Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit ^-/- MEFs escape senescence to become immortal more rapidly than Fhit ^+/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.     

In vitro antimicrobial effect of metallic nanoparticles on phytopathogenic strains of crop plants

In this research, the in vitro antimicrobial effect of zinc oxide (ZnO), copper oxide (CuO) and iron oxide (Fe₂O₃) nanoparticles (NPs)—with average sizes of 20, 46 and 30 nm, respectively—on the root rot disease caused by the fungus Fusarium oxysporum and on blight disease caused by the fungus Alternaria solani were studied. Also, bacterial diseases caused by Clavibacter michiganensis and Pseudomonas syringae that infects a wide range of plant species were assessed. Different concentrations of NPs (0, 100, 250, 500, 700 and 1,000 mg/L) were prepared on PDA agar or King's B medium in a complete randomized design with four replicates. According to the results, ZnO NPs exhibited an outstanding inhibitory effect against fungi and bacteria strains. The above results were associated with the smaller particle size. Fungi strains showed a differential sensitivity depending on the kind of NPs used. A. solani showed the highest sensitivity to ZnO NPs at 1,000 mg/L (99%), followed by CuO NPs at the same dose (95%). Fe₂O₃ NPs at all evaluated doses had no inhibitory effects on the mycelia growth of this strain, although F. oxysporum revealed greater effectiveness of the CuO NPs (96%) compared with ZnO NPs since it only inhibited 91% of the mycelial growth. The antibacterial activity was studied through optical density. C. michiganensis was found to be more sensitive to ZnO NPs because a lesser dose (700 mg/L) was required to reduce the bacterial growth (90%); in comparison, P. syringae required a dose of 1,000 mg/L to inhibit its growth (67%). CuO NPs displayed the smallest growth inhibition against the bacteria strains analysed. The antimicrobial effect of the metallic NPs that were assayed increased with higher doses.     

Asymmetric paternal effect on offspring size linked to parent‐of‐origin expression of an insulin‐like growth factor

Sexual reproduction brings together reproductive partners whose long‐term interests often differ, raising the possibility of conflict over their reproductive investment. Males that enhance maternal investment in their offspring gain fitness benefits, even if this compromises future reproductive investment by iteroparous females. When the conflict occurs at a genomic level, it may be uncovered by crossing divergent populations, as a mismatch in the coevolved patterns of paternal manipulation and maternal resistance may generate asymmetric embryonic growth. We report such an asymmetry in reciprocal crosses between populations of the fish Girardinichthys multiradiatus. We also show that a fragment of a gene which can influence embryonic growth (Insulin‐Like Growth Factor 2; igf2) exhibits a parent‐of‐origin methylation pattern, where the maternally inherited igf2 allele has much more 5′ cytosine methylation than the paternally inherited allele. Our findings suggest that male manipulation of maternal investment may have evolved in fish, while the parent‐of‐origin methylation pattern appears to be a potential candidate mechanism modulating this antagonistic coevolution process. However, disruption of other coadaptive processes cannot be ruled out, as these can lead to similar effects as conflict.     

Chronic Exercise Increases Plasma Brain-Derived Neurotrophic Factor Levels,Pancreatic Islet Size,and Insulin Tolerance in a TrkB-Dependent Manner

Background

Physical exercise improves glucose metabolism and insulin sensitivity. Brain-derived neurotrophic factor (BDNF) enhances insulin activity in diabetic rodents. Because physical exercise modifies BDNF production, this study aimed to investigate the effects of chronic exercise on plasma BDNF levels and the possible effects on insulin tolerance modification in healthy rats.

Methods

Wistar rats were divided into five groups: control (sedentary, C); moderate- intensity training (MIT); MIT plus K252A TrkB blocker (MITK); high-intensity training (HIT); and HIT plus K252a (HITK). Training comprised 8 weeks of treadmill running. Plasma BDNF levels (ELISA assay), glucose tolerance, insulin tolerance, and immunohistochemistry for insulin and the pancreatic islet area were evaluated in all groups. In addition, Bdnf mRNA expression in the skeletal muscle was measured.

