Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

Mice unresponsive to GM-CSF are unexpectedly resistant to cutaneous Leishmania major infection

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.     

Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis

The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1(lacZ)) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis.     

Free filet extremity flap: indications and options for reconstruction

Radical and extended forequarter and hind limb amputations have been used for curative and palliative intents. Concerns regarding wound healing and closure, especially in irradiated fields, have occasionally limited the extent of ablation. This article reports an experience with coverage of these large defects by using the free filet extremity flap. A retrospective review was performed of 11 patients who had undergone immediate reconstruction with free filet extremity flaps between 1991 and 1998. There were nine men and two women with an average age of 43.9 years. All except three patients received preoperative radiotherapy. Resections included four hindquarter and seven forequarter amputations for palliation of intractable pain, tissue necrosis, and infections. Donor vessels included the brachial artery, its venae comitantes, cephalic and basilic veins, and common femoral and popliteal vessels. Immediate reconstruction was successful in all cases by the use of the amputated limb as the free filet flap. All wounds healed despite irradiation inclusive of defects up to 50 cm x 70 cm (3500 cm2). The average follow-up time was 5 months with a mean survival of 3.5 months. Four patients currently are alive, and one patient died within 30 days of surgery. The remaining six patients have died of their disease within 9 months of the palliative procedures. Pain, tissue necrosis, and infections were improved in all patients after hospital discharge. Extensive defects can be reconstructed and healed successfully, even in irradiated wounds, with the use of the free filet extremity flap. Appropriate advanced preoperative and intraoperative planning is essential. Although survival was unchanged, this technique allowed healed wounds with an improvement in the quality of life.     

DNA sequence data reveal a subfamily-level divergence within Thamnophilidae (Aves: Passeriformes)

The Thamnophilidae is a diverse radiation of insectivorous passerine birds that comprises nearly 220 species and is mostly restricted to the lowlands and lower montane forests of the Neotropics. Current classification within Thamnophilidae relies primarily on morphological variation, but recent incorporation of molecular and vocal data has promoted changes at various taxonomic levels. Here we demonstrate that the genus Terenura is polyphyletic because Terenura callinota, T. humeralis, T. spodioptila, and T. sharpei are phylogenetically distant from the type species of the genus, Terenura maculata. More importantly, the former four species are not particularly closely related to any other thamnophilids and represent a clade that is sister to all other members of the family. Because no genus name is available for this previously undetected lineage in the Thamnophilidae, we describe the genus Euchrepomis for callinota, humeralis, spodioptila, and sharpei, and erect the subfamily Euchrepomidinae. We discuss the taxonomic and evolutionary significance of this divergent lineage. This study highlights the importance of taxonomic coverage and the inclusion of type taxa to redefine classifications to reflect accurately evolutionary relationships.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.