Methyl jasmonate affects phenolic metabolism and gene expression in blueberry (Vaccinium corymbosum)

Blueberry (Vaccinium corymbosum) is a fruit very much appreciated by consumers for its antioxidant potential and health‐promoting traits. Its beneficial potential properties are mainly due to a high content of anthocyanins and their amount can change after elicitation with methyl jasmonate. The aim of this work is to evaluate the changes in expression of several genes, accumulation of phenolic compounds and alterations in antioxidant potential in two different blueberry cultivars (‘Duke’ and ‘Blueray’) in response to methyl jasmonate (0.1 mM). Results showed that 9 h after treatment, the expression of phenylalanine ammonium lyase, chalcone synthase and anthocyanidin synthase genes was stimulated more in the ‘Blueray’ variety. Among the phenols measured an increase was recorded also for epicatechin and anthocyanin concentrations. ‘Duke’ is a richer sourche of anthocyanins compared to ‘Blueray’, treatment with methyl jasmonate promoted in ‘Blueray’ an increase in pigments as well as in the antioxidant potential, especially in fully ripe berries, but treated ‘Duke’ berries had greater levels, which were not induced by methyl jasmonate treatment. In conclusion, methyl jasmonate was, in some cases, an effective elicitor of phenolic metabolism and gene expression in blueberry, though with different intensity between cultivars.     

Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF

The current report describes the use of CapLC-ESI-Q/TOF-MS for investigating the proteome profiles of hypertonic saline-induced sputum samples from 56 smokers. The severity of their lung disease ranged from normal (healthy smokers) to chronic bronchitis, chronic obstructive pulmonary disease (COPD), and COPD with emphysema. This pilot study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype and cigarette smoking illness severity. A total of 203 unique proteins were identified. These may represent the most highly expressed proteins in induced sputum. Our results provide evidence that different proteins are expressed, as the disease progresses from health to more advanced stages, and support our contention that a proteomic approach would be beneficial in discovering selective molecules linked to specific COPD stages.     

Gene expression variation in Down's syndrome mice allows prioritization of candidate genes

Background  

Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction.     

Vimentin regulates peripheral nerve myelination

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.     

Hypoxia induces class III beta-tubulin gene expression by HIF-1alpha binding to its 3' flanking region

Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.     

Effective gene flow in a historically fragmented area at the southern edge of silver fir (<Emphasis Type="Italic">Abies alba</Emphasis> Mill.) distribution

Fragmented populations at the edges of a species’ distribution can be highly exposed to the loss of genetic variation, unless sufficient gene flow maintains their genetic connectivity. Gene movements leading to successful establishment of external gametes (i.e. effective gene flow) into fragmented populations can solely be assessed by investigating the origin of natural regeneration. This study is focused on studying gene flow patterns in two silver fir stands in Central Apennines, where the species has a highly fragmented distribution. By using nuclear and chloroplast microsatellite markers, we investigated genetic variation, fine-scale spatial genetic structure, effective gene flow rates and large-scale connectivity characterizing both stands. Similar levels of genetic variation and low genetic differentiation between stands (F _ST = 0.005) and across generations were found, coupled with low inbreeding and weak to absent SGS in the adult cohort (Sp < 0.003). On the other hand, substantial differences between the two stands in terms of gene flow rates were observed. Irrespective of the parentage approach used, higher gene flow rates were found in the stand located at the upper silver fir altitudinal limit, especially for seed-mediated gene flow (0.79 in the upper stand vs. 0.48 in the lower stand). Conversely, the lower stand was characterized by a higher reproductive dominance of local adults. Our findings suggest that, despite similar levels of genetic variation and generally high gene flow rates, different processes may be acting on the two stands, reflecting varying ecological conditions.     

Unfolding and pH studies on manganese peroxidase: role of heme and calcium on secondary structure stability

The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.