The importance of subfragment 2 and C‐terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C‐terminus on thick filament assembly. C‐terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C‐terminus of LMM.     

The developmental ability of vitrified oocytes from different mouse strains assessed by parthenogenetic activation and intracytoplasmic sperm injection

Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 x DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr(2+) treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.     

Pulmonary vascular proliferation and lungworm (Stenurus ovatus) in a bottlenose dolphin (Tursiops turncatus)

A female adult bottlenose dolphin suddenly died at 17 days after the capture. Macroscopically, severe pulmonary congestive edema was found. Histopathology revealed many lungworms in the bronchioli and the worms were identified as Stenurus ovatus. Variously sized vessels proliferated around the lesioned bronchioli. Based on these findings, chronic bronchopneumonia due to the lungworm was diagnosed and vascular proliferation was similar to angiomatosis recently reported in Atlantic bottlenose dolphin.     

Genetic Analyses of an H3N8 Influenza Virus Isolate, Causative Strain of the Outbreak of Equine Influenza at the Kanazawa Racecourse in Japan in 2007

In August 2007, an outbreak of equine influenza occurred among vaccinated racehorses with Japanese commercial equine influenza vaccine at Kanazawa Racecourse in Ishikawa prefecture in Japan. Apparent symptoms were pyrexia (38.2-41.0 degrees C) and nasal discharge with or without coughing, although approximately half of the infected horses were subclinical. All horses had been shot with a vaccine that contained two inactivated H3N8 influenza virus strains [A/equine/La Plata/93 (La Plata/93) of American lineage and A/equine/Avesta/93 (Avesta/93) of European lineage] and an H7N7 strain (A/equine/Newmarket/1/77). Influenza virus, A/equine/Kanazawa/1/2007 (H3N8) (Kanazawa/07), was isolated from one of the nasal swab samples of diseased horses. Phylogenetic analysis indicated that Kanazawa/07 was classified into the American sublineage Florida. In addition, four amino acid substitutions were found in the antigenic sites B and E in the HA1 subunit protein of Kanazawa/07 in comparison with that of La Plata/93. Hemagglutination-inhibition (HI) test using 16 serum samples from recovering horses revealed that 1.4- to 8-fold difference in titers between Kanazawa/07 and either of the vaccine strains. The present findings suggest that Japanese commercial inactivated vaccine contributed to reducing the morbidity rate and manifestation of the clinical signs of horses infected with Kanazawa/07 that may be antigenically different from the vaccine strains.     

Modelling agricultural expansion in Kenya’s Eastern Arc Mountains biodiversity hotspot

The Taita Hills are the northernmost part of the Eastern Arc Mountains of Kenya and Tanzania, which is one of the most important regions for biological conservation in the world. The indigenous cloud forests in this area have suffered substantial degradation for several centuries due to agricultural expansion. In the Taita Hills, currently only 1% of the original forested area remains preserved. In order to create effective policies to preserve the natural resources and biodiversity of the Eastern Arc Mountains it is crucial to understand the causes and interactions involved in the landscape changes in the most degraded areas. The research presented here aimed to understand the role of landscape attributes and infrastructure components as driving forces of agricultural expansion in the Taita Hills. Geospatial technology tools and a landscape dynamic simulation model were integrated to identify and evaluate the driving forces of agricultural expansion and simulate future landscape scenarios. The results indicate that, if current trends persist, agricultural areas will occupy roughly 60% of the study area by 2030. Agricultural expansion will likely take place predominantly in lowlands and foothills throughout the next 20 years, increasing the spatial dependence on distance to rivers and other water bodies. The main factors driving the spatial distribution of new croplands were the distance to markets, proximity to already established agricultural areas and distance to roads. Other driving forces of the agricultural expansion, as well as their implications for natural resources conservation, are discussed. Further studies are necessary to integrate the effects of population pressure and climate change on the sustainability and characteristics of local agricultural systems.     

Size‐dependent stress response in juvenile Arctic charr (Salvelinus alpinus) under prolonged predator conditioning

Predator conditioning can be used to improve post‐release antipredator recognition of hatchery‐reared salmonids. However, possible negative stress‐related effects of prolonged predator conditioning on juvenile fish physiology are poorly understood. We studied the effects of prolonged (91 days) predator odour exposure on whole‐body cortisol level and spleen size in six full‐sib families of juvenile hatchery‐bred Arctic charr (Salvelinus alpinus). Chemical cues from water containing charr‐fed pikeperch (Sander lucioperca) were used as the predator exposure stimuli and lakewater was used as a chemical control. Our study revealed that juvenile body cortisol levels post‐predator conditioning were affected by treatment, fish size and their interaction. Importantly, among the smaller (i.e. slowest growing) charr, the predator‐exposed fish had higher cortisol levels than control fish, while the opposite pattern was true for the larger fish. These results suggest that chemical cues from charr‐fed predators induce a prolonged stress response in juvenile charr. As prolonged predation exposure seems to elevate stress levels in a size‐dependent manner, the larger, faster growing fish could possibly have intrinsically lower stress responses to predation threats than smaller, slower growing fish. Possible coupling between stress sensitivity and growth requires further attention due to the likely implications for the management of unintended domestication among captive‐reared salmonids.     

