Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.     

miR-132 mediates the integration of newborn neurons into the adult dentate gyrus

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.     

Dating an impressive Neotropical radiation: Molecular time estimates for the Sigmodontinae (Rodentia) provide insights into its historical biogeography

With about 400 living species and 82 genera, rodents of the subfamily Sigmodontinae comprise one of the most diverse and more broadly distributed Neotropical mammalian clades. There has been much debate on the origin of the lineage or the lineages of sigmodontines that entered South America, the timing of entrance and different aspects of further diversification within South America. The ages of divergence of the main lineages and the crown age of the subfamily were estimated by using sequences of the interphotoreceptor retinoid binding protein and cytochrome b genes for a dense sigmodontine and muroid sampling. Bayesian inference using three fossil calibration points and a relaxed molecular clock estimated a middle Miocene origin for Sigmodontinae (～12 Ma), with most tribes diversifying throughout the Late Miocene (6.9–9.4 Ma). These estimates together results of analyses of ancestral area reconstructions suggest a distribution for the most recent common ancestor of Sigmodontinae in Central-South America and a South American distribution for the most recent common ancestor of Oryzomyalia.     

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti‐inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A₁ receptor, phosphatidylinositol‐3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin‐sensitive G‐protein‐coupled signaling, blockade of adenosine A_2A receptors (A_2AR), but not via A₁ receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.     

The effects of selective breeding against scrapie susceptibility on the genetic variability of the Latxa Black-Faced sheep breed

Breeding sheep populations for scrapie resistance could result in a loss of genetic variability. In this study, the effect on genetic variability of selection for increasing the ARR allele frequency was estimated in the Latxa breed. Two sources of information were used, pedigree and genetic polymorphisms (fifteen microsatellites). The results based on the genealogical information were conditioned by a low pedigree completeness level that revealed the interest of also using the information provided by the molecular markers. The overall results suggest that no great negative effect on genetic variability can be expected in the short time in the population analysed by selection of only ARR/ARR males. The estimated average relationship of ARR/ARR males with reproductive females was similar to that of all available males whatever its genotype: 0.010 vs. 0.012 for a genealogical relationship and 0.257 vs. 0.296 for molecular coancestry, respectively. However, selection of only ARR/ARR males implied important losses in founder animals (87 percent) and low frequency alleles (30 percent) in the ram population. The evaluation of mild selection strategies against scrapie susceptibility based on the use of some ARR heterozygous males was difficult because the genetic relationships estimated among animals differed when pedigree or molecular information was used, and the use of more molecular markers should be evaluated.     

Patterns of genetic divergence of three Canarian endemic Lotus (Fabaceae): implications for the conservation of the endangered L. kunkelii

We examined data for 11 allozyme loci in 14 populations that represent the distribution of the endangered Lotus kunkelii, the narrowly distributed L. arinagensis (both endemic to Gran Canaria), and the broad-ranging L. lancerottensis (endemic to the easternmost Canary Islands, Fuerteventura and Lanzarote) to explore and construe patterns of genetic variation and use this data to assess the controversial taxonomic status of L. kunkelii relative to L. lancerottensis. While L. kunkelii maintains low levels of variation, presumably as a consequence of prolonged inbreeding due to very low population size and sharp geographic isolation, the other two taxa have much higher indicators of polymorphism than those reported for other oceanic island endemics. Lotus arinagensis has the highest genetic polymorphism and the lowest interpopulation differentiation, presumably because of its considerable antiquity and habitat stability, despite recent fragmentation. The high interpopulation differentiation in L. lancerottensis is attributed to the Atlantic acting as a barrier, reducing gene flow within islands. Evolutionary analysis of the allozyme evidence indicates that L. kunkelii is genetically closer to L. arinagensis than to L. lancerottensis, thereby dispelling the taxonomic uncertainty and supporting L. kunkelii as a distinct species, warranting legal protection in the forthcoming catalog of threatened Canarian species.     

Studies on resources of genetic diversity in conservative flocks of geese using microsatellite DNA polymorphic markers

The studies conducted aimed at evaluating the genetic diversity within and between varieties of conservative flocks of geese, using the polymorphism of 14 microsatellite sequences. The experimental material included conservative flocks of geese the following indigenous breeds and varieties kept in Poland: Kielecka (Ki), Kartuska (Ka), Lubelska (Lu), Suvalska (Su), Rypinska (Ry), Sub-Carpathian (SC), Hunched Beak (HB) and Pomeranian (Po). Among the 14 microsatellite sequences a total of 97 microsatellite alleles were identified. The number of alleles at one locus ranged from 3 to 19. In the overall pool of 97 alleles, 26 (26.8%) were specific for individual breeds and varieties of geese. The values of the expected heterozygosity (He) for individual geese ranged from 0.38 (Sub-Carpathian) to 0.51 (HB). Similarly, the mean values for the observed heterozygosity (Ho) ranged from 0.45 (Po) to 0.55 (Ki and Su). The polymorphic information content reached the highest value of 0.80 at loci CKW21 (Ki) and TTUCG5 (Po and Su). The greatest genetic distance was observed between the HB and Ry (0.44) and between the HB and Po (0.39) varieties, while the smallest–between the Lu and Po as well as Lu and Ki (0.028) varieties. The phylogenetic tree, elaborated on the basis of the genetic distances, clearly confirms the specificity of the HB goose as compared to the remaining breeds and varieties.     

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H(2)O(2) induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H(2)O(2), and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.