Methanol Oxidation at Platinum in Alkaline Media: A Study of the Effects of Hydroxide Concentration and of Mass Transport.

The hydroxide ion concentration dependence of the methanol oxidation reaction at Pt was studied using microelectrode voltammetry and rotating disk electrode voltammetry. Both methods suggest that the rate of methanol oxidation is limited by hydroxide mass transport at low hydroxide concentrations, while it is inhibited by hydroxide adsorption at high concentrations. It was possible to shift from the transport-limited regime to the inhibitory regime by varying the bulk concentration of hydroxide or by varying mass transport to the electrode. Rotating ring-disk electrode voltammetry was employed to qualitatively assess changes in the diffusion layer pH. The results indicated a decrease in the surface pH during methanol oxidation, as expected, but also that the pH reached a steady state during hydroxide transport limited methanol oxidation.

Similarity among the Drosophila (6-4)photolyase, a human photolyase homolog, and the DNA photolyase-blue-light photoreceptor family.

Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase ws cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following gamma-radiation-induced damage to pMTa4 DNA in E. coli.

PURPOSE: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to gamma-radiation in vivo. MATERIALS AND METHODS: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and gamma-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R-)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of gamma-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored. RESULTS: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R- condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of gamma-radiation-induced 'ladder'. The 'ladder' was not observed after repair incubation, UV-irradiation or gamma-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised. CONCLUSIONS: Wild, recF and recA strains of E. coli could repair gamma-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

A new Drosophila ultraviolet light-damaged DNA recognition endonuclease that selectively nicks a (6-4) photoproduct site.

We have previously described the purification of an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, designated D-DDB P1 [Nucleic Acids Res., 23 (1995) 2600-2607]. Here, we obtained highly purified D-DDB P1 from Drosophila Kc cells, and we found that D-DDB P1 is also a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site.

Comparison of somatic reversions between the ivory allele and transposon-caused mutant alleles at the white locus of Drosophila melanogaster after larval treatment with X rays and ethyl methanesulfonate.

Somatic reversion of strains with the ivory (wi) allele, a mutation associated with a tandem duplication of a DNA sequence at the white locus, increased with the age of larvae at the time of X-irradiation as expected from the increase in the number of target cells. In contrast, two independently isolated strains with unstable w+ loci associated with insertion of transposable elements showed higher reversion frequencies after treatment with X rays or ethyl methanesulfonate (EMS) at early larval stages than at late stages. Nevertheless, both the wi strain and the two unstable w+ strains reverted at nearly equal rates after treatment with X rays or EMS at early larval stages. Possible similarity in "hot spot" structure for the high reversibility of the two types of mutations is discussed in relation to production of presumed "mutator-type" cofactors specific to the transposon-caused mutations at early larval stages.

Induced rates of mitotic crossing over and possible mitotic gene conversion per wing anlage cell in Drosophila melanogaster by X rays and fission neutrons.

As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh and flr genes in Drosophila melanogaster strain (mwh +/+ flr) was quantitatively studied. Fission neutrons were five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins and mwh singles-or flr singles, indicating that common processes are involved in the production of crossover and flr singles. The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X 10(-5), whereas that of crossover was 2 x 10(-4); the former and the latter rate are of the same order of magnitude as those of gene conversion and crossover in yeast, respectively. Thus, we conclude that proximal-marker "flr" singles induced in the transheterozygote are gene convertants. Using the model based on yeast that recombination events result from repair of double-strand breaks or gaps, we propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair. Evidence for recombinational misrepair in the fly is given. The relative ratio of radiation-induced mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than in yeast and humans.

Transgenerational transmission of radiation- and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes.

