Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.     

Studies on rat liver argininosuccinate synthetase. Inhibition by various amino acids.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.     

Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.     

Genes affecting the productivity of alpha-amylase in Bacillus subtilis Marburg.

Genetic control of alpha-amylase (alpha-1,4-glucan glucanohydrolase, EC 3.2.1.1.) production by Bacillus subtilis 168 was studied from the standpoint that alpha-amylase production by bacteria is dependent on a long-lived messenger ribonucleic acid and obeys the following equation: E = kappa integral of X-DT where x = cell mass at time t, E = alpha amylase produced, t = culture time, and kappa = productivity constant. So a productivity constand (kappa) is obtained from the slope of the straight line plot of alpha-amylase formed versus the total mass of cells accumulated over that time during the culture process. The following results were obtained. (i) Two sequential mutants, derived from the 168(kappa = 20) strain and having improved alpha-amylase productivity (168 leads to 196), were analyzed for their serine and metal protease production. Strain 128 (kappa = 40) produced half the amount of both proteases, but strain 196 (kappa = 60 similar to 80) produced 20 times that in the original strain. (ii) Amy+ transformants, using the 196 strain as the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71, J47, groups. Seventy-one of 74 Amy+ transformants had a kappa value of 21.0 plus or minus 2.1 and the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71,J47, and J10) produced levels of serine and metal proteases 20 times higher than the other transformants. (iii) Strains 196, J71, J47, and J10 were found to be nonmotile and resistant to phage PBS1, whereas other strains, including strains 168, 128, 3 revertants of strain J71 and 2 revertants of strain 196, were all motile and sensitive to the phage. (iv) Strains 196 and J71 were nonflagellated under electron microscopic observation but strain 168, 128 and a revertant of J71 were flagellated. From the above experimental results, the existence of a quality controlling gene (amyB) was deduced, which is loosely linked to the structural gene and controls productivities of alpha-amylase and proteases, and flagellation. The probable existence of another regulatory gene, amyC, is also discussed.     

Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria

In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency (k _cat/K _m) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

NKT cells are responsible for the clearance of murine norovirus through the virus-specific secretory IgA pathway

Norovirus infection cause epidemic nonbacterial gastroenteritis in patients. The immune mechanisms responsible for the clearance of virus are not completely understood. We examined whether NKT cells are effective against norovirus infection using CD1d KO mice. The body weights of 4-weeks-old CD1d KO mice that were infected with murine norovirus-S7 (MNV-S7) were significantly lower than those of non-infected CD1d KO mice. On the other hand, the body weights of infected WT mice were comparable to those of non-infected WT mice. Correspondingly, CD1d KO mice had an almost 1000-fold higher MNV-S7 burden in the intestine after infection in comparison to WT mice. The mechanism responsible for the insufficient MNV-S7 clearance in CD1d KO mice was attributed to reduced IFN-γ production early during MNV-S7 infection. In addition, the markedly impaired IL-4 production in CD1d KO mice resulted in an impaired MNV-S7-specific secretory IgA production after MNV-S7 infection which is associated with mucosal immunity. Thus, the present results provide evidence that NKT cells play an essential role in MNV-S7 clearance.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Monte Carlo simulation and measurement of radiation leakage from applicators used in external electron radiotherapy

External electron radiotherapy is performed using a cone or applicator to collimate the beam. However, because of a trade-off between collimation and scattering/bremsstrahlung X-ray production, applicators generate a small amount of secondary radiation (leakage). We investigate the peripheral dose outside the radiation field of a Varian-type applicator. The dose and fluence outside the radiation field were analyzed in a detailed Monte Carlo simulation. The differences between the calculation results and data measured in a water phantom in an ionization chamber were less than ±1% in regions more than 3 mm below the surface of the phantom and at the depth of dose maximum. The calculated fluence was analyzed inside and outside the radiation field on a plane just above the water phantom surface. Changing the electron energy affected the off-axis fluence distribution outside the radiation field; however, the size of the applicator had little effect on this distribution. For each energy, the distributions outside the radiation field were similar to the dose distribution at shallow depths in the water phantom. The effect of secondary electrons generation by photon transmission through the alloy making up the lowest scraper was largest in the region from the field edge to directly below the cutout and at higher beam energies. The results of the Monte Carlo simulation confirm that the peripheral dose outside the field is significantly affected by radiation scattered or transmitted from the applicator, and the effect increases with the electron energy.