Inhibition effect of chlorine ion on hydroxyl radical generation in UV-H2O2 process.

UV-H2O2 process is widely used as an advanced oxidation process (AOP) for the treatment of chlorine volatile organic compounds (CVOCs) such as dichloromethane (DCM) with strong oxidativity of hydroxyl radical generated from photolysis of H2O2. The result of DCM degradation rate at different initial concentrations in UV-H2O2 processes indicated the inhibition effect of produced chlorine ions on DCM oxidation processes, because the first-order degradation rate constant increased with lower initial concentrations. A spin trapping adduct of hydroxyl radical with 5,5-dimethyl-1-pyrroline-n-oxide (DMPO) was quantified by ESR spectrometer after UV irradiation in the presence of different amounts of chlorine ion, and as a result, the chlorine ion was found to act as a hydroxyl radical scavenger, which resulted in decreasing DCM degradation rate. An UV-H2O2 reactor equipped with ion exchangers for removing chlorine ion achieved higher DCM degradation rate than that without ion exchangers.

Isolation of a new lytic enzyme for hiochi bacteria and other lactic acid bacteria.

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

Interspecies transmission of feline immunodeficiency virus from the domestic cat to the Tsushima cat (Felis bengalensis euptilura) in the wild.

Feline immunodeficiency virus (FIV) was isolated from a wild-caught Tsushima cat (Felis bengalensis euptilura), an endangered Japanese nondomestic subspecies of leopard cat (F. bengalensis). Phylogenetic analysis of the env gene sequences indicated that the FIV from the Tsushima cat belonged to a cluster of subtype D FIVs from domestic cats. FIVs from both the Tsushima cat and the domestic cat showed similar levels of replication and cytopathicity in lymphoid cell lines derived from these two species. The results indicated the occurrence of interspecies transmission of FIV from the domestic cat to the Tsushima cat in the wild.

Cloning and nucleotide sequence of the alcohol acetyltransferase II gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7.

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery larger yeast have one ATF2 gene, while S. bayanus has no similar gene.

Nucleotide sequences of alcohol acetyltransferase genes from lager brewing yeast, Saccharomyces carlsbergensis.

The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg-ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg-ATF1 protein (75.7%), but the N-terminal region has a relatively low degree of homology. Southern analysis and contour-clamped homogeneous electric field analysis of Saccharomyces strains suggest that the ATF1 gene is located on chromosome XV in S. cerevisiae and that the Lg-ATF1 gene might originate from the "non-S. cerevisiae' genome of S. carlsbergensis, which is similar to that of S. bayanus and S. pastorianus.

Construction of a promoter probe vector autonomously maintained in Aspergillus and characterization of promoter regions derived from A. niger and A. oryzae genomes.

We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMA1) and the E. coli uidA gene as a reporter gene to search the A. oryzae and A. niger genomes for DNA fragments having strong promoter activity. Beta-glucuronidase (GUS)-producing A. oryzae transformants containing the No. 8AN derived from A. niger, or the No. 9AO derived from A. oryzae, were constitutive for the expression of the uidA gene when cultivated in the presence of a variety of carbon and nitrogen sources. When the GUS-producing transformants were grown in liquid culture, the No. 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (alpha-amylase encoding gene) promoter. There was also a corresponding increase in the amount of GUS gene-specific mRNA. When these transformants were grown as rice-koji, the No. 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased. These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

A method for the re-isolation of an autonomously replicating plasmid from Aspergillus transformants.

An autonomously replicating plasmid is expected to increase the frequency of Aspergillus transformation. To construct this type of plasmid, we developed a rapid method of re-isolating autonomously replicating plasmids from Aspergillus transformants. Transformants grown in MM medium under selective pressure for 1-2 days were converted to protoplasts with a cell wall lytic enzyme (e.g. Yatalase). The protoplasts were lysed with phenol/chloroform followed by precipitation with ethanol. The total DNA was treated with RNaseA, re-precipitated with PEG, and then used to transform E. coli. These re-isolated plasmids were mainly the plasmid monomer.