Application of repetitive extragenic palindromic (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis to the identification and classification of Japan and Thai local isolates of Bradyrhizobium japonicum,Shinorhizobium meliloti,Rhizobium leguminosarum

Repetitive extragenic palindromic (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis was applied to the identification and classification of local isolates of 44 Bradyrhizobium japonicum, 7 Sinorhizobium meliloti, 10 Rhizobium leguminosarum strains from Japan and Thai. Using genomic DNA of the 61 strains, both REP and ERIC primers induced reproducible PCR band patterns, although REP-PCR generated more bands and appeared to be more useful for distinguishing the isolates from each other. Using mixed matrix data from both REP- and ERIC-PCR data, it become possible to distinguish all the isolates analyzed in this experiment from each other. When cluster analysis was applied to both PCR matrix data of 44 B. japonicum isolates, only the REP-PCR dendrogram showed a grouping profile corresponding to the exo-polysaccharide phenotype with a exceptions. When the matrix data of R. leguminosarum and S. meliloti were subjected to cluster analysis, S. meliloti appeared to form a different subgroup from R. leguminosarum in the dendrogram of REP-PCR data except for one strain. In the case of ERIC-PCR, isolates of R. leguminosarum from northern Thailand formed a separate subgroup from other R. leguminosarum and S. meliloti which were dispersed in the dendrogram. These data suggest that REP-PCR and ERIC-PCR were effective for the identification of individual isolates even though the isolates showed a wide genetic diversity and the same phenotype. When the data of the local isolates from Japan and Thailand were subjected to cluster analysis, REP- and ERIC-PCR analysis revealed different grouping characteristics.     

Can the Activities of Acetylcholinesterase and Glutathione S-Transferases of Crangon crangon (L.) be Used as Biomarkers of Fuel Oil Exposure?

This work aimed to determine the sensitivity of acetylcholinesterase (AChE) and glutathione S-transferases (GST) activities of Crangon crangon (L.) to the water-accommodated fraction (WAF) of the fuel oil spilled by the oil tanker Prestige, in order to assess their usefulness as markers for this kind of pollution. Laboratory exposure of shrimps to WAF of weathered Prestige fuel oil showed no significant interference with AChE activity. Significant induction of GST activity was observed, potentially as a result of high concentrations of polycyclic aromatic hydrocarbons. However, these results were only obtained with the higher WAF concentrations tested. It was concluded that AChE and GST activities of C. crangon were not useful biomarkers for short-term exposure to the WAF of fuel oil spilled by the tanker Prestige and carried to the beaches of Galicia (NW Spain).     

Biological activities of synthetic grammistins and analogous peptides

Grammistins, peptide toxins isolated from the skin secretions of two species of soapfishes Grammistes sexlineatus and Pogonoperca punctata, are new members of the family of linear membrane-lytic antibacterial peptides characterized by the abundance of amphiphilic α-helices. As the first step toward future use of grammistins to examine the mode of membrane lysis and design new antibiotics, C-terminally free and amidated forms were chemically synthesized for each of five grammistins (Gs 1, Gs 2, Pp 1, Pp 2b and Pp 3), and evaluated for hemolytic and antibacterial activities. No difference in biological activities was observed between C-terminally free and amidated forms of Gs 1, while the C-terminally amidated forms of the other grammistins exhibited much higher activities than the C-terminally free forms. Comparison with the biological activities of natural grammistins, together with our preliminary cloning experiments, showed that natural grammistins, except for Gs 1, are C-terminally amidated. As for Pp 1, six analogous peptides were further synthesized. Determination of their biological activities revealed that the amphiphilic α-helical structure and positive charge are important for the hemolytic and antibacterial activities, respectively, of Pp 1.     

Kinetic changes in glucosinolate-derived volatiles by heat-treatment and myrosinase activity in nakajimana (Brassica rapa L. cv. nakajimana)

Nakajimana (Brassica rapa L. cv. nakajimana), of the family Brassicaceae, is a traditional vegetable in Japan. Three isothiocyanates and five cyanides in the leaves of nakajimana were identified by gas chromatography (GC) and GC-mass spectrometry (GC-MS), and their kinetic changes using heat-treatment (temperature and time) were investigated. In addition, myrosinase activity of extracts prepared from fresh nakajimana leaf was determined. In crushed heat-treated leaves of nakajimana (70 °C for 30 s), formation of isothiocyanates and myrosinase activity increased, whereas formation of 1-cyano-3,4-epithiobutane and 1-cyano-4,5-epithiopentane decreased. Heat-treatment can significantly alter the content of potentially beneficial compounds in nakajimana, and ingestion of suitable isothiocyanates for human health may be better facilitated by mild boiling.