Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population.

We investigated the proviral copies and RNA expression in koala retrovirus (KoRV)-infected koalas. To ascertain any variation in viral load by institution, age, sex, or body condition score, we quantified KoRV proviral DNA and RNA loads in captive koalas (n = 37) reared in Japanese zoos. All koalas were positive for KoRV genes (pol, LTRs, and env of KoRV-A) in genomic DNA (gDNA), and 91.89% were positive for the pol gene in RNA. In contrast, the distribution rates of KoRV-B, KoRV-C, KoRV-D, and KoRV-F env genes in gDNA were 94.59%, 27.03%, 67.57%, and 54.05%, respectively. A wide inter-individual variation and/or a significant inter-institutional difference in proviral DNA (p < 0.0001) and RNA (p < 0.001) amounts (copies/103 koala ß-actin copies) were observed in Awaji Farm England Hill Zoo koalas, which were obtained from southern koala populations, suggesting exogenous incorporation of KoRV in these koalas. Significant (p < 0.05) age differences were noted in KoRV RNA load (p < 0.05) and median total RNA load (p < 0.001), with loads higher in younger koalas (joeys and juveniles). Thus, the current study provides the distribution of KoRV subtypes in Japanese zoo koala populations and identifies several additional risk factors (sex, age, and body condition) associated with KoRV expression.

Factors of choking under pressure in musicians.

Under pressure, motor actions, such as those required in public speech, surgery, or musical performance, can be compromised, even when these have been well-trained. The latter is often referred to as 'choking' under pressure. Although multifaceted problems mediate such performance failure in anxiogenic situations, such as compromised motor dexterity and cognitive disruption, the fundamental set of abnormalities characterizing choking under pressure and how these abnormalities are related have not been elucidated. Here, we attempted, first, to classify behavioural, psychological, and physiological abnormalities associated with choking under pressure in musicians and, second, to identify their relationship based on datasets derived from a questionnaire with 258 pianist respondents. Explorative factor analysis demonstrated eight functional abnormalities related to the musicians' choking, such as attention to the audience, erroneous motor actions, perceptual confusion, and failure of memory recall, which however did not include exaggerated attention to the performance. This suggests distraction of attention away from skill execution, which may underlie the spoiled performance under pressure. A structural equation analysis further inferred causal relationships among them. For instance, while failure of memory recall was influenced by passive behaviours manifesting under pressure, erroneous motor actions during performance were influenced by feeling rushed and a loss of body control. In addition, some specific personal traits, such as neuroticism, public self-consciousness, and a lack of confidence, were associated with the extent to which pressure brought about these abnormalities. These findings suggest that distinct psycho-behavioural abnormalities and personal traits underlie the detrimental effects of pressure on musical performance.

Fabrication of Naftopidil-Loaded Tablets Using a Semisolid Extrusion-Type 3D Printer and the Characteristics of the Printed Hydrogel and Resulting Tablets.

The production of three-dimensional (3D)-printed drugs holds promise for future personalized medicine. Here, we prepared tablets containing naftopidil as a model drug using a semisolid extrusion-type 3D bioprinter applicable for tissue engineering. A hydrogel is typically used as the printer ink for 3D bioprinters, and we incorporated various amounts of hydroxypropyl methylcellulose hydrogel (30%, 40%, and 50% gel) into the printer ink. The resulting 3D-printed gel product was dried to obtain tablets. The rheological properties of the printer ink changed as its composition was changed, and tablets were prepared successfully from several formulations. Increasing the amount of hydroxypropyl methylcellulose hydrogel in the printer ink led to delayed drug dissolution, decreased weight, and decreased hardness of the tablets. Delayed drug dissolution was also observed when the amount of disintegrating agent typically used in powder compression tablets was increased in the ink, and increasing the incorporated amount of the disintegrating agent crospovidone increased the hardness of the tablets. Our results will provide useful information for the preparation of tablets using semisolid extrusion-type 3D printers.

A method for detecting rash and fever illness-associated viruses using multiplex reverse transcription polymerase chain reaction.

In this study, a new multiplex RT-PCR method for detecting various viral genes in patients with rash and fever illnesses (RFIs) was constructed. New primer sets were designed for detection of herpes simplex viruses 1 and 2 (HSV1 and 2), and Epstein-Barr virus (EBV). The newly designed and previously reported primer sets were used to detect 13 types of RFI-associated viruses by multiplex RT-PCR assay systems. Moreover, to eliminate non-specific PCR products, a double-stranded specific DNase was used to digest double-stranded DNA derived from the templates in clinical specimens. RFI-associated viruses were detected in 77.0% of the patients (97/126 cases) by the presented method, multiple viruses being identified in 27.8% of the described cases (35/126 cases). Detected viruses and clinical diagnoses were compatible in 32.5% of the patients (41/126 cases). Sensitivity limits for these viruses were estimated to be 101 -103 copies/assay. Furthermore, non-specific PCR products were eliminated by a double-stranded specific DNase with no influence on sensitivity. These results suggest that this method can detect various RFI-associated viruses in clinical specimens with high sensitivity and specificity.

