Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II)

Quercetin, one of flavonoids, has been reported to be carcinogenic. There have been no report concerning carcinogenicity of kaempferol and luteolin which have structure similar to quercetin. DNA damage was examined by using DNA fragments obtained from the human p53 tumor suppressor gene. Quercetin induced extensive DNA damage via reacting with Cu(II), but kaempferol and luteolin induced little DNA damage even in the presence of Cu(II). Excessive quercetin inhibited copper-dependent DNA damage induced by quercetin. Bathocuproine, a Cu(I)-specific chelator, catalase and methional inhibited the DNA damage by quercetin, whereas free hydroxyl radical scavengers did not. Site specificity of the DNA damage was thymine and cytosine residues. The site specificity and the inhibitory effects suggested that DNA-copper-oxygen complex rather than free hydroxyl radical induced the DNA damage. Formation of 8-oxodG by quercetin increased extensively in the presence of Cu(II), whereas 8-oxodG formation by kaempferol or luteolin increased only slightly. This study suggests a good relationship between carcinogenicity and oxidative DNA damage of three flavonoids. The mechanism of DNA damage by quercetin was discussed in relation to the safety in cancer chemoprevention by flavonoids.     

A novel serum chitinase that is expressed in bovine liver

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.     

Leek Yellow Stripe Virus Can Adjust for Host Adaptation by Trimming the N-Terminal Domain to Allow the P1 Protein to Function as an RNA Silencing Suppressor

In Japan, the P1 protein (S-type) encoded by leek yellow stripe virus (LYSV) isolates detected in Honshu and southward is shorter than the P1 (N-type) of LYSV isolates from garlic grown in Hokkaido due to a large deletion in the N-terminal half. In garlic fields in Hokkaido, two types of LYSV isolate with N- and S-type P1s are sometimes found in mixed infections. In this study, we confirmed that N- and S-type P1 sequences were present in the same plant and that they belong to different evolutionary phylogenetic groups. To investigate how LYSV with S-type P1 (LYSV-S) could have invaded LYSV with N-type P1 (LYSV-N)-infected garlic, we examined wild Allium spp. plants in Hokkaido and found that LYSV was almost undetectable. On the other hand, in Honshu, LYSV-S was detected at a high frequency in Allium spp. other than garlic, suggesting that the LYSV-S can infect a wider host range of Allium spp. compared to LYSV-N. Because P1 proteins of potyviruses have been reported to promote RNA silencing suppressor (RSS) activity of HC-Pro proteins, we analyzed whether the same was true for P1 of LYSV. In onion, contrary to expectation, the P1 protein itself had RSS activity. Moreover, the RSS activity of S-type P1 was considerably stronger than that of N-type P1, suggesting that LYSV P1 may be able to enhance its RSS activity when the deletion is in the N-terminal half and that acquiring S-type P1 may have enabled LYSV to expand its host range.     

Characterization of hepatitis B virus DNA polymerase

Hepatitis B virus (HBV) particles were separated from the blood-plasma containing HBe and HBs antigens (subtype adr) and the nature of the endogenous DNA polymerase in the HBV core particles was studied. The HBV endogenous DNA polymerase activity was examined under the conditions used for preparation of HBV vaccine. The endogenous DNA polymerase activity was reduced slowly upon the heat treatment or the formalin treatment. The reductions of the activity were 65% and 70% upon the heat treatment at 60 C for 10 hr and the formalin treatment at 37 C for 90 hr, respectively. Properties of the HBV endogenous DNA polymerase were studied by utilizing specific inhibitors against the eukaryotic DNA polymerases. Our results showed that the HBV endogenous DNA polymerase is resistant to aphidicolin and N-ethylmaleimide, and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, phosphonoformic acid and 9-beta-D-arabinofuranosyladenosine 5'-triphosphate.     

Essential contribution of germline-encoded lysine residues in Jgamma1.2 segment to the recognition of nonpeptide antigens by human gammadelta T cells.

Human gammadelta T cells display unique repertoires of Ag specificities largely imposed by selective usages of distinct Vgamma and Vdelta genes. Among them, Vgamma2/Vdelta2(+) T cells predominate in the circulation of healthy adults and respond to various microbial small molecular mass nonpeptide Ags. The present results indicate that the primary Vgamma2/Vdelta2(+) T cells stimulated with the distinct groups of nonpeptide Ags, including monoethyl pyrophosphate, isobutyl amine, and aminobisphosphonate, invariably exhibit Jgamma1.2 in the Vgamma2(+) TCR-gamma chains. Gene transfer studies revealed that most of the randomly cloned Vgamma2/Jgamma1.2(+) TCR-gamma genes bearing diverse Vgamma/Jgamma junctional sequences could confer the responsiveness to all these nonpeptide Ags, while none of the Vgamma2/Jgamma1.1(+) or Vgamma2/Jgamma1.3(+) TCR-gamma genes could do so. Furthermore, mutation of the lysine residues encoded by the Jgamma1.2 gene, which are unique in human Jgamma1.2 and absent in other human or mouse Jgamma segments, completely abrogated the responsiveness to all the nonpeptide Ags without affecting the response to anti-CD3 mAb. These results strongly suggested that the positively charged lysine residues in the TCR-gamma chain CDR3 region encoded by the germline Jgamma1.2 gene play a key role in the recognition of diverse small molecular mass nonpeptide Ags.     

Indoor environmental study and post occupancy evaluation in an office environment

1. 1. The objective of this paper is to investigate the indoor environment from the viewpoint of interaction between physical environment and the human responses. The field survey has been conducted over 1 year.

2. 2. A continuous measurement has been carried out for 1 week and distribution of variables have been measured for 1 day.

3. 3. The attitude of workers was investigated by a questionnaire.

4. 4. As the result, average luminance represented more than 1000 lx in the new building, in contrast with less tha 300 lx in the existing building.

5. 5. There was a significant difference of the occupants' response to the light environment between the two buildings.

6. 6. Measured thermal conditions are on the edge of the ASHRAE comfort envelope in summer, and in the neighborhood of the lower dry limit of the envelope in spring.

7. 7. The occupants' evaluations were remarkably changed before and after the moving. The office environment is better than that of the factory.