphological Change Induced by Overexpression of Protein Lunapark,an Integral Membrane Protein of the Endoplasmic Reticulum

N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an N-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.     

Peptidoglycan of Pseudomonas aeruginosa

A practical method for preparing peptidoglycan from Ps. aeruginosa and E. coli was devised. After bacterial cells were dissolved in boiling 4% SDS solution, peptidoglycan was collected and washed with water by centrifugation. Peptidoglycan was treated further with pronase and lyophilized. The final preparation of peptidoglycan from Ps. aeruginosa appeared as a filmy coagulation in electron micrograph and its amino acid composition was determined as follows: Glu/Ala/A₂pm/Mur/GlcN (100/183/104/61/98). The lysozyme digest showed the same pattern as that of E. coli peptidoglycan. N-Terminal analysis suggested that about half of the peptide chains was interbridged by the peptide bond between Ala and A₂pm. The probable ratio of muropeptides in the peptidoglycan was estimated.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.     

AMPKα1‐LDH pathway regulates muscle stem cell self‐renewal by controlling metabolic homeostasis

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self‐renewal) is crucial for tissue repair. Here, we showed that AMP‐activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self‐renewal. AMPKα1^?/? MuSCs displayed a high self‐renewal rate, which impairs muscle regeneration. AMPKα1^?/? MuSCs showed a Warburg‐like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non‐limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1^?/? phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1^?/? MuSC self‐renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.