Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.     

Determination of the volatile fraction of Polygonum bistorta L. at different growing stages and evaluation of its antimicrobial activity against two major honeybee (Apis mellifera) pathogens

The composition of the volatile fraction of Polygonum bistorta L. (also known as bistort or snakeroot) was investigated. Fresh aerial parts of this plant species were collected in the Western Italian Alps during the summer at three different phenological stages, namely vegetative, flowering, and fruiting, and steam-distilled in a Clevenger-type apparatus. The oils accounted for 0.004 to 0.010% of the fresh plant material, and their compositions were determined by GC/FID and GC/MS. The composition of the oils during the vegetative period varied both in quantity and quality; several classes of compounds were found with a predominance of alcohols in the vegetative phase, terpenes and linear-chained saturated hydrocarbons in the flowering phase, while saturated aliphatic acids and their methyl esters were predominant in fruiting phase. The most abundant compounds were 3-methylbut-3-en-1-ol in the vegetative phase, linalool in the flowering phase, and dodecanoic acid and its methyl ester in the fruiting phase. The obtained essential oils were then tested against two major bee pathogens, i.e., Paenibacillus larvae and Melissococcus plutonius, and against a reference bacterial species, Bacillus subtilis. Data were compared to those obtained with reference standards used against those pathogens such as the essential oils obtained from leaves and bark of Cinnamomum zeylanicum (cinnamon), and the antibiotic oxytetracyclin.     

Characterization of selectivity and pharmacophores of type 1 sea anemone toxins by screening seven Na(v) sodium channel isoforms

During their evolution, animals have developed a set of cysteine-rich peptides capable of binding various extracellular sites of voltage-gated sodium channels (VGSC). Sea anemone toxins that target VGSCs delay their inactivation process, but little is known about their selectivities. Here we report the investigation of three native type 1 toxins (CGTX-II, δ-AITX-Bcg1a and δ-AITX-Bcg1b) purified from the venom of Bunodosoma cangicum. Both δ-AITX-Bcg1a and δ-AITX-Bcg1b toxins were fully sequenced. The three peptides were evaluated by patch-clamp technique among Nav1.1-1.7 isoforms expressed in mammalian cell lines, and their preferential targets are Na(v)1.5>1.6>1.1. We also evaluated the role of some supposedly critical residues in the toxins which would interact with the channels, and observed that some substitutions are not critical as expected. In addition, CGTX-II and δ-AITX-Bcg1a evoke different shifts in activation/inactivation Boltzmann curves in Nav1.1 and 1.6. Moreover, our results suggest that the interaction region between toxins and VGSCs is not restricted to the supposed site 3 (S3-S4 linker of domain IV), and this may be a consequence of distinct surface of contact of each peptide vs. targeted channel. Our data suggest that the contact surfaces of each peptide may be related to their surface charges, as CGTX-II is more positive than δ-AITX-Bcg1a and δ-AITX-Bcg1b.     

Association of inflammatory markers elevation with aggressive behavior in domestic dogs

We tested the hypothesis that elevations of inflammatory markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) could be associated with the presence of aggressive behavior in domestic dogs. Serum concentrations of CRP and IL-6 were determined by ELISA in eighteen adult male German Shepherd dogs showing no clinical signs but aggression. Eighteen healthy male dogs with a negative history of behavioral and neurological disorders were used as controls. Compared with normal dogs, those with aggression had significantly higher levels of CRP (P < 0.05) and IL-6 (P < 0.01) after adjustment for age, body weight, creatinine, blood urea nitrogen, total protein, total bilirubin and cholesterol. Our pilot data suggest for the first time that an activation of systemic inflammatory processes may contribute to the pathophysiology of aggression in domestic dogs. Further investigations are needed regarding the impact of our findings on treatment strategies.     

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.     

Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8⁺ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8⁺ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8⁺ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8⁺ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection.