Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9) gene are not linked with significant plasma MMP-9 variations in healthy subjects

Background: Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of the extracellular matrix during physiological and pathological processes. Two functional polymorphisms [C(-1562)T and microsatellite (CA)(13-25)] in the promoter region of the MMP-9 gene have been associated with several diseases. The aim of this study was to examine whether these MMP-9 polymorphisms and haplotypes are linked with plasma MMP-9 variations in healthy subjects. Methods: We studied 177 healthy male white volunteers (age range 20-55 years) who were non-smokers and not taking any medication. Genomic DNA was extracted from whole blood and genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. MMP-9 levels were measured in plasma samples by gelatin zymography. Results: The frequency of the alleles C and T for the C(-1562)T polymorphism were 90% and 10%, respectively. The frequency of the alleles with less than 21 CA repeats Q and with 21 repeats or higher (H) were 47% and 53%, respectively. We found no differences in plasma MMP-9 levels among the genotype groups or among different haplotypes (all p > 0.05). Conclusions: These findings suggest that functional polymorphisms in the promoter of the MMP-9 gene are not linked with significant plasma MMP-9 variations in healthy subjects.

Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ER alpha (alpha ERKO) or ER beta (beta ERKO) knockout mice, and their wild-type (alpha WT and beta WT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ER beta was more abundant. Estradiol benzoate (EB) decreased ER alpha-positive cells in WT and beta ERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ER beta expression. ER beta deletion increased ER alpha while ER alpha deletion did not alter ER beta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alpha ERKO animals but to a lesser extent, suggesting that ER alpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in beta ERKO mice were similar to those in the alpha ERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alpha ER, beta ER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

Molecular phylogenetics of soricid shrews (Mammalia) based on mitochondrial cytochrome b gene sequences: with special reference to the Soricinae

Molecular phylogeny of soricid shrews (Soricidae, Eulipotyphla, Mammalia) based on 1140 bp mitochondrial cytochrome b gene (cytb) sequences was inferred by the maximum likelihood (ML) method. All 13 genera of extant Soricinae and two genera of Crocidurinae were included in the analyses. Anourosorex was phylogenetically distant from the main groupings within Soricinae and Crocidurinae in the ML tree. Thus, it could not be determined to which subfamily Anourosorex should be assigned: Soricinae, Crocidurinae or a new subfamily. Soricinae (excluding Anourosorex) should be divided into four tribes: Neomyini, Notiosoricini, Soricini and Blarinini. However, monophyly of Blarinini was not robust in the present data set. Also, branching orders among tribes of Soricinae and those among genera of Neomyini could not be determined because of insufficient phylogenetic information of the cytb sequences. For water shrews of Neomyini (Chimarrogale, Nectogale and Neomys), monophyly of Neomys and the Chimarrogale-Nectogale group could not be verified, which implies the possibility of multiple origins for the semi-aquatic mode of living among taxa within Neomyini. Episoriculus may contain several separate genera. Blarinella was included in Blarinini not Soricini, based on the cytb sequences, but the confidence level was rather low; hence more phylogenetic information is needed to determine its phylogenetic position. Furthermore, some specific problems of taxonomy of soricid shrews were clarified, for example phylogeny of local populations of Notiosorex crawfordi, Chimarrogale himalayica and Crocidura attenuata.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. VIDEO ABSTRACT.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chitosan nanoparticles for non-viral gene therapy

The cationic polysaccharide chitosan has been widely used for non-viral transfection in vitro and in vivo and has many advantages over other polycations. Chitosan is biocompatible and biodegradable and protects DNA against DNase degradation. However following administration the ChitosanDNA polyplexes must overcome a series of barriers before DNA is delivered to the cell nucleus. This paper describes the most important parameters involved in the chitosan-DNA interaction and their effects of on the condensation, shape, size and protection of DNA. Strategies developed for chitosanDNA polyplexes to avoid non-specific interaction with blood components and to overcome intracellular obstacles as the crossing of die cell membrane, endosomal escape and nuclear import are presented. © 2006 American Chemical Society.

