Mammalian base excision repair and DNA polymerase beta

OBJECTIVE: To describe six dogs with congenital abnormalities involving the portal vein, caudal vena cava, or both. ANIMALS: Six client-owned dogs with congenital interruption of the portal vein or the caudal vena cava, or both. METHODS: Portal vein and caudal vena cava anatomy was evaluated by contrast radiography and visualization at surgery. Vascular casts or plastinated specimens were obtained in three animals. RESULTS: Portal blood shunted into the caudal vena cava in four dogs and the left hepatic vein in one. Two of these five dogs also had interruption of the caudal vena cava with continuation as azygous vein, as did an additional dog, in which the portal vein was normally formed. Portal vein interruption was present in 5 of 74 (6.8%) dogs with congenital portosystemic shunts evaluated at the Veterinary Teaching Hospital during the study period. CONCLUSIONS: Serious malformations of the abdominal veins were present in more than 1 in 20 dogs with single congenital portosystemic shunts. CLINICAL RELEVANCE: Veterinarians involved in diagnosis and surgery for portosystemic shunts should be aware of these potential malformations, and portal vein continuity should be evaluated in all dogs before attempting shunt attenuation.     

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.     

Mammalian base excision repair by DNA polymerases delta and epsilon

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.     

Mammalian 3-methyladenine DNA glycosylase protects against the toxicity and clastogenicity of certain chemotherapeutic DNA cross-linking agents

DNA repair status is recognized as an important determinant of the clinical efficacy of cancer chemotherapy. To assess the role that a mammalian DNA glycosylase plays in modulating the toxicity and clastogenicity of the chemotherapeutic DNA cross-linking alkylating agents, we compared the sensitivity of wild-type murine cells to that of isogenic cells bearing homozygous null mutations in the 3-methyladenine DNA glycosylase gene (Aag). We show that Aag protects against the toxic and clastogenic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C (MMC), as measured by cell killing, sister chromatid exchange, and chromosome aberrations. This protection is accompanied by suppression of apoptosis and a slightly reduced p53 response. Our results identify 3-methyladenine DNA glycosylase-initiated base excision repair as a potentially important determinant of the clinical efficacy and, possibly, the carcinogenicity of these widely used chemotherapeutic agents. However, Aag does not contribute significantly to protection against the toxic and clastogenic effects of several chemotherapeutic nitrogen mustards (namely, mechlorethamine, melphalan, and chlorambucil), at least in the mouse embryonic stem cells used here. We also compare the Aag null phenotype with the Fanconi anemia phenotype, a human disorder characterized by cellular hypersensitivity to DNA cross-linking agents, including MMC. Although Aag null cells are sensitive to MMC-induced growth delay and cell cycle arrest, their sensitivity is modest compared to that of Fanconi anemia cells.     

Mammalian mitochondrial DNA evolution: a comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.     

Mammalian fuel utilization during sustained exercise

The 'crossover' and 'lactate shuttle' concepts of substrate utilization in humans during exercise are extended to describe metabolic responses on other mammalian species. The 'crossover concept' is that lipid plays a predominant role in sustaining efforts requiring half or less aerobic capacity (VO2max); however, greater relative efforts depend increasingly on blood glucose and muscle glycogen as substrates. Thus, as exercise intensity increases from mild to moderate and hard, fuel selection switches (crosses over) from lipid to carbohydrate dependence. Glycogen and glucose catabolic rates are best described as exponential functions of exercise intensity, but with a greater gain in slope of the glycogen than glucose response. In contrast, plasma free fatty acid flux is described as an inverted hyperbola with vertex at approximately 50% VO2max. Both endocrine and intra-cellular factors play critical roles in determining substrate balance during sustained exercise. Moreover, genotypic adaptation for aerobic capacity as well as phenotypic adaptations to short- and long-term chronic activity affect the balance of substrate utilization during exercise. The concept of a 'lactate shuttle' is that during hard exercise, as well as other conditions of accelerated glycolysis, glycolytic flux in muscle involves lactate formation regardless of the state of oxygenation. Further, according to the lactate shuttle concept, lactate represents a major means of distributing carbohydrate potential energy for oxidation and gluconeogenesis. In humans and other mammals, the formation, distribution and disposal of lactate (not pyruvate) represent key steps in the regulation of intermediary metabolism during sustained exercise.     

Mammalian coronary perfusion--an improved laboratory device

An improvised device for coronary perfusion of isolated rat heart consisted of a reservoir of buffer pressurised with continuous bubbling of O2 connected to two double condensors in series. The outer jacket of each condenser was connected to a temperature controlled motorised water bath to ensure steady temperature of buffer. Isolated hearts could be tested with known agents directly through a syringe connected to the aortic cannula.     