This article provides a broad overview of our earlier studies on the induction of tumors and congenital anomalies in the progeny of X-irradiated or chemically treated mice and our subsequent (published, hitherto unpublished and on-going) investigations aimed at identifying potential relationships between genetic changes induced in germ cells and the adverse effects manifest as tumors and congenital anomalies using cytogenetic and molecular approaches. The earlier studies document the fact that tumors and congenital anomalies can be induced by irradiation or treatment with certain chemicals such as urethane and that these phenotypes are heritable i.e., transmitted to generations beyond the first generation. These findings support the view that transmissible induced genetic changes are involved. The induced rates of congenital abnormalities and tumors are about two orders of magnitude higher than those recorded in the literature from classical mutation studies with specific locus mutations. The cytogenetic studies addressed the question of whether there were any relationships between induced translocations and induced tumors. The available data permit the inference that gross chromosomal changes may not be involved but do not exclude smaller induced genetic changes that are beyond the resolution of the techniques used in these studies. Other work on possible relationship between visible chromosomal anomalies (in bone marrow preparations) and tumors were likewise negative. However, there were indications that some induced cytogenetic changes might underlie induced congenital anomalies, i.e., trisomies, deletions and inversions were observed in induced and transmissible congenital anomalies (such as dwarfs, tail anomalies). Studies that explored possible relationships between induction of minisatellite mutations at the Pc-3 locus and tumors were negative. However, gene expression analysis of tumor (hepatoma)-susceptible offspring of progeny descended from irradiated male mice showed abnormal expression of many genes. Of these, only very few were oncogenes. This lends some support to our hypothesis that cumulative changes in gene expression of many genes, which perform normal cellular functions, may contribute to the occurrence of tumors in the offspring of irradiated or chemically treated mice.

Identification of cellular factors that recognize UV-damaged DNA in Drosophila melanogaster.

Using a gel electrophoresis DNA band-shift assay, we have identified 2 DNA-binding protein complexes in wild-type Drosophila embryonic cells which have high affinity for UV-irradiated, double-stranded DNA. Screening of Drosophila mutants deficient in DNA repair led to the identification of 5 mutants which lacked either one of the 2 protein complexes. Four excision repair-deficient mutants (mus-201, phr, mus-308 and mus-205) lacked one protein complex (Factor 2). The other protein complex (Factor 1) was not detectable in the post-replication repair-deficient mutant mus-104. These findings might suggest the possible involvement of these gene products in lesion recognition and repair of UV-induced photoproducts in DNA.

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females.

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.

Enhanced susceptibility of a transposable-element-bearing strain of Drosophila melanogaster to somatic eye-color mutations by ethyl nitrosourea, methyl nitrosourea, and X-rays.

A strain of Drosophila with the genes z and w+ plus a transposable element (TE) is about 3 times more sensitive than a strain without TE toward somatic eye-color mutations after larval exposure to ethyl nitrosourea, methyl nitrosourea and X-rays. The assay system with TE is simple, reliable, and sensitive for detecting somatic mutations induced in vivo by mutagens.

Purification and characterization of Drosophila melanogaster photolyase.

Animal-type photolyases have very limited sequence homology to microbial-type photolyases. We wanted to find out whether the two types of enzymes have different or similar biochemical and photochemical properties. In particular, the chromophore/cofactor composition of animal photolyases is of special interest since the presence and nature of a second chromophore in these enzymes are not known in contrast to the microbial photolyases which contain FAD cofactor, and folate or deazaflavin as second chromophores. We overproduced the Drosophila melanogaster photolyase in Escherichia coli using the cloned gene. The enzyme contains FAD and folate and thus belongs in the folate class of enzymes but with an action spectrum peak at 420 nm.

Differential antimutagenic effects of caffeine and the protease inhibitor antipain on mutagenesis by various mutagens in Escherichia coli.

The effects of caffeine (2 mg/ml) and the protease inhibitor antipain (1.75 mg/ml) in the plating agar medium on the yields of prototrophic revertants induced by 10 mutagens in E. coli uvrA- strains were tested. Mutagenesis by 4-nitroquinoline 1-oxide was greatly diminished by both caffeine and antipain. UV mutagenesis was decreased moderately by caffeine, and greatly by antipain. X-Ray mutagenesis was decreased very slightly by both caffeine and antipain. Mutagenesis by N-hydroxyurethan was inhibited moderately by caffeine, and greatly by antipain; that by methyl methanesulfonate was inhibited moderately by both caffeine and antipain, and that by N-methyl-N'-nitro-N-nitrosoguanidine was not suppressed by caffeine but was inhibited moderately by antipain. Mutagenesis by ethyl methanesulfonate was inhibited greatly by caffeine, but only slightly by antipain. The antimutagenic effect of caffeine was strong on furylfuramide (AF-2) mutagenesis, moderate on that of mitomycin C (tested with B2r type strain) and negligible on that of N-methyl-N-nitrosourea. These diverse antimutagenesis patterns are briefly discussed in relation to the current idea that antipain antimutagenesis is due to inhibition of inducible error-prone repair.