Modification of plant cell wall structure accompanied by enhancement of saccharification efficiency using a chemical, lasalocid sodium.

The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture.

Semi-automatic organelle detection on transmission electron microscopic images.

Recent advances in the acquisition of large-scale datasets of transmission electron microscope images have allowed researchers to determine the number and the distribution of subcellular ultrastructures at both the cellular level and the tissue level. For this purpose, it would be very useful to have a computer-assisted system to detect the structures of interest, such as organelles. Using our original image recognition framework CARTA (Clustering-Aided Rapid Training Agent), combined with procedures to highlight and enlarge regions of interest on the image, we have developed a successful method for the semi-automatic detection of plant organelles including mitochondria, amyloplasts, chloroplasts, etioplasts, and Golgi stacks in transmission electron microscope images. Our proposed semi-automatic detection system will be helpful for labelling organelles in the interpretation and/or quantitative analysis of large-scale electron microscope imaging data.

Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth.

[The roles of medical support center nurses and the timing for resuming nursing support for outpatients receiving chemotherapy].

In a Medical Support Center (MSC), nurses care for both inpatients and outpatients. For outpatients receiving chemotherapy, nursing support is ended at PS1 or PS0. We also clarified the roles of MSC nurses and the timing for resuming nursing support for outpatients. We found that support was resumed between 53 and 153 days after support was first administered. By considering the services provided at the first instance of support, MSC nurses were able to build a system of medical and nursing care that the patients and their families desired.

[Role of nurses at the Medical Support Center in the discharge and in-home supports].

The Medical Support Center(MSC)of the A university hospital encourages inpatients to leave the hospital and outpatients to live better at home. The medical social workers and the nurses cooperate to achieve these aims. The role of the nurses at the Medical Support Center is to collect information regarding the medical services and the welfare systems, and to provide the patients and their families with this information. We also educate the hospital staff to better understand the lives of the patients who leave hospital. We also provide cancer patients with information regarding the medical treatment available in the area, social welfare services, hospices, and palliative care, such that they can choose the method of treatment and an appropriate place to live.

Arabidopsis mitochondrial protein TIM50 affects hypocotyl cell elongation through intracellular ATP level.

The plant hypocotyl is an excellent model for the analysis of cell elongation. We have characterized a knockout mutant of the Arabidopsis TIM50 gene that showed a reduction in the hypocotyls length of etiolated seedlings. We also found that a knockout of TIM50 caused enlargement and deformation of the mitochondrial structure and a reduction in intracellular ATP levels. TIM50 is a component of the mitochondrial TIM23 inner membrane protein complex and is involved in the import of mitochondrial proteins. The short hypocotyl phenotype was recovered by the addition of Compound C, an inhibitor of AMPK. Thus, the mitochondrial ATP level controls cell elongation in Arabidopsis hypocotyls through possible signaling via AMPK.

A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis.

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.

Increased level of polyploidy1, a conserved repressor of CYCLINA2 transcription, controls endoreduplication in Arabidopsis.

Endoreduplication is a type of cell cycle in which DNA replication continues without cell division. We have isolated several dominant mutants from Arabidopsis thaliana activation tagging lines by flow cytometry. One of the mutants, increased level of polyploidy1-1D (ilp1-1D), showed increased polyploidy in both light- and dark-grown hypocotyls. The corresponding gene of ilp1-1D encodes a protein homologous to the C-terminal region of mammalian GC binding factor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The expression of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing line, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DNA insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 expression. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demonstrate that this protein regulates endoreduplication through control of CYCA2 expression in Arabidopsis.

An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response.

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

IL-18 induces apoptosis of adherent bone marrow cells in TNF-alpha mediated osteoclast formation in synergy with IL-12.