Mammalian parathyroids: morphological and functional implications

Fixation with aldehydes is achieved either by immersion or perfusion. The parenchyma of parathyroid glands fixed by immersion consists of dark cells containing a lot of membranes of these organelles which are concerned with hormone secretion, light cells which are poor in these organelles, intermediate forms between the two, and multinuclear syncytial cells. They have been attributed to represent different functional stages of secretory activity, the dark cell being in an active form, the light cell in a resting form. Studies of the parathyroids of mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, and horses employing various fixation protocols clearly demonstrate that light cell variants and multinuclear syncytial cells are formed during improper immersion fixation as a result of membrane disintegration. Parathyroids fixed by perfusion or by immersion in an appropriate fixation medium comprise only one cell type which correspond to the dark chief cell. Parathyroid cells are polar cells bearing some of the rough endoplasmic reticulum in the basal pole, the rest of it, the Golgi complex, and secretory granules in the apical pole. The secretory product is released by exocytosis at the apicolateral domain of the plasma membrane into the intercellular space. Secretory activity can be altered experimentally, leading to drastic changes in the amount of cell membrane related to hormone synthesis, intracellular transport, exocytic release, and secretion coupled membrane retrieval. The sensitive reaction of parathyroid cells to both the mode of fixation and to fixation media demands careful evaluation of the fixation protocol. This and the polarity of parathyroid cells have to be borne in mind for estimating secretory activity on the basis of morphological criteria.     

Mammalian glial cells in culture synthesize acetylcholine

OBJECTIVE: To evaluate the postoperative outcome and long-term results of patients who underwent iterative and extended pulmonary resection leading to completion pneumonectomy for pulmonary metastases. METHODS: From January 1985 to December 1995, 12 patients (mean age 45 years) underwent completion pneumonectomy for pulmonary metastases. These patients represent 1.5% of all pulmonary metastases operated on. There were 5 sarcoma and 7 carcinoma patients. Before completion pneumonectomy, 8 patients had only one pulmonary resection (wedge resection, 2; segmentectomy, 2; lobectomy, 4), 3 patients had two operations and finally, 1 patient had multiple bilateral wedge resections and 1 lobectomy. The median interval time between the last pulmonary resection and completion pneumonectomy was 13.5 months (range 1-24 months). RESULTS: There were 10 left and two right completion pneumonectomies. Three patients had an extended resection (1 carina; 1 chest wall; 1 pleuropneumonectomy). Intrapericardial dissection was used in 3 patients. Two patients died within 30 days of the operation: 1 died of postoperative complications (8.3%) whereas the other died of rapidly evolving metastatic disease. The remaining 10 patients had an uneventful postoperative course. Only 1 patient is still alive and free of disease 69 months after completion pneumonectomy. One patient is alive with disease, another was lost to follow-up; 9 patients died of metastatic disease. The median survival time after completion pneumonectomy was 6 months (range 0-69 months). The estimated 5-year probability of survival was 10% (95% CI: 2-40%). CONCLUSIONS: Indications for both iterative and extended pulmonary resection for PM may be discussed only in highly young selected patients; the extremely poor outcome of our subgroup of patients should lead to even more restrictive indications of CP for pulmonary metastatic disease.     

Mammalian Bax triggers apoptotic changes in yeast

The extracellular loop of P2X channel proteins contains a sequence stretch (positions 170-330) that exhibits similarities with the catalytic domains of class II aminoacyl-tRNA synthetases as shown by secondary structure predictions and sequence alignments. The arrangement of several conserved cysteines (positions 110-170) shows similarities with metal binding regions of metallothioneins and zinc finger motifs. Thus, for the extracellular part of P2X channel proteins a metal binding domain and an antiparallel six-stranded beta-pleated sheet containing the ATP binding site are very probable. The putative channel forming H5 part (positions 320-340) shows similarities with the enzyme motif 1 responsible for aggregation of subunits to the holoenzyme.     

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.     

Corticosterone Influences on Mammalian Neonatal Sensitive-Period Learning.

Infant rats exhibit sensitive-period odor learning characterized by olfactory bulb neural changes and odor preference acquisitions critical for survival. This sensitive period is coincident with low endogenous corticosterone (CORT) levels and stress hyporesponsivity. The authors hypothesized that low corticosterone levels modulate sensitive-period learning. They assessed the effects of manipulating CORT levels by increasing and removing CORT during (Postnatal Day 8) and after (Postnatal Day 12) the sensitive period. Results show that (a) exogenous CORT prematurely ends sensitive-period odor-shock-induced preferences; (b) adrenalectomy developmentally extends the sensitive period as indicated by odor-shock-induced odor-preference learning in older pups, whereas CORT replacement can reinstate fear learning; and (c) CORT manipulation modulates olfactory bulb correlates of sensitive-period odor learning in a manner consistent with behavior. (PsycINFO Database Record (c) 2010 APA, all rights reserved)