Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

Discovery of novel DNA viruses in small mammals from Kenya

Emergence and re-emergence of infectious diseases of wildlife origin have led pre-emptive pathogen surveillances in animals to be a public health priority. Rodents and shrews are among the most numerically abundant vertebrate taxa and are known as natural hosts of important zoonotic viruses. Many surveillance programs focused more on RNA viruses. In comparison, much less is known about DNA viruses harbored by these small mammals. To fill this knowledge gap, tissue specimens of 232 animals including 226 rodents, five shrews and one hedgehog were collected from 5 counties in Kenya and tested for the presence of DNA viruses belonging to 7 viral families by PCR. Diverse DNA sequences of adenoviruses, adeno-associated viruses, herpesviruses and polyomaviruses were detected. Phylogenetic analyses revealed that most of these viruses showed distinction from previously described viruses and formed new clusters. Furthermore, this is the first report of the discovery and full-length genome characterization of a polyomavirus in Lemniscomys species. This novel polyomavirus, named LsPyV KY187, has less than 60% amino acid sequence identity to the most related Glis glis polyomavirus 1 and Sciurus carolinensis polyomavirus 1 in both large and small T-antigen proteins and thus can be putatively allocated to a novel species within Betapolyomavirus. Our findings help us better understand the genetic diversity of DNA viruses in rodent and shrew populations in Kenya and provide new insights into the evolution of those DNA viruses in their small mammal reservoirs. It demonstrates the necessity of ongoing pathogen discovery studies targeting rodent-borne viruses in East Africa.     

Soil and vegetation differences across ecological boundaries in an arid South African ecosystem

Boundaries are the most reactive nodes in landscapes and may be hypersensitive to global change in climate and land use. Investigations on how soils govern vegetation boundaries are scant, particularly in arid and semiarid ecosystems. The Tankwa Karoo National Park (TKNP) is a unique arid biodiversity hotspot with an unrivalled aridity gradient from < 100 mm MAP to about 700 mm in < 10 km. We investigated the abruptness of four soil‐vegetation boundaries separating eight communities. Two 50 m transects were established across four boundaries for 24 descending point transects, in which the cover‐abundance of each plant encounter at 1 m intervals was recorded. In addition, three soil samples were collected from the top 5 cm in each of the four boundaries and twelve patches. Soil and vegetation parameters altogether indicated three boundary syndromes that were context dependent: (a) a sharp boundary, (b) a gradual boundary or (c) no boundary exists. Soil respiration recorded here, and perhaps other ecosystem processes, was mediated by the soil‐vegetation boundaries. These nodes should be the focus of ecological studies since they reveal much more than the constituent patches themselves.     

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a “detuned” structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the “Y-model”) in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model (“I-model”). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.     

Bacterial diversity obtained by culturable approaches in the gut of Glossina pallidipes population from a non sleeping sickness focus in Tanzania: preliminary results

Background

Glossina pallidipes is a haematophagous insect that serves as a cyclic transmitter of trypanosomes causing African Trypanosomiasis (AT). To fully assess the role of G. pallidipes in the epidemiology of AT, especially the human form of the disease (HAT), it is essential to know the microbial diversity inhabiting the gut of natural fly populations. This study aimed to examine the diversity of G. pallidipes fly gut bacteria by culture-dependent approaches.

Results

113 bacterial isolates were obtained from aerobic and anaerobic microorganisms originating from the gut of G. pallidipes. 16S rDNA of each isolate was PCR amplified and sequenced. The overall majority of identified bacteria belonged in descending order to the Firmicutes (86.6%), Actinobacteria (7.6%), Proteobacteria (5.5%)and Bacteroidetes (0.3%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to four phyla (83% Firmicutes, 15% Actinobacteria, 1% Proteobacteria and 0.5% Bacteroidetes, whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to two phyla only (90% Firmicutes and 10% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes.

Conclusion

This study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, which are likely to be important for ecological microbial well being of the fly and possibly on differing vectorial competence and refractoriness against AT epidemiology.

     

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

Cell culture media are potent antioxidants that interfere during LDL oxidation experiments

In vitro cell-induced low-density lipoprotein (LDL) oxidation is a model frequently used for studies on antioxidant compounds which may be potentially antiatherogens. Using Cu2+ or the free radical generator 2,2'-azobis-[2-amidinopropane] dihydrochloride (AAPH) to oxidize human LDL, we showed that the cell culture media Ham's F10 and RPMI are potent antioxidants which reduce LDL-protective effect of various thyroid compounds. The culture media interfered with the compounds depending on their mechanism of action, and RPMI had the greatest antioxidant effect, completely hiding antioxidant efficiency of the compounds whatever the prooxidant agent was. We suggest some recommendations for study of antioxidant compounds using cell-induced LDL oxidation models.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

AlkB mystery solved: oxidative demethylation of N1-methyladenine and N3-methylcytosine adducts by a direct reversal mechanism

All organisms have multiple DNA repair pathways to protect against alkylation-induced mutation and cell death. For nearly two decades, we have known that the Escherichia coli alkB gene product protects against cell killing by S(N)2-alkylating agents, probably through DNA repair. Despite numerous attempts, a specific DNA repair activity could not be assigned to AlkB. Now, a breakthrough in biology and biochemistry, coupled with the discovery of an in silico protein structure, has uncovered a novel direct reversal DNA repair mechanism that is catalyzed by AlkB, namely the oxidative demethylation of N1-methyladenine or N3-methylcytosine DNA lesions. This reaction occurs on both single- and double-stranded DNA, and requires AlkB-bound non-heme Fe(2+), O(2) and alpha-ketogluterate to oxidize the offending methyl group. This is followed by the release of succinate, CO(2) and formaldehyde, and the restoration of undamaged A or C in DNA.     

Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization.The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.