JEAP, a novel component of tight junctions in exocrine cells.

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.     

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. on their root colonization and growth promotion of cucumber (Cucumis sativus L.)

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. was studied for its effect on their root colonization and plant growth of cucumber. Two isolates of Phoma sp. (GS8-2 and GS8-3) were tested with G. mosseae. The percent root length colonized by G. mosseae was not adversely affected by the presence of Phoma isolates. In contrast, the root colonization of both isolates GS8-2 and GS8-3 in 4-week-old plants was significantly reduced (80.7% and 84.3%, respectively) by added G. mosseae. Inoculating plants with each Phoma isolate significantly increased the shoot dry weight. However, dual inoculation of each Phoma isolate with G. mosseae had no significant effect on growth enhancement.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.     

Isomerization and/or racemization at Asp23 of Abeta42 do not increase its aggregative ability, neurotoxicity, and radical productivity in vitro

Aggregation of the 42-mer amyloid β peptide (Aβ42) plays a pivotal role in the pathogenesis of Alzheimer’s disease. Recent investigations suggested the isomerization and/or racemization of Asp at position 1, 7, or 23 to be associated with the pathological role of Aβ42. Our previous study indicated that the turn at positions 22 and 23 of Aβ42 is closely related to its neurotoxicity through the formation of radicals. To clarify the contribution of these modifications at Asp23 to the pathology, three isomerized and/or racemized Aβ42 mutants were prepared. l-isoAsp23- and d-Asp23-Aβ42 showed moderate aggregative ability similar to the wild type. However, d-Asp23-Aβ42 was less neurotoxic than the wild type, while l-isoAsp23-Aβ42 was as toxic as the wild type. In contrast, d-isoAsp23-Aβ42 showed weak aggregative ability without neurotoxicity. These results suggest the isomerization and/or racemization of Asp23 not to be related to the pathogenesis, but to be a consequence of chemical reactions during the long-term deposition of fibrils.     

Essential oil of lavender inhibited the decreased attention during a long-term task in humans

This study examined the effects of odors on sustained attention during a vigilance task. Two essential oils (lavender and eucalyptus) and two materials (l-menthol and linalyl acetate) were compared with a control. The increase in reaction time was significantly lower with lavender than with the control. The results suggest that the administration of lavender helped to maintain sustained attention during the long-term task.     

Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

Use of single-protoplast isolates in the study of the mating phenomena of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> (<Emphasis Type="Italic">Thanatephorus cucumeris</Emphasis>) AG-1 IC and IA

This study evaluates the effectiveness of using single-protoplast isolates (SPIs) to study the mating phenomena of Rhizoctonia solani AG-1 IC and IA. SPIs obtained from three field isolates (F-1, Rh28, and RO2) of AG-1 IC were paired with representative single-basidiospore isolate (SBI)-M1/-M2 testers, each from their own field isolates, or paired in all possible combinations. Tufts were formed between SPIs and SBI-M1/-M2 testers and between SPIs-M1 and -M2. The separation ratios of SPIs-M1 and -M2 were approximately 1:1, which were similar to the results obtained with SBIs. SPIs obtained from three isolates (GNSD, R59, and Tr8) of AG-1 IA, which failed to form basidiospores, were paired in all possible combinations. Although no tufts formed among SPIs from Tr8 and R59, tufts did form between SPIs from GNSD. SPIs from GNSD were separated into homokaryotic (-M1 or -M2) and heterokaryotic isolates, and the separation ratio of -M1 and -M2 was also around 1:1. Amplified fragment length polymorphism (AFLP) phenotypes of the tuft isolates formed between GNSD SPIs-M1 and -M2 suggested that these tuft isolates were all heterokaryotic. These results indicate that all three isolates of AG-1 IC and one isolate GNSD of AG-1 IA are heterokaryotic, and that the other two isolates of Tr8 and R59 of AG-1 IA are homokaryotic. Single-protoplast isolates are effective for studies of the mating phenomena of isolates belonging to different AGs of R. solani that could not form a perfect stage.