Prevalence of Toxoplasma gondii and Neospora caninum in sika deer from eastern Hokkaido, Japan

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.     

Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity

For mass production of lactic acid, we newly constructed a transgenic wine yeast strain that included six copies of the bovine L-lactate dehydrogenase gene on the genome. On fermentation in inexpensive cane juice-based medium, L-lactate production of this recombinant reached 122 g/liter and the optical purity was 99.9% or higher.     

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.     

Root hydraulic conductivity and whole-plant water balance in tropical saplings following a shade-to-sun transfer

We hypothesized that pioneer and late successional species show different morphological and physiological responses in water use after gap formation. The magnitude of the responses was compared between two pioneer species (Macaranga gigantea and Trema orientalis) and four late successional species (Shorea sp.), in an experiment in which saplings were transferred from shade to sun. Although transpiration demand increased following the transfer, root hydraulic conductivity (Lp_r) decreased. Lp_r was sensitive to brief treatments with HgCl₂ (a specific inhibitor of aquaporins). This allows Lp_r to be divided into two components: cell-to-cell and apoplastic pathways. The Lp_r of cell-to-cell pathway decreased in all species following the transfer, relating to aquaporin depression in roots. Following the transfer, leaf osmotic potentials at full hydration decreased and both leaf mass per area [leaf mass/leaf area (LMA)] and fine-root surface area/leaf surface area (root SA/leaf SA) increased in almost all species, allowing saplings to compensate for the decrease in Lp_r. Physiologically, pioneer species showed larger decreases in Lp_r and more effective osmotic adjustment than late successional species, and morphologically, pioneer species showed larger increases in root SA/leaf SA and LMA. Water balance at the whole-plant level should be regulated by coupled responses between the aboveground and the belowground parts. Interspecific differences in responses after gap formation suggest niche differentiation in water use between pioneer and late successional species in accordance with canopy-gap size.     

Involvement of circadian clock gene Clock in diabetes-induced circadian augmentation of plasminogen activator inhibitor-1 (PAI-1) expression in the mouse heart

Diabetes is associated with an excess risk of cardiac events, and one of the risk factors for infarction is the elevated-levels of plasminogen activator inhibitor-1 (PAI-1). To evaluate how the molecular clock mechanism is involved in the diabetes-induced circadian augmentation of PAI-1 gene expression, we examined the expression profiles of PAI-1 mRNA in the hearts of Clock mutant mice with streptozotocin-induced diabetes. Circadian expression of PAI-1 mRNA was blunted to low levels under both normal and diabetic conditions in Clock mutant mice, although the expression rhythm was augmented in diabetic wild-type (WT) mice. Furthermore, plasma PAI-1 levels became significantly higher in WT mice than in Clock mutant mice after STZ administration. Our results suggested that the circadian clock component, CLOCK, is involved in the diabetes-induced circadian augmentation of PAI-1 expression in the mouse heart.     

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP-ribose) polymerase-1 and 2 (PARP-1) and (PARP-2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP-1 and PARP-2, quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2. SBDD analysis via a combination of X-ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains

Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1-linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C-->T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called "puratrophin-1" (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the spectrin repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1--normally expressed in a wide range of cells, including epithelial hair cells in the cochlea--was aggregated in Purkinje cells of the chromosome 16q22.1-linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5' untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5' UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans.     

Generation and application of a monoclonal antibody that detects a wide variety of protein tyrosine kinases

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.     

Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.