Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

Toxicity of free proline revealed in an arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase

The toxicity of proline (Pro) to plant growth has raised questions despite its protective functions in response to environmental stresses. To evaluate Pro toxicity, we isolated an Arabidopsis T-DNA-tagged mutant, pdh, that had a defect in Pro dehydrogenase (AtProDH), which catalyzes the first step of Pro catabolism. The pdh mutant showed hypersensitivity to exogenous application of < or =10 mM L-Pro, at which wild-type plants grew normally. A dose-dependent increase in internal free Pro accumulation was observed in pdh plants during external Pro supply. These results do not just prove the toxicity of Pro, but also suggest that AtProDH is the only enzyme acting as a functional ProDH in ARABIDOPSIS: To further analyze the targets of Pro toxicity, we compared the expression of thousands of genes by pdh plants with that by wild-type plants by cDNA microarray analysis. Most genes were unaffected. Here we demonstrate Pro toxicity by using the pdh mutant and discuss a cause-and-effect action between an excess of free Pro and growth inhibition in ARABIDOPSIS.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

West Nile fever/encephalitis

West Nile virus (WNV), a member of the family Flaviviridae (genus Flavivirus), is a mosquito-borne virus first isolated in 1937 in the West Nile district of Uganda. The disease in humans is characterized by a dengue-like illness with fever, and a more severe form is characterized by central nervous system involvement, including encephalitis, meningitis, and myelitis. WN encephalitis was first reported in the Western Hemisphere in the summer of 1999, there was an outbreak in New York City. Epidemic WNV strains in North America are severely pathogenic, however, attenuated WNV strains were found in Texas and Mexico in 2003. The principal vectors of WNV transmission in North America are Culex. pipiens, Cx. Quinquefasciatus, Cx. restuans, Cx salinarius and Cx talsalis. The number of WN fever case has exceeded 27,000 since 1999 in the United States and 4,600 since 2002 in Canada. The first imported case of West Nile fever in Japan was confirmed in September, 2005. The patient had returned to Japan from the United States and developed symptoms the next day. There is currently no WN vaccine for use in humans. An inactivated WNV vaccine for use in horses has been available since 2001. A DNA vaccine, a chimeric live attenuated vaccine, and a recombinant vaccine have also been licensed for use in horses.     

A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication

Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.     

Clinicopathological assessment of cancer/testis antigens NY-ESO-1 and MAGE-A4 in osteosarcoma

The cancer/testis antigens (CTAs), New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and melanoma antigen gene (MAGE)-A4 are normally restricted to male germ cells but are aberrantly expressed in several cancers. Considering the limited information regarding their significance in osteosarcoma (OS), the purpose of this study was to determine the clinical significance of NY-ESO-1 and MAGE-A4 expression in OS. Nine patients with OS treated at Kindai University Hospital were included in the study. The median age was 27 years, and median follow-up period was 40 months. The specimens obtained at the time of biopsy were used to perform immunostaining for NY-ESO, MAGE-A4, p53, and Ki-67. The positive cell rates and positive case rates of NY-ESO, MAGE-A4, p53, and Ki-67 were calculated. The correlation between the positive cell rate of immunohistochemical markers was also calculated. The correlation between the positive cell rate of NY-ESO-1 or MAGE-A4 and tumor size or maximum standardized uptake (SUV-max) was also determined. The positive cell rates of NY-ESO-1 or MAGE-A4 in continuous disease-free (CDF) cases were also compared with those in alive with disease (AWD) or dead of disease (DOD) cases. The average positive cell rates of NY-ESO, MAGEA4, p53, and Ki-67 were 71.7%, 85.1%, 16.2%, and 14.7%, and their positive case rates were 33.3%, 100%, 44.4%, and 100%, respectively. The positivity rates of NY-ESO-1 and p53 were strongly correlated, whereas those of NY-ESO-1 and Ki-67 were moderately correlated. The MAGE-A4 and p53 positivity rates and the MAGE-A4 and Ki-67 positive cell rates were both strongly correlated. The NY-ESO-1 and MAGE-A4 positivity rates were moderately correlated. The positive correlation between the NY-ESO-1 positive cell rate and tumor size was medium, and that between the MAGE-A4 positivity rate and SUV-max was very strong. There was no significant difference in the positive cell rates of NY-ESO-1 or MAGE-A4 between CDF cases and AWD or DOD cases. Overall, our results suggest that NY-ESO-1 and MAGE-A4 may be involved in the aggressiveness of OS.Key words: New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma antigen gene (MAGE)- A4, osteosarcoma, prognosis, cancer/testis antigen (CTA), immunohistochemistry     

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.     

LSD1 Overexpression Is Associated with Poor Prognosis in Basal-Like Breast Cancer,and Sensitivity to PARP Inhibition

LSD1, a lysine-specific histone demethylase, is overexpressed in several types of cancers and linked to poor outcomes. In breast cancer, the significance of LSD1 overexpression is not clear. We have performed an in silico analysis to assess the relationship of LSD1 expression to clinical outcome. We demonstrate that LSD1 overexpression is a poor prognostic factor in breast cancer, especially in basal-like breast cancer, a subtype of breast cancer with aggressive clinical features. This link is also observed in samples of triple negative breast cancer. Interestingly, we note that overexpression of LSD1 correlates with down-regulation of BRCA1 in triple negative breast cancer. This phenomenon is also observed in in vitro models of basal-like breast cancer, and is associated with an increased sensitivity to PARP inhibitors. We propose therefore that high expression levels of the demethylase LSD1 is a potential prognostic factor of poor outcome in basal-like breast cancer, and that PARP inhibition may be a therapeutic strategy of interest in this poor prognostic subtype with overexpression of LSD1.     

Does understory vegetation reflect the history of fluvial disturbance in a riparian forest?

We have examined the relationship between the history of fluvial disturbance and understory vegetation in a riparian forest. The study site was divided into three sites, by use of aerial photographs and topographical maps, with different histories of fluvial disturbance: (1) Fagus-type on land that has not been flooded for the last 39 years, at least; (2) Populus-type on land that has not been flooded since debris flow occurred 34 years ago; and (3) Salix-type on land that has been flooded periodically from an abandoned channel since debris flow occurred 34 years ago. Species richness in the Salix-type was significantly higher than in the other types. Detrended correspondence analysis revealed obvious floristic differences among the three canopy types. Canonical correspondence analysis showed that herbaceous species were mainly found on lower plots with high moss cover, implying that moss layers may capture seeds transported by the stream. Tall herbs occurred in less shaded plots and on higher plots, suggesting that their rapid growth prevented the occurrence of other species. Fagus-type was dominated by species with ingested fruits which depended on animals for their dispersal. Populus and Salix-types were dominated by species with wind dispersal or no dispersal mechanism, which depended on physical phenomena for dispersal. Attributes of current understory vegetation were connected with historical events, suggesting that riparian vegetation reflects the history of fluvial disturbance.