Reactivation of Inactivated d-Glucose Dehydrogenase of a Bacillus Species by Pyridine and Adenine Nucleotides

d-Glucose dehydrogenase [β-d-glucosc: NAD(P) oxidoreductase (EC 1.1.1.47)] was synthesized derepressively in a mutant of a Bacillus species which was isolated as an improved strain for d-ribose production. The enzyme was very unstable and inactivated during storage or column chromatography. The inactivation was prevented in the presence of NAD⁺, NADP⁺ or certain salts. The inactive enzyme was reactivated by the addition of NAD⁺, NADH, NADP⁺, NADPH, AMP, ADP, ATP or certain salts. The molecular weights of the inactive and active form of the enzyme were estimated to be about 45,000 and 80,000, respectively, by Sephadex G–150 gel filtration. Thus, it seems that the enzyme activity is regulated by monomer-dimer interconversion of the enzyme molecule.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Role of Pyridine Nucleotides in the Control of Respiration in Ultraviolet-Irradiated Escherichia coli B/r Cells

Escherichia coli B/r cells grown on glycerol-containing medium and irradiated with ultraviolet light to about 1% survival respire for about 1 hr and then cease completely for several hours. The results of studies on cell-free respiration and analyses of pyridine nucleotide levels at various times after ultraviolet irradiation show that the cessation of respiration is associated with two changes—loss of glycerol kinase activity and complete disappearance of pyridine nucleotides. Under other cultural conditions in which respiratory inhibition is less complete and more transitory, the losses of pyridine nucleotides are smaller and the rises which follow are correlated with increases in respiratory activity.     

Inhibition of CA2+ Efflux by Pyridine Nucleotides

Abstract

The effects of pyridine nucleotides on the Mg-dependent ATP-stimulated Ca²⁺ pump and on the ATP-independent Na⁺-Ca²⁺ exchanger were investigated in rat brain synaptic plasma membranes. Both Ca²⁺ efflux mechanisms are inhibited by pyridine nucleotides, in the order NADPH>NADP>NADH>NAD with IC₅₀ = ca. 3–4 mM for NADP or NADPH and ca. 5 mM for the other pyridine nucleotides in the case of the ATP-driven Ca²-pump, and with IC₅₀ = 8 to 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP or FeCN also affect the Ca²⁺-efflux mechanisms. DCIP and FeCN inhibit the ATP-driven Ca²⁺ pump but not the Na⁺-Ca²⁺ exchanger. Inhibition of the ATP-dependent Ca²⁺ pump is optimal when both a reduced pyridine nucleotide and an oxidizing agent (e.g. DCIP or FeCN) were added together. Under similar experimental conditions the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger is partially removed. Therefore Ca²⁺-efflux mechanisms appear to be controlled in part through the redox environnement, probably by means of transplasma membrane dehydrogenases.     

Multiple Strategies of Bloom-Forming Microcystis to Minimize Damage by Solar Ultraviolet Radiation in Surface Waters

The occurrence of bloom-forming cyanobacteria is one of the most obvious sign of eutrophication in freshwaters. Although in eutrophic lakes water transparency in the ultraviolet (UV) region is strongly reduced, bloom-forming cyanobacteria are exposed to high solar UV radiation at the surface. Here, we show that, in a natural phytoplankton community from a very eutrophic lake, Microcystis synthesizes UV sunscreen compounds identified as mycosporine-like amino acids (MAAs). The biomass-specific MAA concentration was significantly correlated with the occurrence of Microcystis but not with other algal groups, even though they were dominant in terms of biomass. Based on a photo-optical model, we estimated that the maximum MAA concentration per cell observed (2.5% dry weight) will confer only ~40% of internal screening to a single layer of Microcystis cells. Thus, the formation of a colony with several layers of cells is important to afford an efficient UV screening by internal self-shading. Overall, we propose that Microcystis uses a combination of photoprotective strategies (MAAs, carotenoids) to cope with high solar UV radiation at the water surface. These strategies include also the screening of UV radiation by d-galacturonic acid, one of the main chemical components of the slime layer in Microcystis.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD⁺, NADH) of NAD⁺-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD⁺ was the varied substrate. However, the NAD⁺-linked malic enzyme exhibited the weakest affinity for NAD⁺ and the lowest sensitivity for NADH. It is suggested that NAD⁺-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.     

Postreplication Repair of Ultraviolet Damage in Haemophilus influenzae

The deoxyribonucleic acid (DNA) synthesized following ultraviolet (UV) irradiation of wild-type (Rd) and recombination-defective strains of Haemophilus influenzae has been analyzed by alkaline sucrose gradient sedimentation. Strain Rd and a UV-resistant, recombination-defective strain Rd(DB117) (rec-) are able to carry out postreplication repair, i.e., close the single-strand gaps in the newly synthesized DNA; in the UV-sensitive, recombination-defective strain DB117, the gaps remain open. The lack of postreplication repair in this strain may be the result of degradation of the newly synthesized DNA.     

