Ryoji Nakagawa

Mechanisms of copper toxicity in Saccharomyces cerevisiae determined by microarray analysis.

The effect of the heavy metal copper on the expression of a wide spectrum of genes was analyzed by using a DNA microarray. The gene expression profile of bakers yeast Saccharomyces cerevisiae grown in a medium containing a sublethal concentration of cupric sulfate was compared with that of yeast grown in a normal medium. Among ∼6000 yeast ORFs, 143 ORFs were induced more than twofold to resist copper toxicity after exposure to copper. Copper metallothionein CUP1‐1 and CUP1‐2 were induced more than 20‐fold. Some genes related to sulfur metabolism and oxidative stress response were also up‐regulated. This DNA microarray analysis identified several molecular targets of copper toxicity.

Toxicity of methanol and formaldehyde towards Saccharomyces cerevisiae as assessed by DNA microarray analysis.

To assess the toxicity of the C1 compounds methanol and formaldehyde, gene expression profiles of treated baker’s yeast were analyzed using DNA microarrays. Among approximately 6,000 open reading frames (ORFs), 314 were repressed and 375 were induced in response to methanol. The gene process category “energy” comprised the greatest number of induced genes while “protein synthesis” comprised the greatest number of repressed genes. Products of genes induced by methanol were mainly integral membrane proteins or were localized to the plasma membrane. A total of 622 and 610 ORFs were induced or repressed by formaldehyde, respectively. More than one-third of the genes found to be strongly repressed by formaldehyde belonged to the “protein synthesis” functional category. Conversely, genes in the subcategory of “nitrogen, sulfur, and selenium metabolism” within “metabolism” and in the category of “cell rescue, defense, and virulence” were up-regulated by exposure to formaldehyde. Our data suggest that membrane structure is a major target of methanol toxicity, while proteins were major targets of formaldehyde toxicity.

Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression.

Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein.

Cloning, Sequencing, and Heterologous Expression of a Cellobiohydrolase cDNA from the Basidiomycete Corticium rolfsii

From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64% identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.

Stimulated Accumulation of Lectin mRNA and Stress Response in Helianthus tuberosus Callus by Methyl Jasmonate

Callus from Helianthus tuberosus expresses a mannose-specific lectin (HTA). The level of HTA mRNA significantly increased one hour after treatment of the callus with 20 mg/l methyl jasmonate. Following this, fragmentation of the callus DNA at regular intervals was observed together with strong self-fluorescence emission in the callus cells.

Development and Technology Transfer of Functional Foods Using Lactobacillus plantarum HOKKAIDO

We have isolated a new strain of Lactobacillus plantarum from Japanese pickles produced by a farmer in Hokkaido, and have named the strain HOKKAIDO. Our investigation of HOKKAIDO strain revealed several characteristics. The strain exhibits digestive juice tolerance and can survive in the intestine. The strain strongly adheres to human enterocyte-like Caco-2 cells, and cells of HOKKAIDO strain competed with E. coli O-157 cells for adhesion to Caco-2 cells. From several examinations with human dendritic cells, this strain may act to improve immune function. It was found that HOKKAIDO strain is superior in the fermentation of vegetables, fruits, and cereals. These characteristics were exploited to produce foods such as fermented soybean milk, alcoholic beverages using sake lees, and fermented carrot drink. In addition, HOKKAIDO strain as a probiotic was contained in commercial products such as yogurt and a milk substitute for calves. (Accepted Feb. 16, 2018)