Hyperthermostable endoglucanase from Pyrococcus horikoshii.

An endoglucanase homolog from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, and its enzymatic characteristics were examined. The expressed protein was a hyperthermostable endoglucanase which hydrolyzes celluloses, including Avicel and carboxymethyl cellulose, as well as beta-glucose oligomers. This enzyme is the first endoglucanase belonging to glycosidase family 5 found from Pyrococcus species and is also the first hyperthermostable endoglucanase to which celluloses are the best substrates. This enzyme is expected to be useful for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products.     

Instantaneous determination via bimolecular recognition: usefulness of FRET in phosphorylcholine group enriched nanoparticles

This paper deals with smart bimolecular recognition for instantaneous determination. In particular, we installed the fluorescence resonance energy transfer (FRET) system in phosphorylcholine (PC) group enriched nanoparticles (NPs). The most favorable characteristics were as follows: (i) the suppression of nonspecific protein adsorption by the PC group enriched surface and (ii) simple bioassay protocol relative to the conventional enzyme-linked immunosorbent assay (ELISA). In the case of immunoassays, nonspecific interaction and complex protocols are known dominant problems. To address these issues, we designed FRET-installed NPs. Agglutination of NPs is a fundamental immunoassay technique; however, it is not quantitative. By evaluating the degree of agglutination based on the fluorescence intensity, the resulting information can be used for diagnosis. Therefore, we installed the FRET system on the surface of the NPs. In this paper, C-reactive protein (CRP) and osteopontin (OPN) were the target biomarkers for instantaneous determination, and the resulting fluorescence intensity correlated well with changes in the concentrations of the target molecules. The immunoassay protocol was quite simple, involving only the mixing of FRET-installed NPs and target molecules, such as CRP and OPN antigens. We successfully evaluated the concentration of the target biomarkers, even when human serum albumin was present as an interference molecule.     

Oocyte Mitochondria: Strategies to Improve Embrbryogenesis

Abstract  Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.     

Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.     

Influence of synchronization between adult emergence and host plant phenology on the population density of Pseudasphondylia neolitseae (Diptera: Cecidomyiidae) inducing leaf galls on Neolitsea sericea (Lauraceae)

Synchronization between the appearance of herbivorous insects and their host-plant phenology is a critical event, especially for short-lived insects such as gall midges. We studied a natural population of Pseudasphondylia neolitseae (Diptera: Cecidomyiidae) that induces leaf galls on Neolitsea sericea (Lauraceae) to evaluate the effect of synchronization on gall density in the subsequent generation. To do so, we combined quantitative data on host resources with time lag between emergence and host-available seasons. The gamma distribution model was applied to the emergence curve of P. neolitseae and the normal distribution model to the daily changes in the number of host buds suitable for oviposition; the latter model was transformed into an available-resource curve based on the mean number of host buds required for a single female to realize her eggs. By superimposing the emergence curve on the available-resource curve and calculating overlapped area, the degree of synchronization was evaluated more accurately than previous studies, which had treated only the time lag. The number of females that synchronized with host buds affected gall density in the next generation.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

5-hydroxytryptamine synthesis in HL-1 cells and neonatal rat cardiocytes

Some reports showed that serotonergic system might have existed and that 5-hydroxytryptamine (5-HT) was detected in the hamster heart. The source of 5-HT in the heart, however, remains to be fully elucidated. So the present study was designed to define serotonergic system and to clarify which cell could produce 5-HT in the heart. As a result, 5-HT was detected in homogenates of HL-1 cardiomyocytes by high performance liquid chromatography with fluorescence detection, but not in those of neonatal rat non-cardiomyocytes (NMCs). And TPH and AADC mRNAs were expressed in HL-1 cardiomyocytes and neonatal rat cardiomyocytes (MCs), not in NMCs. mRNAs of 5-HT(2A) receptor were detected in both MCs and NMCs, and those of 5-HT(2B) receptor in NMCs. These findings definitively demonstrate that 5-HT is secreted from the myocytes of the heart and strongly implied that 5-HT might play a certain role in cardiac physiology.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.