DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.     

Bone marrow cells repair cigarette smoke-induced emphysema in rats

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.     

Conserved domains in DNA repair proteins and evolution of repair systems.

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

DNA repair in mammalian nerve cells. I. DNA synthesis in the neocortex of rats induced by gamma irradiation

A study was made of the DNA synthesis in cerebral cortex of rats, aged 14 and 60 days, after gamma-irradiation in vivo in a dose of 7 Gy, the 3H-thymidine incorporation into DNA being determined.137 Cs-radiation induces additional DNA synthesis in the neocortex tissue and in neurons. In the cortex of 14 day-old rats, the induced DNA synthesis stops 2 hours after irradiation, whereas in the cortex of 60 day-old rats and in neurons of rats of both the age groups DNA synthesis is proceeding for 3-3.5 hours. Specificity of DNA reparation processes in non-dividing cells is discussed.     

Sensory perturbations using suture and sutureless repair of transected median nerve in rats

The effects of changes to cold, mechanical, and heat thresholds following median nerve transection with repair by sutures (Su) or Rose Bengal adhesion (RA) were compared to sham-operated animals. Both nerve-injured groups showed a transient, ipsilateral hyposensitivity to mechanical and heat stimuli followed by a robust and long-lasting hypersensitivity (6–7 weeks) with gradual recovery towards pre-injury levels by 90 days post-repair. Both tactile and thermal hypersensitivity were seen in the contralateral limb that was similar in onset but differed in magnitude and resolved more rapidly compared to the injured limb. Prior to injury, no animals showed any signs of aversion to cold plate temperatures of 4–16?°C. After injury, animals showed cold allodynia, lasting for 7 weeks in RA-repaired rats before recovering towards pre-injury levels, but were still present at 12 weeks in Su-repaired rats. Additionally, sensory recovery in the RA group was faster compared to the Su group in all behavioural tests. Surprisingly, sham-operated rats showed similar bilateral behavioural changes to all sensory stimuli that were comparable in onset and magnitude to the nerve-injured groups but resolved more quickly compared to nerve-injured rats. These results suggest that nerve repair using a sutureless approach produces an accelerated recovery with reduced sensorimotor disturbances compared to direct suturing. They also describe, for the first time, that unilateral forelimb nerve injury produces mirror-image-like sensory perturbations in the contralateral limb, suggesting that the contralateral side is not a true control for sensory testing. The potential mechanisms involved in this altered behaviour are discussed.     

8-Oxoguanosine and uracil repair of nuclear and mitochondrial DNA in red and white skeletal muscle of exercise-trained old rats.

Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.     

脱细胞羊膜与小肠黏膜下层促进大鼠皮肤缺损修复和血管形成

目的观察脱细胞羊膜(HAM)与小肠黏膜下层(SIS)促进大鼠皮肤缺损修复和血管形成的作用。方法SD大鼠24只,在两侧背部各做1个直径为1.8cm圆形全层皮肤缺损。创面随机分为A组、B组和C组。A组HAM覆盖,B组SIS覆盖,C组纱布覆盖。在2周时处死动物取材,HE染色观察皮肤缺损修复情况。免疫组织化学染色检测K19和VEGF,RT-PCR检测VEGF mRNA的表达。结果A组、B组愈合较好。C组愈合较差。免疫组织化学染色显示,A、B组K19、VEGF阳性细胞显著多于C组,其中A组最多;RT-PCR结果显示,A、B组比C组表达更多的VEGF mRNA其中A组最多,差异显著,有统计学意义(P<0.05)。结论HAM和SIS均能增加皮肤创面组织中K19阳性细胞数,上调VEGF mRNA的表达、增加VEGF的分泌,其中HAM具有更强的促进皮肤缺损修复和血管形成作用。     

Serum lipidomics-based study of electroacupuncture for skin wound repair in rats

Lipid metabolism plays an important role in the repair of skin wounds. Studies have shown that acupuncture is very effective in skin wound repair. However, there is little knowledge about the mechanism of electroacupuncture. Thirty-six SD rats were divided into three groups: sham-operated group, model group and electroacupuncture group, with six rats in each group. After the intervention, orbital venous blood was collected for lipid metabolomics analysis, wound perfusion was detected and finally the effect of electroacupuncture on skin wound repair was comprehensively evaluated by combining wound healing rate and histology. Lipid metabolomics analysis revealed 11 differential metabolites in the model versus sham-operated group. There were 115 differential metabolites in the model versus electro-acupuncture group. 117 differential metabolites in the electro-acupuncture versus sham-operated group. There were two differential metabolites common to all three groups. Mainly cholesteryl esters and sphingolipids were elevated after electroacupuncture and triglycerides were largely decreased after electroacupuncture. The electroacupuncture group recovered faster than the model group in terms of blood perfusion and wound healing (p < 0.05). Electroacupuncture may promote rat skin wound repair by improving lipid metabolism and improving local perfusion.     

Heat injury and repair in Campylobacter jejuni.

A procedure for detecting and quantitating heat injury in Campylobacter jejuni was developed. Washed cells of C. jejuni A7455 were heated in potassium phosphate buffer (0.1 M, pH 7.3) at 46 degrees C. Samples were plated on brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate and on a medium containing brilliant green, bile, Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate. Colonies were counted after 5 days of incubation at 37 degrees C in an atmosphere containing 5% O2, 10% CO2, and 85% N2. After 45 min at 46 degrees C, there was virtually no killing and ca. two log cycles of injury. Cells grown at 42 degrees C were more susceptible to injury than cells grown at 37 degrees C. The addition to brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate of three different antibiotic mixtures used in the isolation of C. jejuni from foods or clinical specimens did not prevent recovery of heat-injured C. jejuni. Cells lost 260 nm of absorbing materials during heat injury. The addition of 5% NaCl or 40% sucrose to the heating buffer prevented leakage but did not prevent injury. Of the additional salts, sugars, and amino acids tested for protection, only NH4Cl, KCl, and LiCl2 prevented injury. Heat-injured C. jejuni repaired (regained dye and bile tolerance) in brucella broth supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate within 4 h. Increasing the NaCl in this medium to 1.25% inhibited repair, and increasing it to 2% was lethal. Heat-injured C. jejuni will repair at 42 degrees C but not at 5 degrees C.     

Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect.

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders.     

DNA repair and mutant frequency in schizophrenia.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes was determined with a cloning assay. T-lymphocytes were obtained from 14 individuals diagnosed with schizophrenia and 5 controls. No significant difference in mutant frequency was observed between the 2 groups. In addition, DNA-repair capacity was measured with the unscheduled DNA synthesis technique in lymphocytes from 7 individuals diagnosed with schizophrenia and 7 controls. Repair capacity was determined following treatment with MMS, MNNG, and 20 J/m2 ultraviolet light. No significant differences in DNA repair were observed between the patient and control groups in response to any of the 3 DNA-damaging agents. These results argue against differences between normal and schizophrenic individuals with respect to in vivo mutant frequency or their capacity to repair DNA lesions induced by MMS, MNNG, or ultraviolet radiation.     

Mismatch repair and recombination in E. coli

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.