Mitochondrial DNA deletions associated with aging and presbyacusis

BACKGROUND: The membrane hypothesis of aging proposes an association between reactive oxygen metabolites and aging processes. Reactive oxygen metabolites are a normal by-product of oxidative phosphorylation and are also formed under conditions of ischemia, hypoperfusion, and as a result of environmental contaminants. Among the many detrimental activities of reactive oxygen metabolites, also known as free oxygen radicals, is direct damage to mitochondrial DNA. Progressive accumulation of mitochondrial DNA damage renders cells unable to conduct oxidative phosphorylation reactions effectively, thereby leading to a bioenergetically deficient cell. Over time, mitochondrial DNA damage accumulates and leads to cellular dysfunction with subsequent organ failure, aging, and ultimately, death. This sequence forms the basis of the membrane hypothesis of aging. OBJECTIVE: To determine if the membrane hypothesis of aging may be involved in the development of presbyacusis. DESIGN: Fischer rats from 4 age groups were tested for auditory sensitivity using the auditory brainstem response. Brain, stria vascularis, and auditory nerve tissues were harvested and mitochondrial DNA was amplified to identify the highly conserved cytochrome b and ND1-16S ribosomal RNA segment of the NADH genes, as well as a 4834-base pair (bp) deletion associated with aging. SUBJECTS: Fischer rats (n=28) from 4 age groups were used: young (2-4 months [n=9]), mid-young (9-11 months [n=5]), mid-old (18-20 months [n=5]), and old (30-34 months [n=9]). RESULTS: The results demonstrate a progressive reduction in auditory sensitivity with age. The mitochondrial DNA studies identify a significant increase in the presence of the 4834-bp deletion in the aged subjects compared with the young. CONCLUSIONS: These findings raise the possibility that the 4834-bp deletion may be associated with presbyacusis, as well as with aging.     

Distance and end configuration effects on VDJP-mediated DNA joining

We have previously reported the cloning of a protein, VDJP, that is capable of binding the nonamer element of the V(D)J Recombinational Signal Sequence (RSS) as well as joining linear DNA fragments containing RSS elements in vitro. We show here that the linearized DNA molecules must contain a 5' extension or blunt end in order to be joined by VDJP. DNAs with 3' extensions are not efficiently joined by VDJP. Furthermore, the joining activity of DNAs with 5' extensions is significantly increased as the distance between the end and the RSS decreases. It is not yet clear what role VDJP plays in vivo, because our assay may not mimic exactly the in vivo DNA intermediates.     

Use of DNA end joining activity of a Xenopus laevis egg extract for construction of deletions and expression vectors for HIV-1 Tat and Rev proteins

Blood gases were analyzed in dogs with pulmonary heartworm (HW) disease. The arterial oxygen tension (PaO2) in dogs with mild signs of dirofilariasis (mildly affected group, n = 48, 85.7 +/- 8.2 mmHg) and in dogs with signs of right heart failure (severely affected group, n = 13, 76.4 +/- 11.6 mmHg) was lower (p < 0.01) than in dogs without HW infection (HW-free group, n = 19, 91.5 +/- 7.3 mmHg). Only 2 dogs in the severely affected group had a PaO2 less than 60 mmHg. The arterial carbon dioxide tension (PaCO2, p < 0.01) and mixed venous O2 (p < 0.01) and CO2 (p < 0.01) tensions were lower, and alveolar-arterial oxygen difference (AaDO2, p < 0.01) was greater in the severely affected group than in the HW-free and mildly affected groups. Arterial pH and bicarbonate (HCO3-) concentrations were lower (p < 0.01) in both affected groups than in the HW-free groups. The anion gap level was not different among the 3 groups. Serum lactic acid level in the severely affected group was higher (p < 0.01) than in the HW-free and mildly affected groups. However, a slightly higher serum lactic acid concentration was found only in 2 dogs of the severely affected group (3.84 mmol/l and 3.82 mmol/l). The PaO2 (r = -0.62) and AaDO2 (r = 0.66) correlated significantly (p < 0.01) with mean pulmonary arterial pressure. One week after HW removal, blood gases, pH and HCO3- concentration remained unchanged in the mildly affected group. In the severely affected group, blood gas values were the same, but pH and HCO3- concentration improved slightly.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

Association of myopathy with large-scale mitochondrial DNA duplications and deletions: which is pathogenic?

We identified large-scale heteroplasmic mitochondrial DNA (mtDNA) rearrangements in a 50-year-old woman with an adult-onset progressive myopathy. The predominant mtDNA abnormality was a 21.2-kb duplicated molecule. In addition, a small population of the corresponding partially deleted 4.6-kb molecule was detected. Skeletal muscle histology revealed fibers that were negative for cytochrome c oxidase (COX) activity and had reduced mtDNA-encoded COX subunits. By single-fiber polymerase chain reaction analysis, COX-negative fibers contained a low number of wild-type or duplicated mtDNA molecules (ie, nondeleted). In situ hybridization demonstrated that the abnormal fibers contained increased amounts of mtDNA compared with normal fibers and that most of the genomes were deleted. We concluded that deleted mtDNA molecules were primarily responsible for the phenotype in this patient.     

