BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

Fatty Acid Composition of Human Myelin Proteolipid Protein in Peroxisomal Disorders

Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester-bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP-acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.     

Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

Background

Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs.

Methodology/Principal Findings

Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol.

Conclusions/Significance

The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.     

Thiodisaccharides with galactofuranose or arabinofuranose as terminal units: Synthesis and inhibitory activity of an exo β-d-galactofuranosidase

Thiodisaccharides having β-d-Galf or α-l-Araf units as non-reducing end have been synthesized by the SnCl₄- or MoO₂Cl₂-promoted thioglycosylation of per-O-benzoyl-d-galactofuranose (1), its 1-O-acetyl analogue 4, or per-O-acetyl-α-l-arabinofuranose (16) with 6-thioglucose or 6-thiogalactose derivatives. After convenient removal of the protecting groups, the free thiodisaccharides having the basic structure β-d-Galf(1→6)-6-thio-α-d-Glcp-OMe (5) or β-d-Galf(1→6)-6-thio-α-d-Galp-OMe (15) were obtained. The respective α-l-Araf analogues 18 and 20 were prepared similarly from 16. Alternatively, β-d-Galf(1→4)-4-thio-3-deoxy-α-l-Xylp-OiPr was synthesized by Michael addition to a sugar enone of 1-thio-β-d-Galf derivative, generated in situ from the glycosyl isothiourea derivative of 1. The free S-linked disaccharides were evaluated as inhibitors of the β-galactofuranosidase from Penicillium fellutanum, being 15 and 20 the more active inhibitors against this enzyme.     

Ultraviolet light sensitivity of carotene-and cytochrome-c-accumulating strains of the smut fungusUstilago violacea

White, pink, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were exposed to ultraviolet light. The survival curves for all strains were of exponential decay form, but the carotene-accumulating strains were generally more resistant to UV than those strains with no carotenes at all. The UV exposure time leading to 90% loss in viability, LD₉₀, was quantitatively related to the carotene content in the form of a power function, where LD₉₀ increased as the fifth root of the total carotene content per cell. We also determined that the ratio of total carotenes to total cytochrome c per cell was quantitatively related to the rate of viability loss during UV exposure.     

Antioxidant activity of twenty five plants from Colombian biodiversity

The antioxidant activity of the crude n-hexane, dichloromethane, and methanol extracts from 25 species belonging to the Asteraceae, Euphorbiaceae, Rubiaceae, and Solanaceae families collected at natural reserves from the Eje Cafetero Ecorregión Colombia, were evaluated by using the spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging method. The strongest antioxidant activities were showed by the methanol and dichloromethane extracts from the Euphorbiaceae, Alchornea coelophylla (IC50 41.14 mg/l) and Acalypha platyphilla (IC50 111.99 mg/l), respectively. These two species had stronger DPPH radical scavenging activities than hydroquinone (IC50 151.19 mg/l), the positive control. The potential use of Colombian flora for their antioxidant activities is discussed.     

Detoxification of olefinic epoxides and nucleotide excision repair of epoxide-mediated DNA damage: Insights from animal models examining human sensitivity to 1,3-butadiene

1,3-Butadiene (BD) is a well-documented mutagen and carcinogen in rodents and is currently classified as a probable carcinogen in humans. Studies investigating workers exposed to BD indicate that, in some plants, there may be an increased genetic risk, and that polymorphisms in biotransformation and DNA repair proteins may modulate genetic susceptibility. To investigate the role of genetic polymorphisms in microsomal epoxide hydrolase (mEH) or nucleotide excision repair (NER) in contributing to the mutagenicity of BD, we conducted a series of experiments in which mice lacking mEH or NER activity were exposed to BD by inhalation or to the reactive epoxide metabolites of BD (epoxybutene-EB or diepoxybutane-DEB) by i.p. injection. Genetic susceptibility was measured using the Hprt cloning assay. Both deficient strains of mouse were significantly more sensitive to the mutagenic effects of BD and the injected epoxides. These studies provide support for the critical role that mEH plays in the biotransformation of BD, and the role that NER plays in maintaining genomic integrity following exposure to BD. Additional studies are needed to examine the importance of base excision repair (BER) in maintaining genomic integrity, the differential formation of DNA and protein adducts in deficient strains, and the potential for enhanced sensitivity to BD genotoxicity in mice either lacking or deficient in both biotransformation and DNA repair activity.