Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

The MntC crystal structure suggests that import of Mn2+ in cyanobacteria is redox controlled

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 A resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended alpha-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.     

The effects of storage condition on starch metabolism and regeneration potentials of twin-scales and inflorescence stem explants of <Emphasis Type="Italic">Narcissus tazetta</Emphasis>

Summary The natural propagation rate of Narcissus is very slow. In vitro micropropagation of Narcissus is more efficient than conventional propagation for rapidly building up aseptic stocks of varieties, especially for the establishment of new cvs. and the production of pathogen-free stock material. In the present study, Narcissus tazetta cv. ‘Ziva’ bulbs were used as the source of mother plants. The bulbs were kept at 30°C in a dark chamber until the start of the experiments. Prior to explant preparation, the bulbs were subjected to a cold treatment at 15°C in the dark for 6 wk to break dormancy. Twin-scales and inflorescence stem discs were isolated from aseptic bulb parts and were used as the initial explants. The polar orientation affected the regeneration of the inflorescence stem. Storage duration at 30°C followed by cold treatment were found to affect starch levels, adenosine diphosphate glucose pyrophosphorylase (AGPase) activities, and regeneration potentials. Starch levels were reduced significantly during a 10 mo. storage period at 30°C in both twin-scales and inflorescence stem disc explants. Regeneration was followed by an efficient acclimatization system with 98–99% survival. More than 500 highly uniform young bulbs were propagated from one mother bulb in a 12 mo. period.     

Development and validation of a method for the quantitative determination of aflatoxin contaminants in Maytenus ilicifolia by HPLC with fluorescence detection

A method for the quantification of aflatoxins B1, G1, B2 and G2 in the medicinal herb Maytenus ilicifolia was developed and validated. The method used immunoaffinity columns for sample clean-up and HPLC with fluorescence detection without any derivatisation step. The method showed good inter-day accuracy (bias values in the range 4.5-10.7%) and precision (5-16% RSD) when applied to the determination of levels of aflatoxins ranging from 7 to 20 ppb in the plant material. The detection limits for samples of the plant material spiked with aflatoxins were 3.5 ng/g for B1 and G1 and 0.1 ng/g for B2 and G2. The method was successfully applied to commercial samples of Maytenus ilicifolia for the screening of aflatoxin contaminants.     

Reciprocal regulation of expression of pore‐forming KATP channel genes by hypoxia

The ATPsensitive potassium (K_ATP) channel is thought to play an important role in the protection of heart and brain against tissue hypoxia. The genetic regulation of the components of the channel by hypoxia has not been previously described. Here, we investigated the regulation of the two poreforming channel proteins, Kir6.1 and Kir6.2, in response to hypoxia in vivo and in vitro. We find that these two structurallyrelated inwardly-rectifying potassium channel proteins are reciprocally regulated by hypoxia in vivo, with upregulation of Kir6.1 and downregulation of Kir6.2, thereby resulting in a significant change in the composition of the channel complex in response to hypoxia. In vitro we describe neuronal and cardiac cell lines in which Kir6.1 is upregulated by hypoxia, demonstrating that Kir6.1 is a hypoxiainducible gene. We conclude that the heart and brain display genetic plasticity in response to hypoxic stress through specific genetic reprograming of cytoprotective channel genes.     

Microbiota of Cow’s Milk; Distinguishing Healthy,Sub-Clinically and Clinically Diseased Quarters

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.     

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag ^−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.     

The cycad genotoxin MAM modulates brain cellular pathways involved in neurodegenerative disease and cancer in a DNA damage-linked manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O ⁶-methyldeoxyguanosine lesions, O ⁶-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O ⁶-mG DNA methyltransferase (MGMT) showed elevated O ⁶-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer''s disease.     

Secondary forest fragments offer important carbon and biodiversity cobenefits

Tropical forests store large amounts of carbon and high biodiversity, but are being degraded at alarming rates. The emerging global Forest and Landscape Restoration (FLR) agenda seeks to limit global climate change by removing carbon dioxide from the atmosphere through the growth of trees. In doing so, it may also protect biodiversity as a free cobenefit, which is vital given the massive shortfall in funding for biodiversity conservation. We investigated whether natural forest regeneration on abandoned pastureland offers such cobenefits, focusing for the first time on the recovery of taxonomic diversity (TD), phylogenetic diversity (PD) and functional diversity (FD) of trees, including the recovery of threatened and endemic species richness, within isolated secondary forest (SF) fragments. We focused on the globally threatened Brazilian Atlantic Forest, where commitments have been made to restore 1 million hectares under FLR. Three decades after land abandonment, regenerating forests had recovered ~20% (72 Mg/ha) of the above‐ground carbon stocks of a primary forest (PF), with cattle pasture containing just 3% of stocks relative to PFs. Over this period, SF recovered ~76% of TD, 84% of PD and 96% of FD found within PFs. In addition, SFs had on average recovered 65% of threatened and ~30% of endemic species richness of primary Atlantic forest. Finally, we find positive relationships between carbon stock and tree diversity recovery. Our results emphasize that SF fragments offer cobenefits under FLR and other carbon‐based payments for ecosystem service schemes (e.g. carbon enhancements under REDD+). They also indicate that even isolated patches of SF could help to mitigate climate change and the biodiversity extinction crisis by recovering species of high conservation concern and improving landscape connectivity.