Transformation of intact Aspergillus niger by electroporation.

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene.

The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.

The purification, properties and internal peptide sequences of alcohol acetyltransferase isolated from Saccharomyces cerevisiae Kyokai No. 7.

Alcohol acetyltransferase (AATase) catalyzes the esterification of isoamyl alcohol by acetyl coenzyme A. The enzyme was solubilized from the microsomal fraction of Saccharomyces cerevisiae Kyokai No. 7, using Triton X-100 and then purified by a series of chromatographic separations: Poly Buffer Exchanger 94 (PBE94), DEAE Toyopearl, Toyopearl HW60, hydroxyapatite, Octyl-Sepharose CL-4B, and hexanol-affinity column chromatography. When the solubilized enzyme was put on PBE94, two active fractions were obtained. The enzyme obtained after affinity column chromatography had a single band on an SDS polyacrylamide gel, and its molecular mass was estimated to be 60 kDa. The enzyme was most active at pH 8.0 and 25 degrees C. It was stable between pH 7.5 and 8.5, but was unstable at temperatures above 10 degrees C. The activity was markedly inhibited by heavy metal ions such as Cd2+, Cu2+, Zn2+, and Hg2+, and sulfhydryl reagents. The Km for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.

Analysis of the molecular mimicry between HLA-B27 and a bacterial OmpA protein using synthetic peptides.

In spite of a lack of sequence 'homology' between HLA-B27 and the bacterial OmpA outer membrane proteins, they both react with the Ye-2 monoclonal anti-HLA-B27 antibody. The Ye-2 antibody also reacted positively in ELISA with a synthetic peptide derived from the segment spanning residues 63-84 of B*2705. The critical peptide residues were determined by testing first with overlapping peptides, followed by a replacement set made according to the determined epitope. The results were compared with those with overlapping eight mers made to span a carboxyl fragment of the Escherichia coli OmpA protein. They indicate the reason why Ye-2 reacts with both sets of peptides is because it has a preference for polymers of arginine.

Epitope analysis of an HLA-B27-derived synthetic peptide: a possible approach to analyzing HLA class I antigens.

A monoclonal antibody, F3H7, was generated by immunizing mice with a synthetic peptide corresponding to residues 63-84 of the B*2705 allele of the HLA-B27 antigens. The reactive epitope and the contact residues on the peptide were localized by ELISA using a large panel of overlapping peptides as well as peptides with substituted amino acids. Residues corresponding to R75, D77 and L78 on the HLA-B27 protein appeared to be critical. The clarity of these results indicate that this is a potentially useful approach to the study of HLA class I epitopes.

Epitope mapping of an HLA-B27 monoclonal antibody that also reacts with a 35-kD bacterial outer-membrane protein.

Ye-3 is an HLA-B27-specific murine monoclonal antibody recognizing the heat-modifiable protein of Enterobacteriaceae. Here we used recombinant hybrid molecules between the HLA-B7 and HLA-B27 antigens to delineate the epitope recognized by Ye-3. Results of these experiments indicated that the segment of HLA-B*2705 spanning residues 77-81 was critical to the reactive epitope. It is known to be a major serological and functional recognition site of HLA-B*2705 and our data give support for its involvement also in the serological cross-reactivity with bacterial antigens.

The bacterial outer membrane protein that reacts with anti-HLA-B27 antibodies is the OmpA protein.

A bacterial outer membrane protein of 35-kDa Mr has been reported to react with several anti-HLA-B27 mAb. Here, we demonstrated that this protein showed the heat-modifiability of the OmpA protein during SDS-PAGE. Further, the protein was not detected in mutants of Escherichia coli in which the expression of the OmpA protein has been suppressed. The protein would be reexpressed when one of the mutants was transformed with an expression vector carrying the OmpA gene. Finally, the identity of the reactive protein to OmpA protein was verified by homology in amino acid sequences. An NH2-terminal fragment of this protein was generated by tryptic digestion. Inasmuch as this was unreactive with the anti-HLA-B27 antibody, we concluded that the carboxyl-terminus contributed directly or indirectly to the reactive domain.