Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae

Abstract Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB ) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.     

Restoration of gene function by homologous recombination: from PCR to gene expression in one step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na₂SeO₃, Na₂SeO₄, Na₂Se, seleno-dl-cystine, seleno-l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na₂SeO₃ was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na₂SeO₃ was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na₂SeO₃, may be the critical enzyme.     

Selective degradation of altered tomato β-fructofuranosidase molecules by neutral protease

Evidence is presented for the selective breakdown of altered tomato β-fructofuranosidase molecules by a neutral protease from Bacillus subtilis.     

Studies on Flavor Components of Foodstuffs

Distribution of tetramethylpyrazine (T.M.P.) in Japanese fermented foodstuffs was investigated by more accurate analytical method.

Namely, the method was successful when trapping T.M.P. with picric acid after flash evaporation of the foodstuffs, followed by analysis with gas chromatography.

T.M.P. was detected in many Japanese fermented foodstuffs, especially in Miso, Soy sause and Natto, which suggests that alkylpyrazines may play an important role as flavor of those foodstuffs.     

Microorganism Capable of Decomposing N-Acetylglucosaminyl Ribitol Teichoic Acid of Staphylococcus aureus

Enzyme(s) capable of decomposing N-acetylglucosaminyl ribitol teichoic acid prepared from the cell wall of Staphylococcus aureus FDA 209 P was obtained from the culture supernatant of a gram-negative, rod-shaped, spore-forming soil bacterium. Properties of the bacterium were very similar to those of Bacillus circulans.     

Phytomelatonin in the leaves and fruits of wild perennial plants

Phytomelatonin has been documented in numerous flowering plants, mostly in cultivated species consumed by humans. Although frugivorous animals feed on fruits, the phytomelatonin content of these organs has hardly ever been tested in wild plants. The aim of this study was to determine the levels of phytomelatonin in the leaves and fleshy fruits of 31 wild perennial species known to be eaten by herbivorous and frugivorous mammals and birds. Considerable levels of phytomelatonin were found in the leaves of all the tested species, and some contained melatonin in their fruits as well. The melatonin content was found to vary significantly in different life forms (trees, shrubs, and climbers), with trees possessing the highest levels. The analysis revealed a significant positive correlation between the phytomelatonin levels in the leaves and the fruits of various species. However, the concentration found in the fruits was generally lower than that found in the leaves of the same species. Despite the presence of phytomelatonin in the fleshy fruits of different families, there was no noticeable common attribute among them. Phytomelatonin was exhibited in both the seeds and the pulp, with no obvious preference for either one. Although it was determined that ingested melatonin enters the bloodstream of birds and mammals, its specific role is still not certain. The potential impact of edible phytomelatonin on the circadian rhythm of herbivores and frugivores is discussed on the basis of these findings.     

Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.     

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.