Influence of anoxic sensitizers on the radiation damage in biologically active DNA in aqueous solution

The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime.     

Heat-sensitive radiation damage in single-stranded DNA irradiated in a bacterial extract

The effect of heat treatment (45 degrees C) on gamma-irradiated biologically active single-stranded phi X174 DNA, dissolved in a bacterial extract, was studied. The results show that under these circumstances heat-sensitive damage is found, which is absent after irradiation in pure buffer. The damage is non-lethal and probably becomes an apurinic/apyrimidinic site, which is lethal, upon the post-irradiation heat treatment. Prolonged heating converts it into a break. The amount of the initial damage depends on the conditions of irradiation.     

Effects of sulphydryl compounds on the radiation damage in biologically active DNA

The effect of sulphydryl compounds on the induction of alkali-labile sites and on the contribution of such sites to the inactivation of single-stranded phi X174 DNA was studied. Cysteamine is capable of reacting with DNA radicals, thereby modifying the radiation damage in such a way that the induction of immediate and latent breaks is reduced. This depends on the pH of the solution. With cysteine only, a pH dependent protection, against lethal alkali-labile potential breaks could be observed. The damage other than breaks is not influenced by the presence of sulphydryl compounds.     

Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c.

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).     

Effect of oxygen on radiation-induced DNA damage in isolated nuclei.

In intact mammalian cells, ionizing radiation causes substantially less damage to DNA in the absence of oxygen than in the presence of oxygen. In contrast, when DNA is isolated (usually from viruses) and irradiated in solution, the absence of oxygen does not lead to a decrease in damage unless low-molecular-weight thiols are also present. We investigated an intermediate condition: that of DNA irradiated in isolated nuclei. Using an HPLC-based assay of thiols with electrochemical detection, we have determined that the nuclear isolation procedure leads to the elimination of virtually all low-molecular-weight thiols (predominantly glutathione and cysteine). Thus it was our expectation that the thiol-depleted state would concurrently eliminate the OER, and thereby mimic the isolated DNA system, while retaining structural characteristics of chromosomal DNA. We evaluated radiation-induced DNA damage in isolated nuclei by measuring single-strand breaks using alkaline elution and by measuring double-strand breaks using neutral elution and pulsed-field gel electrophoresis. Despite the removal of low-molecular-weight thiol compounds, the oxygen dependence of radiation-induced damage more closely paralleled that of whole cells than that of DNA in solution. Thus damage of DNA irradiated in isolated nuclei is dependent on oxygen.     

Hydrogen exchange in a bacterial cytochrome c: a fingerprint of the cytochrome c fold

The hydrogen exchange rates of backbone amides in a minimal (71 amino acid long) monoheme cytochrome c were determined as a function of pH in the absence and in the presence of guanidinium chloride. These data permitted the identification of units undergoing the opening reaction that precedes hydrogen exchange through a common mechanism. The opening units broadly correlate with the secondary structure elements of the protein. It is found that, despite the significant difference in primary sequence, the distribution of the opening units within the three-dimensional structure of the cytochrome studied here closely resembles that determined in mitochondrial c-type cytochromes. It is proposed that the observed distribution represents a fingerprint of the cytochrome c fold and has a role in directing the folding/unfolding of the protein.     

Free and membrane-bound forms of bacterial cytochrome c4.

Cytochrome c4 was isolated from cells of Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii. The dihaem nature, Mr of approx. 20,000 and ferrohaem spectra in the region of the alpha- and beta-peaks define this family of cytochromes c. The behaviour of the holocytochromes in SDS was atypical, but removal of the haem groups resulted in a normal migration. In all three organisms most of the cytochrome c4 was tightly bound to the membrane, but some free cytochrome was detected. The membrane-attached cytochrome could be extracted with butanol, and this solubilized form was then indistinguishable in properties from the free form. Denitrifying rather than aerobic growth conditions hardly affected the total cytochrome c4 in the two pseudomonads, but there was slightly more free form and less membrane-attached form in denitrifying growth. The nature of the attachment of cytochrome c4 to the membrane is discussed and a model is proposed for the process of solubilization.     

Reactive oxygen and DNA damage in mitochondria.

During the last decade the importance of reactive oxygen species as major contributors to various types of cancer, heart diseases, cataracts, Parkinson's and other degenerative diseases that come with age, and to natural aging has become apparent. Mitochondria are the most important intracellular source of reactive oxygen. Mitochondrial DNA is heavily damaged by reactive oxygen at the bases, as indicated by the high steady-state level of 8-hydroxydeoxyguanosine, the presence of which causes mispairing and point mutations. Mitochondrial DNA is also oxidatively fragmented to a certain extent. Conceivably, such fragmentation relates to deletions found in mitochondrial DNA. Point mutations and deletions have recently been shown to be etiologically linked to several human diseases and natural aging. Future studies should address the causal relationship between mitochondrial dysfunction, production of reactive oxygen species, and aging.     

渗透胁迫对杧果叶片活性氧伤害的影响

杧果叶片经渗透胁迫处理后,叶水势Ψ_L下降,O₂^·产生速率和MDA含量增加,SOD、POD和CAT的活性水平与O₂^·和MDA的变化相一致。结果表明,杧果叶片的渗透胁迫损伤,是由O₂^·引发的膜脂过氧化,致使MDA含量增加,破坏细胞膜系统所致。渗透胁迫处理过程中,GSH和AsA含量下降。     

Adduct formation is involved in radiosensitization, mediated by cytochrome c, of phi X174 DNA by misonidazole

Radiosensitization by misonidazole of biologically active phi X174 DNA, mediated by cytochrome c, is most probably at least partly due to formation of an adduct between sensitizer and DNA, which can be removed from the DNA by a mild alkaline treatment thereby restoring the activity of the DNA.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa(3) (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a(3), and time-resolved experiments indicate that even transient binding to Cu(B) does not occur. Only at very high (approximately 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu(B). In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of approximately 1 microM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Effect of low-dose radiation on repair of DNA and chromosome damage

In this report results of studies on the effect of different doses of low LET (linear energy transfer) radiations on the unscheduled DNA synthesis (UDS) and DNA polymerase activity as well as the induction of adaptive response in bone marrow cells (BMC) by low dose radiation were presented. It was found that whole-body irradiation (WBI) with X-ray doses above 0.5 Gy caused a dose-dependent depression of both UD5 and DNA polymerase activity, while low dose radiation below 250 mGy could stimulate the DNA repair synthesis and the enzyme activity. WBI of mice with low doses of X-rays in the range of 2-100 mGy at a dose rate of 57.3 mGy per minute induced an adaptive response in the BMC expressed as a reduction of chromosome aberrations following a second exposure to a larger dose (0.65 mGy). It was demonstrated that the magnitude of the adaptive response seemed to be inversely related to the induction dose. The possibility of induction of adaptive response in GO phase of the cell cycle and the possibility of a second induction of the adaptive response were discussed.     

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.