The Role of Iron and Dissolved Organic Carbon in the Absorption of Ultraviolet Radiation in Humic Lake Water

Absorption of solar ultraviolet radiation (UVR) in aquatic ecosystems is primarily controlled by dissolved organic carbon (DOC). The role of iron (Fe) has also been suggested to contribute to UVR attenuation either directly or by interactions with DOC. Here we present findings from three laboratory manipulations of Fe and DOC on changes to the dissolved UVR absorption (a_d,320) in a mid-latitude, dimictic, humic lake. In a laboratory simulation of lake turnover where anoxic, hypolimnetic water was oxygenated a_d,320 significantly increased from 23.3 to 81.7 m⁻¹ (p<0.0001). In a second laboratory experiment, addition of ferrous Fe to deoxygenated lake water increased a_d,320 upon reoxygenation up to a concentration of 1.0 mg l⁻¹ Fe, where a solubility saturation threshold may have been reached. In situ lake experiments were designed to simulate release of UV absorbing substances from anoxic sediments by placing 20-l carboys (open at the bottom, sealed at the top) onto the lake bottom. UV absorption at 320 nm increased over time for samples from within the experimental carboys. Finally, samples from several lake profiles and sediment experiments were analyzed for a_d,320, total Fe, and DOC. UV absorption of dissolved substances at 320 nm and total Fe concentration both increased with depth, however DOC remained relatively constant over depth. Furthermore, total Fe and spectral slope showed tight coupling up to 1 mg l⁻¹ total Fe in our survey analysis. Our results provide evidence for the importance of anoxic sediments as a source of ferrous iron and UV absorbing substances and suggest a role for ferric iron in increasing UVR and PAR absorption in lake water. We suggest that as this ferrous Fe oxidizes, its absorptive properties increase, and it may bind with dissolved organic matter, enabling it to remain in solution and thus increasing the dissolved absorption of lake water for extended periods of time.     

Susceptibility of Legionella pneumophila to Ultraviolet Radiation

Distilled water suspensions of Legionella pneumophila were found to be sensitive to low doses of germicidal ultraviolet radiation.     

Role of Telomerase in Reactivation of Macrophage Nuclei in Heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

Synthesis of 5-Mercaptocytosine Nucleosides and Nucleotides

Abstract

The synthesis of a series of new nucleosides and nucleotides, including ribo-, 2-deoxyribo- and arabinofuranosides of 5-sulfur-substituted cytosines, is described. The synthetic methods employed involve 5-thiolation of the appropriate cytosine or 5-bromocytosine nucleosides and nucleotides, or alternatively, 4-thiation followed by amination of the corresponding protected 5-(S-benzyl)mercaptouracil nucleosides and subsequent deblocking with sodium and liquid ammonia.     

紫外线辐射对蓝藻细胞活性的影响

紫外线辐射 2种蓝藻Anabaena 6 3l和Anabaenaazollae不同时间 ,用UV30 0 0分光光度计测定活体连续吸收光谱 ,并测定细胞存活率。结果发现紫外线辐射对这 2种蓝藻的光合色素有不同程度的破坏作用 ;紫外线辐射 2min左右就可以杀死全部的A .6 3l和A .azollae细胞。实验还证实了小剂量刺激效应的存在。     

紫外辐射的细胞生物学效应及其机制

地球表面的生物体会受到来自阳光紫外辐射的照射。两极臭氧层空洞的形成使地球表面的紫外辐射水平,特别是UVB的水平增加,进而对生物体的危害日益加重。因此,深入研究紫外辐射生物学效应的信号转导机制并阐明其损伤机制和生物体的反应机制等具有重要意义,也可为紫外辐射损伤防护的研究提供新思路。本文综述了本领域近年来的相关研究,从紫外辐射所致生物学效应的信号转导机制(包括对DNA的直接损伤作用、细胞膜激酶和非激酶受体通路、细胞质通路以及microRNA)等方面对紫外辐射的损伤机制进行了比较系统的阐述和总结。     

肿瘤睾丸抗原 CT45-5在 DNA 损伤应答中的作用

目的:用建立的 CT45-5-siRNA 研究肿瘤睾丸抗 CT45-5在 DNA 损伤应答中的作用.方法:通过靶向CT45-5基小干扰 RNA(siRNA)下调 CT45-5的表达,用 MTT 比色法检测经喜树碱和托泊苷处理后细胞对 DNA 损伤诱导剂的敏感性,用流式细胞技术检测电离辐射后细胞周期的变化.结果:在 DNA 损伤诱导剂处理后,在脆性组氨酸三联体(Fhit)高表达细胞中下调 CT45-5的表达可以使细胞表现出更强的 G2期阻滞及对 DNA 损伤诱导剂更耐受,而在 Fhit 缺失表达的细胞中却没有此现象.结论:CT45-5可能是以 Fhit 赖的途径参与了 DNA 损伤应答.