Caracterização funcional dos elementos de transcrição do gene da glicogênio sintase (gsn) de Neurospora crassa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Common matrix metalloproteinase 2 gene haplotypes may modulate left ventricular remodelling in hypertensive patients

Matrix metalloproteinases (MMPs) are involved in cardiac remodelling. We examined whether MMP-2 genetic polymorphisms are associated with hypertension and left ventricular (LV) remodelling in hypertensive patients. We studied 160 hypertensive patients and 123 healthy controls. Echocardiography was performed in all patients and the C-1306T (rs243865) and C-735T (rs 2285053) MMP-2 polymorphisms were analysed. Haplo.stats analysis was used to evaluate whether MMP-2 haplotypes are associated with hypertension and with extremes in LV mass index (LVMI). Multiple linear regression analysis was performed to assess whether MMP-2 genotypes or haplotypes affect LVMI and other echocardiography parameters. The C-1306T 'CC' genotype was associated with reduced LVMI and LV end-diastolic diameter (EDD) (P=0.0365 and P=0.0438, respectively). The haplotype 'C, C' was associated with reduced LVMI and EDD (P=0.0278 and P=0.0322, respectively). The comparison of upper and lower extremes of the LVMI phenotype showed that the 'C, C' haplotype was more common in the lower LVMI group (P=0.0060), whereas the 'T, C' haplotype was more common in the higher quartile of LVMI (P=0.0187), and this haplotype was associated with increased risk of higher LVMI values (odds ratio=3.5121, 95% confidence interval 1.3193-9.3494). The findings suggest that MMP-2 polymorphisms affect hypertension-induced LV remodelling. Journal of Human Hypertension (2012) 26, 171-177; doi:10.1038/jhh.2011.8; published online 10 February 2011

Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.

Ground-penetrating radar (GPR) point measurements of ice thickness in Austria

Glacier thickness is an important factor in the course of glacier retreat in a warming climate. Thiese study data presents the results (point data) of GPR surveys on 66 Austrian mountain glaciers carried out between 1995 and 2014. The glacier areas range from 0.001 to 18.4 km**2, and their ice thickness has been surveyed with an average density of 36 points/km**2 . The glacier areas and surface elevations refer to the second Austrian glacier inventory (mapped between 1996 and 2002). According to the glacier state recorded in the second glacier inventory, the 64 glaciers cover an area of 223.3±3.6 km**3. Maps of glacier thickness have been calculated by Fischer and Kuhn (2013) with a mean thickness of 50±3 m and contain an glacier volume of 11.9±1.1 km**3. The mean maximum ice thickness is 119±5 m. The ice thickness measurements have been carried out with the transmitter of Narod and Clarke (1994) combined with restively loaded dipole antennas (Wu and King, 1965; Rose and Vickers, 1974) at central wavelengths of 6.5 (30 m antenna length) and 4.0 MHz (50 m antenna length). The signal was recorded trace by trace with an oscilloscope. 168 m/µs as used by Haeberli et al. (1982), Bauder (2001), and Narod and Clarke (1994), the signal velocity in air is assumed to be 300 m/µs. Details on the method can be are found in Fischer and Kuhn (2013), as well as Span et al. (2005) and Fischer et al. (2007).

Effects of CO2 and iron availability on rbcL gene expression in Bering Sea diatoms

Iron (Fe) can limit phytoplankton productivity in approximately 40% of the global ocean, including in high-nutrient, low-chlorophyll (HNLC) waters. However, there is little information available on the impact of CO2-induced seawater acidification on natural phytoplankton assemblages in HNLC regions. We therefore conducted an on-deck experiment manipulating CO2 and Fe using Fe-deficient Bering Sea water during the summer of 2009. The concentrations of CO2 in the incubation bottles were set at 380 and 600 ppm in the non-Fe-added (control) bottles and 180, 380, 600, and 1000 ppm in the Fe-added bottles. The phytoplankton assemblages were primarily composed of diatoms followed by haptophytes in all incubation bottles as estimated by pigment signatures throughout the 5-day (control) or 6-day (Fe-added treatment) incubation period. At the end of incubation, the relative contribution of diatoms to chlorophyll a biomass was significantly higher in the 380 ppm CO2 treatment than in the 600 ppm treatment in the controls, whereas minimal changes were found in the Fe-added treatments. These results indicate that, under Fe-deficient conditions, the growth of diatoms could be negatively affected by the increase in CO2 availability. To further support this finding, we estimated the expression and phylogeny of rbcL (which encodes the large subunit of RuBisCO) mRNA in diatoms by quantitative reverse transcription polymerase chain reaction (PCR) and clone library techniques, respectively. Interestingly, regardless of Fe availability, the transcript abundance of rbcL decreased in the high CO2 treatments (600 and 1000 ppm). The present study suggests that the projected future increase in seawater pCO2 could reduce the RuBisCO transcription of diatoms, resulting in a decrease in primary productivity and a shift in the food web structure of the Bering Sea.

Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.