Detection of specific DNA from crude extracts of rice seed grains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

To rapidly detect specific genes, crude extracts prepared from rice seed grains were used as templates for PCR, the PCR products were digested with restriction enzymes or urasil-DNA glycosylase, and then matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) was used to detect amplified DNA. It was possible to amplify small DNA fragments (50–60 bp), but not large ones (>200 bp), using crude extracts as the PCR template. This method can be completed within 1 h, including extractions, and is well suited to automation for high-throughput analyses.     

Synthetic Analogues of the Termite Trail-following Substance

The effect of tripropyltin chloride (TPT) on some functional reactions in E. coli was investigated. The inhibition on respiration and protein, DNA and RNA syntheses were examined in vivo. Oxygen consumption by E. coli cells was scarcely inhibited even at the concentration of 30 µg/ml TPT. The incorporations of ¹⁴C-labeled amino acids into protein fraction were inhibited. Especially, in the case of l-leucine, it was inhibited 60% even at 10 µg/ml TPT. Both incorporations of ¹⁴C-adenine into DNA and RNA fraction were inhibited 50–60% at 20 µg/ml TPT. RNA polymerase was prepared from E. coli cells and the effect of organotin compounds on the enzyme activity was examined. Organotin compounds inhibited the enzyme activity only at high concentrations (5-10mm). and dialkyltin chlorides which possess no antimicrobial action showed the inhibition more intensely than trialkyltin chlorides. The effect on membrane-bound ATPase was also examined in vitro. We found that TPT has high inhibitory action on membrane-bound ATPase. However, it slightly inhibited the activity of ATPase separated from membrane.     

Stereoselective Synthesis of Ectocarpene and Its Antipode via Microbiological Asymmetric Hydrolysis

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (?)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(?)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(?)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.     

Leaf Alcohol

The diethylamine-catalyzed aldol condensation of E-2-hexenal yielded a mixture of 2-E,4-E,6-E- (IV-a) and 2-E,4-Z,6-E-4-ethyldeca-2,4,6-triene-1-al (IV-b). Structual and geometrical elucidation of both alcohols were made by means of spectral evidence as well as by the catalytic hydrogenation leading to the same 4-ethyldecanol (VI). The “b-peak substance” detected in the leaf alcohol reaction products was proved to be identical with 4-ethyldecanol (VI). The treatment of the trienal containing the central Z-double bond with sodium under the leaf alcohol reaction condition failed to afford ethyl-propyl-benzyl alcohol, but gave 4-ethyldecanol (VI). This result safely excludes the operation of the previously suspected valence tautomerism (Cope rearrangement) in the leaf alcohol reaction, and accounts for the pathway of the formation of (VI).     

Isolation of Z-3-Hexenal in Tea Leaves,Thea sinensis,and Synthesis Thereof

Z-3-Hexenal, a precursor in the biosynthesis of leaf alcohol and leaf aldehyde, was first isolated from fresh tea leaves, Thea sinensis and its structure was confirmed by unequivocal synthetic evidence.     

Cloning of Lipoxygenase Genes from a Cyanobacterium, Nostoc punctiforme, and Its Expression in Eschelichia coli

Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.     

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.     

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains

Mycopathologia - Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms’ lipase...     

Role of interleukin-17 in a murine community-associated methicillin-resistant Staphylococcus aureus pneumonia model

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.     

Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production

The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.