A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events

Eukaryotic repair of double-strand DNA breaks can occur either by homologous recombination or by nonhomologous DNA end joining (NHEJ). NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and DNA-dependent protein kinase. NHEJ involves a synapsis step in which the two ends are maintained in proximity, processing steps in which nucleases and polymerases act on the ends, an alignment step in which a few nucleotides of terminal homology guide the ends into preferred alignments, and a ligation step. Some of the steps, such as ligation, rely on a single enzymatic component. However, the processing steps begin and end with a wide array of alternative substrates and products, respectively, and likely involve multiple nucleases and polymerases. Given the alternative pathways that can be catalyzed by the remaining nucleases and polymerases, no one of these processing enzymes is likely to be essential. The only requirement for the processing enzymes, as a collective, is to generate a ligatable configuration, namely a ligatable nick on each strand. Here, we have tested the two major known 5'-specific nucleases of Saccharomyces cerevisiae for involvement in NHEJ. Whereas EXO1 does not appear to be involved to any detectable level, deleting RAD27 (FEN-1 of yeast) leads to a 4.4-fold reduction specifically of those NHEJ events predicted to proceed by means of 5' flap intermediates. Because Rad27/FEN-1 acts specifically at 5' flap structures, these results suggest that the NHEJ alignment step precedes nucleolytic processing steps in a significant fraction of NHEJ events.     

An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3

Human adenoviruses (Ads) are attracting considerable attention because of their potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of Ad5 DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb.     

Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells

We have previously presented evidence that hydroxyurea treatment of synchronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Walters, R.A., Tobey, R.A. and Hildebrand, C.E. (1976) Biochem. Biophys. Res. Com. 69, 212-217). In view of the reported effects of hydroxyurea on deoxyribonucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248,3904-3909 and Walters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336-347), we examined the fate of DNA synthesized during and shortly after hydroxyurea treatment to determine if this DNA exhibited any kinetic behavior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 +/- 0.12 - 10(6) dalton/min (0.98 +/- 0.12 mum/min) for about 2.3h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 - 10(8) was very rapidly integrated into bulk DNA of greater than or equal to 3.5 - 10(8) daltons. The apparent growth rate of the 1.4 - 10(8) dalton DNA was approx. 10.6 mum/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and once begun, occurred very rapidly. The possibility of inegration of replicon clusters is considered.     

Alpha B-crystallin is associated with intermediate filaments in astrocytoma cells

To achieve the optimal management program for the diabetic vasculopathic patient, a multidisciplinary approach incorporating the necessary major elements is required. The endocrinologist is essential in tight metabolic control of blood sugars and diet modifications. The podiatrist is indispensable in the early detection of foot ulcerations and preventive care. Visiting nurses function as a vital component in outpatient wound assessment and daily care. With this approach, the vascular surgeon is ensured of the most favorable outcome with conservative measures.     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

Neurotoxicity associated with acyclovir in end stage renal failure

AIMS: To alert practitioners to the danger of acyclovir neurotoxicity occurring in the presence of renal failure. METHODS: Two case reports of acyclovir neurotoxicity in the patients on continuous ambulatory peritoneal dialysis. RESULTS: In one case neurotoxicity resulted from the use of a dosage regimen that would be appropriate in patients with normal renal function. In the other case, neurotoxicity occurred even though a reduced dose of acyclovir was given. Supportive management resulted in a complete recovery. CONCLUSIONS: In patients with end stage renal failure with varicella zoster infections, when acyclovir is prescribed the loading dose should be 400 mg and the maintenance dose should be 200 mg twice daily.     

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.     

Large-scale deletions in a Chinese infant associated with a variant form of Werdnig-Hoffmann disease

A Chinese male infant with arthrogryposis multiplex congenita (AMC), ventricular and atrial septal defects, and Werdnig-Hoffmann disease (WHD) had deletions of the telomeric copy of the survival motor neuron (SMN(T)) and neuronal apoptosis inhibitory protein genes. Children with AMC or congenital heart disease, or both, and motor neuron disease should undergo testing for SMN(T) deletion. This rare association further illustrates the variable phenotypic expressions of WHD.     

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.     

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.     

The radioprotective effect of BBI is associated with the activation of DNA repair-relevant genes

We previously reported that S100b protein (homodimer of S100 beta subunit) can bind copper ions with a submicromolar dissociation constant (T. Nishikawa et al., J. Biol. Chem. 272, 23037-23041, 1997). In this study, a question was addressed as to whether this protein can sequester copper ions in an in vivo situation. Escherichia coli cells that had been rendered able to produce a fusion protein of rat S100 beta subunit with glutathione S-transferase displayed a marked resistance to cellular damage induced by copper alone or its combination with H2O2, compared with control cells expressing the transferase moiety only. A study by gel chromatography showed that about half of the expressed S100 beta fusion protein in the cytosol of copper-treated cells was eluted in the void volume fraction (molecular mass > 200 kDa), which contained most of the copper incorporated. The S100 beta fusion protein purified from the void volume fraction was found to contain 82% of the total copper in the fraction, while in a parallel experiment with the control cells, the glutathione S-transferase eluted in the void volume fraction contained only 18% of the total copper. Thus, it is clear that extraneously expressed S100b protein can acts as a "copper sink," thereby protecting E. coli cells from copper-induced cellular damage.     

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.