Non-mutagenic repair of (6-4)photoproducts by (6-4)photolyase purified from Drosophila melanogaster.

The (6-4)photoproduct DNA photolyase ((6-4)photolyase) repairs UV-induced pyrimidine (6-4) pyrimidone photoproduct ((6-4)photoproduct, pyr[6,4]pyr) in a light dependent manner. Drosophila (6-4)photolyase was purified to near homogeneity from Drosophila embryonic cells and is shown to be a 62 kDa protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified (6-4)photolyase repairs (6-4)photoproducts induced at 5'-CC-3' site (C[6,4]C) as well as T[6,4]T and T[6,4]C. Photoreactivation of (6-4)photoproduct constructed in M13 phage eliminates the replication block and abolishes induced mutagenesis in E. coli cells, suggesting that the (6-4)photolyase repairs the photoproduct to the unmodified form.

High-level expression of the photorepair gene in Drosophila ovary and its evolutionary implications.

DNA photolyase catalyzes light-dependent repair of cis, syn-cyclobutane dipyrimidines (pyrimidine dimers); its apoenzyme is encoded by the photorepair (phr) gene. The phr cDNA was cloned from D. melanogaster; it has an open reading frame to encode a 61,483-Da protein. The phr cDNA hybridized to band 44C-D of Drosophila polytene chromosome, equivalent to the locus of the phr- gene. Drosophila photolyase is made of an apoenzyme with a molecular weight of 62 kDa. Drosophila photolyase is extraordinarily abundant in the embryo and adult ovary, whereas mRNA of the phr gene is abundant only in the ovary. The action spectrum of Drosophila photolyase for photoreactivation has a maximum at 440 nm. The phr gene of Drosophila has about 60% identical amino acid sites with that of goldfish but only 13-18% with those of microorganisms. Implications of the unique characteristics of the Drosophila phr gene are discussed overviewing the diversified characteristics of phr genes in various organisms that have presumably evolved from a common ancestral gene.

Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide.

A standard method for determining mutant frequencies per survivor was used to study the detailed kinetics of reverse mutations of Ames tester strains of Salmonella typhimurium induced by UV and by 4NQO. After UV irradiation, strain TA1538 was non-mutable, but its plasmid-containing derivative TA98 was mutable, whereas TA1535 was mutable and its plasmid-bearing derivative TA100 was about 10-fold more mutable. After treatment with 4NQO, TA98 was less mutable than TA1538, whereas TA100 was more mutable than TA1535 by a factor of 10-50. TA1537 was slightly less mutable than TA1535 by either UV or 4NQO. The differential mutabilities of these strains are briefly discussed in relation to the "hot spot" base sequences for reversion and the nature of DNA damage caused by UV and 4NQO.

Genotoxic activities in vivo of cobaltous chloride and other metal chlorides as assayed in the Drosophila wing spot test.

A series of metal chlorides were subjected to the wing spot test of Drosophila melanogaster. In the test, larvae trans-heterozygous for the wing-hair mutations mwh and flr were orally treated at the third instar stage with a test compound and the wings were inspected at the adult stage for spots expressing phenotypes of the markers. CoCl2, MnCl2, MoCl3, NiCl2 and ZnCl2 were clearly effective in inducing spots with one or two mutant hairs (small spots). CoCl2 was clearly effective in inducing spots with three or more mutant hairs (large spots) as well. CrCl3, FeCl2 and FeCl3 were negative under the conditions used. Based on estimated frequencies of small spots induced at the LD50, the genotoxic effectiveness of the positive metal salts were ranked in a sequence of CoCl2 > ZnCl2 > MoCl3 > (MnCl2, NiCl2). Since CoCl2 did not induce large spots in the wings of the mwh/TM3 flies with a suppressed ability of mitotic crossing-over, the large spots induced by this compound in the mwh/flr system were ascertained as mutant clones due to mitotic crossing-overs.

