Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading

The actomyosin cytoskeleton plays prominent roles in cell spreading and migration. To address the roles of myosin II isoforms and to estimate the region where the myosin IIs are activated in spreading cells, we examined the immunolocalization of myosin II isoforms and phosphorylated RLCs in the spreading MRC-5 cells. We observed the formation of actin ring-like structure at the base of the lamella. Both myosin IIA and IIB were predominantly localized there. Myosin IIA and diphosphorylated RLC were distributed outside of the region where myosin IIB and monophosphoryated RLC were distributed predominantly. Inhibition of Rho-kinase resulted in the disappearance of the diphosphorylation of RLC, moreover, it accelerated the rate of cell spreading and induced an aberrant cell shape at later stage of spreading. These results indicate that diphosphorylation of RLCs of myosin IIA by Rho-kinase in lamella is responsible for the cell to spread properly.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

Brief Communication: Orphaned male Chimpanzees die young even after weaning

If a social‐living animal has a long life span, permitting different generations to co‐exist within a social group, as is the case in many primate species, it can be beneficial for a parent to continue to support its weaned offspring to increase the latter's survival and/or reproductive success. Chimpanzees have an even longer period of dependence on their mothers' milk than do humans, and consequently, offspring younger than 4.5–5 years old cannot survive if the mother dies. Most direct maternal investments, such as maternal transportation of infants and sharing of night shelters (beds or nests), end with nutritional weaning. Thus, it had been assumed that a mother's death was no longer critical to the survival of weaned offspring, in contrast to human children, who continue to depend on parental care long after weaning. However, in theory at least, maternal investment in a chimpanzee son after weaning could be beneficial because in chimpanzees' male‐philopatric society, mother and son co‐exist for a long time after the offspring's weaning. Using long‐term demographic data for a wild chimpanzee population in the Mahale Mountains, Tanzania, we show the first empirical evidence that orphaned chimpanzee sons die younger than expected even if they lose their mothers after weaning. This suggests that long‐lasting, but indirect, maternal investment in sons continues several years after weaning and is vital to the survival of the sons. The maternal influence on males in the male‐philopatric societies of hominids may be greater than previously believed. Am J Phys Anthropol, 153:139–143, 2014. © 2013 Wiley Periodicals, Inc.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Differences in the heat stress associated with white sportswear and being semi-nude in exercising humans under conditions of radiant heat and wind at a wet bulb globe temperature of greater than 28 °C

This study investigated whether wearing common white sportswear can reduce heat stress more than being semi-nude during exercise of different intensities performed under radiant heat and wind conditions, such as a hot summer day. After a 20-min rest period, eight male subjects performed three 20 min sessions of cycling exercise at a load intensity of 20 % or 50 % of their peak oxygen uptake (VO_2peak) in a room maintained at a wet bulb globe temperature (WBGT) of 28.7?±?0.1 °C using two spot lights and a fan (0.8 m/s airflow). Subjects wore common white sportswear (WS) consisting of a long-sleeved shirt (45 % cotton and 55 % polyester) and short pants (100 % polyester), or only swimming pants (SP) under the semi-nude condition. The mean skin temperature \( \left(\overline{T} sk\right) \) was greater when subjects wore SP than WS under both the 20 % and 50 % exercise conditions. During the 50 % exercise, the rating of perceived exertion (RPE) and thermal sensation (TS), and the increases in esophageal temperature (ΔTes) and heart rate were significantly higher (P?<?0.001–0.05), or tended to be higher (P?<?0.07), in the WS than SP trials at the end of the third 20-min exercise session. The total sweat loss (m _sw,tot) was also significantly higher in the WS than in the SP trials (P?<?0.05). However, during the 20 % exercise, the m _sw,tot during exercise, and the ΔTes, RPE and TS at the end of the second and third sessions of exercise did not differ significant between conditions. The heat storage (S), calculated from the changes in the mean body temperature (0.9Tes + 0.1 \( \overline{T} sk \) ), was significantly lower in the WS trials than in the SP trials during the 20 min resting period before exercise session. However, S was similar between conditions during the 20 % exercise, but was greater in the WS than in the SP trials during 50 % exercise. These results suggest that, under conditions of radiant heat and wind at a WBGT greater than 28 °C, the heat stress associated with wearing common WS is similar to that of being semi-nude during light exercise, but was greater during moderate exercise, and the storage of body heat can be reduced by wearing WS during rest periods.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.