Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1

Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.     

Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.     

Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses

A DExD/H protein, RIG-I, is critical in innate antiviral responses by sensing viral RNA. Here we show that RIG-I recognizes two distinct viral RNA patterns: double-stranded (ds) and 5'ppp single-stranded (ss) RNA. The binding of RIG-I with dsRNA or 5'ppp ssRNA in the presence of ATP produces a common structure, as suggested by protease digestion. Further analyses demonstrated that the C-terminal domain of RIG-I (CTD) recognizes these RNA patterns and CTD coincides with the autorepression domain. Structural analysis of CTD by NMR spectroscopy in conjunction with mutagenesis revealed that the basic surface of CTD with a characteristic cleft interacts with RIG-I ligands. Our results suggest that the bipartite structure of CTD regulates RIG-I on encountering viral RNA patterns.     

Forest edge creates small-scale variation in reproductive rate of sika deer

Habitat edges are considered to have an important role in determining the abundance of deer in forest landscapes, but to our knowledge there are few lines of evidence indicating that forest edge enhances the vital rate of deer. We examined pregnancy of female sika deer in Boso peninsula, central Japan, and explored how forest edges, food availability in forests, and local population density influence the pregnancy rate of sika deer. Local deer density was estimated by the number of fecal pellets, and food availability in forests was estimated by combining GIS data of vegetation distribution and the relationship between vegetation biomass and local deer density. Forest edge length was also determined by GIS data. Model selection was performed with multiple logistic regression analyses using the AIC to find the best model for accounting for the observed variation in pregnancy rates of the deer. Multiple logistic regression analysis showed that the length of forest edge had a positive effect on the pregnancy rate of females, whereas food availability in forests and local deer density had little effect. This forest edge effect was detected in a 100–200-m radius from deer captured locations, indicating that deer pregnancy is primarily determined by habitat quality within a 10-ha area. This result was confirmed by tracking females with GPS telemetry, which found that the core areas of the home range were less than 12 ha. The positive effect of edges and the lack of density dependence could be a result of high plant productivity in open environments that produces forages not depleted by high deer densities. Our results support the view that land management is the cause of the current problem of deer overabundance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. The exposure causes structural change, resulting in the generation of a variety of photomodified products having different bioactivities compared with the parent compounds. In this study, we found that benzo[a]pyrene (BaP) exposed to solar-simulated light (SSL)-induced phosphorylation of histone H2AX (gamma-H2AX), which was recently identified as an early event after the induction of DNA double strand breaks (DSBs). Although BaP itself did not produce gamma-H2AX, SSL-exposed BaP significantly generated gamma-H2AX depending on the period of exposure. Furthermore, we revealed that reactive oxygen species produced by the SSL-exposed BaP mainly contributed to the generation of gamma-H2AX. The appearance of gamma-H2AX means the induction of the most serious form of DNA damage, DSBs, suggesting the potential risk of carcinogenesis.     

Limited distribution of natural cyanamide in higher plants: occurrence in Vicia villosa subsp. varia, V. cracca, and Robinia pseudo-acacia

Cyanamide (NH2CN) has recently been proven to be a natural product, although it has been synthesized for over 100 years for agricultural and industrial purposes. The distribution of natural cyanamide appears to be limited, as indicated by our previous investigation of 101 weed species. In the present study, to investigate the distribution of natural cyanamide in Vicia species, we monitored the cyanamide contents in V. villosa subsp. varia, V. cracca, and V. amoena during their pre-flowering and flowering seasons. It was confirmed that V. cracca was superior to V. villosa subsp. varia in accumulating natural cyanamide, and that V. amoena was unable to biosynthesize this compound under laboratory condition examined. The localization of cyanamide in the leaves of V. villosa subsp. varia seedlings was also clarified. In a screening study to find cyanamide-biosynthesizing plants, only Robinia pseudo-acacia was found to contain cyanamide among 452 species of higher plants. We have investigated 553 species to date, but have so far found the ability to biosynthesize cyanamide in only three species, V. villosa subsp. varia, V. cracca and R. pseudo-acacia.     

Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)

Analysis of collagen degradation remains an important but cumbersome task. Traditional methods with dansyl chloride derivatization of collagen have been used to quantify collagen damage. Fluorescent labeling reagents have been developed that offer advantages such as greater solubility in water and low background emission. One such reagent is o-phthalaldehyde (OPA). In this study, we used OPA as a means of detecting small amounts of degraded collagen. Collagen samples isolated from skin or heart were used for OPA conjugation to exposed amino termini (“opalation”). Experiments utilizing small samples aliquoted in microtiter plates were performed to evaluate effects of increasing concentrations of OPA, varying concentrations of collagen, and effects of matrix metalloproteinase (MMP) digestion. Results indicate that within 10 min of reaction, OPA can be used to detect relative differences in cleaved vs. uncleaved collagen from skin or heart. Heart samples obtained from regions of high MMP activity correlated with increased OPA fluorescence relative to tissue with lower MMP activity. On the basis of these results, we conclude that OPA has valuable practical advantages for analytical use in detecting cleaved collagen in small tissue samples.     

The relationship between health-related quality of life and social networks among Japanese family caregivers for people with disabilities

Aims

The purpose of this study was to examine HRQOL depending on whether the participants have family members with disabilities or not. In addition, we examined the relationship between HRQOL and social networks among family caregivers in Japan.

Methods

The study has a cross-sectional design. Survey forms were distributed to 9205 people aged 30 and older who visited a dispensing pharmacy within fifteen areas of Japan. We collected data on gender, age, job status, and care giving status for persons with disabilities. Moreover, we assessed support size, social support, and HRQOL. Out of the 2029 questionnaires returned, 1763 (male: 663, female: 1100, mean age = 63.06 ± 13.34) were valid for statistical analyses (the available response rate was 19.15%).

Results

A significant difference in HRQOL was identified between family caregivers and non-family caregivers. Further, in males (N = 101), the results confirmed that only social support predicted the PCS and MCS scores, while other variables did not predict either score. On the other hand, in females (N = 144), it was found from the second step of hierarchical multiple regression analysis that only age explained the PCS score, while job status and support size explained the MCS score.

Conclusion

It is reasonable to conclude that the HRQOL of family caregivers was lower than that of non-family caregivers, and that the HRQOL of family caregivers was estimated by their social networks.

     

Combination of the anti-tumour cell ether lipid edelfosine with sterols abolishes haemolytic side effects of the drug

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is an anti-tumour cell ether lipid with surface-active properties. Pure edelfosine can be dispersed in aqueous media in the form of micelles. One important, negative side effect of edelfosine is that it is highly haemolytic. In this paper, we show that edelfosine can be co-dispersed in water with certain lipids (particularly cholesterol, campesterol or β-sitosterol) so that it gives rise to liposomes. Surface pressure measurements demonstrate that edelfosine is slowly released from these liposomes. In liposomal form, edelfosine remains apoptogenic for a variety of leukemia cell lines, while its haemolytic effect is abolished. The phenomenon is explained on the basis of the complementarity of the molecular geometries of sterols and edelfosine.