[Functions of large granular lymphocytes--a case of large granular lymphocytosis with characteristic cell surface antigens].

A 58-year-old male visited the hematological clinic of Surugadai Nihon University Hospital, Tokyo, complaining of numbness around both elbows. The peripheral leukocyte count was 12,400/microliters, and large granular lymphocytes (LGL) occupied 79% of the leukocytes. The cell surface antigen studied by flow cytometry were the positive CD 2, 3, 5, 8, 11, and Leu-7, and the negative CD1, 4, 10, 16 (Leu-11), 19, 20, and OKTIa1. IgG-FCR checked by mean of the EA-rosette formation was positive. The LGL showed the negative NK cell activity and the positive ADCC and LAK cell activities. It was interesting that LGL was negative for CD16 (Leu-11) while they had ADCC activity. Since the rearrangement of the receptor gene in T-cells was demonstrated by the southern blot analysis, the proliferation of LGL was considered to be a clonal one. LGL did not inhibit the colony formation of granulocyte and erythrocyte precursors in the plasma clot culture. It was thus considered that this might partially explain the fact that the patient's neutrophil, Hb and platelet levels remained normal.

Mutations induced in Drosophila during space flight.

To examine the possible effects of space radiation on living organisms, fruit flies Drosophila melanogaster were loaded on the US Space Shuttle Endeavour, and after the flight we have analyzed two types of mutations, sex-linked recessive lethal mutations induced in male reproductive cells and somatic mutations which give rise to morphological changes in hairs growing on the surface of wing epidermal cells. Wild type strains and a radiation-sensitive strain mei-41 were used. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-41 strains, respectively, than those in ground control groups. By contrast, the frequencies of wing-hair somatic mutations differed little between flight and control groups. The possibility that the space environment causes mutations in certain types of cells such as male reproductive cells, is discussed.

[Bone metastasis of primary lung cancer].

To improve the treatment of patients with bone metastasis from lung cancer, 178 (23.8%) of 748 patients with primary lung cancer admitted to our hospital during an 11-year period were studied. The 178 patients were mainly women. The frequency of bone metastasis was significantly higher among patients with adenocarcinoma than among those with small cell or squamous cell cancer. The concentration of carcinoembryonic antigen in serum was significantly higher in patients with bone metastasis than in those with stage IV lung cancer without bone metastasis. The bone lesions were symptomatic in 116 patients (65%) and were treated in 67. Symptomatic improvement was achieved in 50 patients (74%) and pain was alleviated in 94%. However, neurological disorders improved in only 41%. Differences in survival did not depend on the presence or absence of symptomatic bone metastasis or no whether the metastases were treated. The median survival time of patients who responded to treatment tended to be longer than that of patients in whom treatment was not effective. The median survival time calculated from the start of treatment was 5 months for patients with bone metastasis and 7.3 months for patients with stage IV disease without bone metastasis. Aggressive local treatment may be effective for painful bone metastasis.

[Rheumatoid nodule diagnosed by thoracoscopy using flexible fiberoptic bronchoscope].

A 51-year-old man had been treated at a nearby hospital since 1993 for rheumatoid arthritis. Right pectoralgia developed in December 1994, and the patient consulted a nearby hospital, which detected right pleural effusion retention was pointed out on chest x-ray films. The patient was referred and admitted to our hospital. Rheumatic pleurisy was suspected because of a high serum rheumatoid factor(RF)level and high RF and high rheumatoid arthritis hemagglutination levels in the pleural effusion. However, due to a high adenosine deaminase level in the pleural effusion tuberculous pleurisy could not be ruled out. After drainage through a trocar catheter, the thoracic cavity was examined by thoracoscopy through the site of catheter insertion. As a result, sporadic bluish white nodular lesions were observed on the pleura. Granuloma formations presenting a palisade arrangement of giant cells were also observed, and pathologically diagnosed as rheumatoid nodules, thus providing the basis for a diagnosis of rheumatic pleurisy. Treatment with an increased dose of prednisolone achieved a rapid remission of the pleural effusion. Our experience underscored the usefulness of thoracoscopy as a means diagnosing of rheumatic pleurisy.