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.     

Isolation of a bacterium that causes anaaki disease of the red algae Porphyra yezoensis

Anaaki disease causes severe damage to the red algae Porphyra yezoensis from which the Japanese traditional food 'nori'is produced. The causative agent of anaaki disease was isolated by several repeats of single-colony isolation and infection experiments, and was identified as Flavobacterium sp. LAD-1. The bacterium showed hydrolytic activity toward porphyran but not toward other polysaccharides composing the thallus of Porphyra , such as β-1,3-xylan or β-1,4-mannan. The bacterium also showed β-D-galactosidase activity.     

Evolution of restriction sites of ribosomal DNA in natural populations of the field mouse,Apodemus speciosus

An analysis by restriction endonuclease digestion of ribosomal DNA (rDNA) was carried out in natural populations of Apodemus speciosus, a field mouse that is endemic to Japan. Two restriction sites, the EcoRI (E3) and DraI (D4) sites, in the nontranscribed spacer region downstream from the gene for 28S RNA showed polymorphism within and between individuals in the populations from the Japanese main islands. By contrast, populations from the small adjoining islands which are thought to have separated from the main islands 1–2 × 10⁴ years ago showed relatively low levels of polymorphism within and between individuals, i.e., one of the polymorphic bands in the case of each enzyme was predominant in these populations, irrespective of the variants. These results indicate that the rate of fixation of site variations depends on population size and that the direction of fixation is random. Furthermore, each polymorphic restriction site seems to be fixed independently.Correspondence to: H. Suzuki     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

FK506 and Cyclosporin A Enhance IL-6 Production in Monocytes: A single-Cell Assay

The effect of FK506 and cyclosporin A (CsA) on the production of interleukin 6 (IL-6) in adherent monocytes was studied at a single-cell level by the avidinbiotin- peroxidase complex methods. The percentage of IL-6-producing monocytes increased when stimulated with lipopolysaccharide (LPS) at concentrations between 10 ng/ml and 10 mug/ml, in a dose dependent manner. Both FK506 and CsA enhanced the percentage of IL-6- producing monocytes stimulated with 100 pg/ml-1 mug/ml of LPS up to values near those obtained with 10 mug/ml of LPS. The enhancement by FK506 and CsA was not seen when monocytes were stimulated with a high concentration of LPS (10 mug/ml). When monocytes were stimulated with a low concentration of LPS (10 ng/ml), FK506 and CsA enhanced IL-6 production in a dose dependent manner, at a drug concentration of 0.12 nM-1.2 muM (0.1-1 000 ng/ml) for FK506 and 0.83 nM-8.3 muM (1-10 000 ng/ml) for CsA. The optimal effect of FK506 was achieved at a concentration 7-fold lower than that of CsA. In contrast, production of turnout necrosis factor-alpha (TNFalpha and interleukin 1beta (IL-1beta) was slightly suppressed by FK506 and CsA at the concentrations tested. Moreover, pretreatment of monocytes with FK506 and CsA had a significant enhancing effect on LPS-induced IL-6 production, while treatment with FK506 or CsA after LPS stimulation had no effects on IL-6 production, suggesting that the enhancing effect of each drug is exerted before LPS stimulation or at an early stage of the post-receptor pathway after LPS stimulation. These experiments demonstrate that FK506 and CsA can selectively enhance IL-6 production in monocytes under certain conditions in vitro and, possibly, also in vivo.     

Additive Induction of Egr-1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108-15 Cells via Cholinergic Muscarinic, α2-Adrenergic, and Bradykinin Receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α₂-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.     

Population dynamics,productivity and biomass allocation ofZizania latifolia in an aquatic-terrestrial ecotone

The population and production ecology of aZizania latifolia stand at a sheltered shore of the Hitachi-Tone River were investigated. Shoot emergence was observed twice a year; the fist was a synchronized shoot emergence in April and the second was from August to October. Aboveground biomass was mostly occupied by leaves and peaked at 1500 g dry weight m⁻² in August. The belowground biomass also reached its peak, 750 g dry weight m⁻², in August. The secondary shoots were small in spite of their high density. Leaves were produced continuously throughout the season. The leaf life span was as short as 55.6 days for cohorts that emerged from May through to September. Total annual net production ofZ. latifolia could be more than 3400 g dry weight m⁻². Shoot clusters of several centimeters were observed in April. The following self-thinning caused a regular distribution of the remaining shoots in August. Most shoots produced in August to October were found near a shoot persisting since April. They showed more concentrated distribution than shoots in April. A large biomass allocation to leaves and the ability to produce many clump shoots during the late growing period may facilitate dominance ofZ. latifolia in relatively sheltered sites.     

Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.