Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Purification and characterization of microbial gellan lyase.

Gellan lyase was purified from the culture fluid of soil samples incubated in a medium containing gellan as a sole carbon source. The enzyme was a monomer with a molecular mass of 140 kDa and was most active at pH 7.5 and 45 degrees C. The enzyme was highly specific to gellan and lowered the viscosity of the polymer.     

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.     

An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII--and their population frequency.

Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. We have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations we have found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis was used to localize exons with possible mutations. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, delta 5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 (delta C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for all illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, we found only one delta 5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

Enhanced production of rat interleukin-8 by in vitro and in vivo infections with influenza A NWS virus.

We investigated the interleukin-8 (IL-8)-producing activity of influenza A NWS virus in cultured rat kidney NRK-52E cells and a rat influenza model. The production of rat IL-8 increased significantly in the virus-infected cells but not in UV-inactivated virus- or split-product-treated cells. The increase in IL-8 production could be detected in the bronchoalveolar lavage of infected rats. These data suggest that infectious virus has the potential to accelerate the production of IL-8 in cultured cells and in vivo in airway-lining cells.     

Gene structure and expression of the MboI restriction--modification system.

The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli. Three open reading frames were found in the sequence containing the genes. These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome. Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively. The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues. Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI. R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis. The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems. The protein sequences of the MboI system had 38-49% homology with those of the DpnII system.