It has recently been reported that TNF-alpha has the ability to accelerate osteoclastogenesis. We previously reported that the proinflammatory cytokine IL-12 induced apoptosis in TNF-alpha-mediated osteoclastogenesis in mouse bone marrow culture through an interaction of Fas and Fas ligand (FasL). In this study, the effect of IL-18 was investigated, which is also a proinflammatory cytokine, on TNF-alpha-mediated osteoclastogenesis. When mouse bone marrow cells were cultured with both TNF-alpha and IL-18, the number of adherent cells in the culture decreased. Apoptotic effects, indicated by nuclear, cellular and DNA fragmentation, were observed in the adherent cells. The apoptosis was inhibited by an anti-FasL antibody. Apoptosis of the adherent bone marrow cells might be caused by Fas-FasL interactions. Furthermore, IL-18 and IL-12 synergistically induced apoptosis of adherent bone marrow cells in the presence of TNF-alpha, and up-regulated FasL transcription in non-adherent cells. The results suggested that FasL synergistically up-regulated by IL-12 and IL-18 increased apoptosis of the adherent cells.

The FOX hunting system: an alternative gain-of-function gene hunting technique.

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.

TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis.

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.

Inhibition of RANKL-induced osteoclast formation in mouse bone marrow cells by IL-12: involvement of IFN-gamma possibly induced from non-T cell population.

IL-12 was shown to have the potential to inhibit osteoclast formation in mouse bone marrow cells treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). When bone marrow macrophages (BMM) were used as osteoclast precursors, IL-12 failed to inhibit M-CSF/RANKL-induced osteoclast formation from BMM. In coculture experiments using transwells, IL-12 did inhibit osteoclast formation from BMM cocultured with whole bone marrow cells. These results indicated that IL-12 indirectly affected M-CSF/RANKL-induced osteoclastogenesis in bone marrow cells and that the inhibition of IL-12 on osteoclast formation was caused by a humoral factor from bone marrow cells treated with IL-12. Experiments with anti-interferon (IFN)-gamma antibody and bone marrow cells from IFN-gamma receptor knockout mice revealed that IFN-gamma might be involved in the inhibition of osteoclast formation in this system. The expression of osteoprotegerin mRNA in bone marrow cells was not affected by treatment with IL-12. The inhibitory effect of IL-12 on osteoclast formation was also seen in the T cell-depleted bone marrow cells of normal mice and the whole bone marrow cells of athymic nude mice, while the inhibitory effect of IL-12 was partially suppressed in the B cell-depleted bone marrow cells. The inhibitory effect of IL-12 on M-CSF/RANKL-induced osteoclastogenesis was not accompanied with cell death, in contrast with our previous finding that the inhibitory effect of IL-12 on M-CSF/TNF-alpha-induced osteoclastogenesis is attributable to Fas and FasL-mediated apoptosis.

Interleukin-4 directly inhibits tumor necrosis factor-alpha-mediated osteoclast formation in mouse bone marrow macrophages.

Recently it has been found that osteoclast differentiation is induced by tumor necrosis factor (TNF)-alpha. Interleukin (IL)-4 was reported to suppress osteoclast differentiation and bone resorption. However, no study has investigated the effect of IL-4 on TNF-alpha-induced osteoclast formation. In this study, we investigated whether IL-4 inhibits TNF-alpha-mediated osteoclast formation in mouse bone marrow derived macrophages (BMM). First, IL-4 suppresses RANKL-induced osteoclast formation and bone resorption. Next, when BMM were cultured with TNF-alpha, osteoclast-like cells were formed. When they were cultured with both TNF-alpha and IL-4, osteoclast formation and bone resorption was suppressed by IL-4 in a dose-dependent manner. It has been recently found that TNF-alpha and RANKL synergistically promote osteoclastogenesis. Finally, we investigated whether IL-4 had the ability to inhibit synergistic TNF-alpha and RANKL-induced osteoclastogenesis, with the result that it effectively inhibited the synergistic osteoclast formation in a dose-dependent manner. We conclude that IL-4 can strongly inhibit osteoclast formation that is related to both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-alpha.

Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant.

To study the functions of the nuclear genes involved in chloroplast development, we systematically analyzed albino and pale-green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as a mutagen. One of the pale-green mutants, albino or pale green mutant 1 (designated as apg1), did not survive beyond the seedling stage, when germinated on soil. The chloroplasts of the apg1 plants contained decreased numbers of lamellae with reduced levels of chlorophyll. A gene encoding a 37 kDa polypeptide precursor of the chloroplast inner envelope membrane was disrupted by insertion of the Ds transposon in apg1. The 37 kDa protein had partial sequence similarity to the S-adenosylmethionine-dependent methyltransferase. The apg1 plants lacked plastoquinone (PQ), suggesting that the APG1 protein is involved in the methylation step of PQ biosynthesis, which is localized at the envelope membrane. Our results demonstrate the importance of the 37 kDa protein of the chloroplast inner envelope membrane for chloroplast development in Arabidopsis.