Principal Findings

Chronic treadmill exercise significantly increased plasma BDNF levels and insulin tolerance, and both effects were attenuated by TrkB blocking. In the MIT and HIT groups, a significant TrkB-dependent pancreatic islet enlargement was observed. MIT rats exhibited increased liver glycogen levels following insulin administration in a TrkB-independent manner.

Conclusions/Significance

Chronic physical exercise exerted remarkable effects on insulin regulation by inducing significant increases in the pancreatic islet size and insulin sensitivity in a TrkB-dependent manner. A threshold for the induction of BNDF in response to physical exercise exists in certain muscle groups. To the best of our knowledge, these are the first results to reveal a role for TrkB in the chronic exercise-mediated insulin regulation in healthy rats.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Microscopic and histochemical study of odontoclasts in physiologic resorption of teeth of the polyphyodont lizard, Liolaemus gravenhorsti

Using tartrate-resistant acid phosphatase (TRAP), we examined the cytodifferentiation of odontoclast cells in resorbing areas of dental tissues during the replacement of teeth in a polyphyodont lizard, Liolaemus gravenhorsti. We also report, by means of Lectin-HRP histochemistry, the distribution pattern of some specific sugar residues of TRAPase-positive cells. For detection of TRAPase activity, the azo dye-coupling technique was used. Lectin binding sites were demonstrated by means of specific HRP-lectins. The process of tooth resorption was divided into four stages: 1) preresorption-the wall of the dental pulp is covered with an odontoblast layer, and no TRAP-positive cells are in the dental pulp; 2) early resorption-TRAP-positive multinucleate odontoclasts are present on the dental wall, but the rest of the pulp surface is still covered with an odontoblast layer; 3) later resorption-the entire surface of the pulp chamber is lined with multinucleate odontoclasts; and 4) final resorption-the tooth has been totally resorbed. Odontoclasts are usually detached from the resorbed surface, and show signs of degeneration. Of the six lectins used, PNA, ECA, and UEA-1 bind to multinucleated but not mononuclear cells. All the remaining lectins, BS-1, RCA(120), and LTA showed no binding to any cells of the teeth. The significance of saccharidic moieties such as acetyl-galactosamine, acetyl-glucosamine, and fucose sugar residues is difficult to ascertain. Perhaps these oligosaccharides might be borne on molecules associated with odontoclastic resorption or associated with multinucleation of odontoclasts after attachment to the dentine surface.     

Ethanol effect on the chick embryo ossification: a macroscopic and microscopic study

This study attempts to analyze anomalies in avian embryos induced macroscopically and microscopically when exposed to ethanol (EtOH) during the first stages of development. Fertilized chicken eggs were employed in this study. The eggs were incubated at 37.8 degrees C. Some of the eggs were treated on day 0 with EtOH (20%, 40% and 60%) by instillation in the air sac. The control group was instilled with 0.1 ml of NaCl at 0.9%. Other eggs were treated on the 4th post-incubation day, employing the same methodology. The embryos in both groups were removed from the eggs on the 11th incubation day and examined using a dissecting binocular microscope. After macroscopic analysis, the samples obtained were fixed in 10% formol, photographed and processed according to common histological techniques and the Picrosirius method. Embryos treated with EtOH demonstrated a significant weight decrease. Microscopic analysis by means of the Picrosirius method revealed that the intra-membranous ossification process presents less development, and therefore there was less type I collagen in trabecular bone in the embryos post-exposure to EtOH with respect to the control.     

Effect of cholinergic agonists on muscular tonus of the lizard small intestine and esophagus.

The underlying mechanisms of acetylcholine-induced intestinal relaxation in the lizard Liolaemus tenuis tenuis are still unknown. By using a classical model of intestinal recording of isometric contraction and relaxation in conjunction with specific pharmacological tools, this article studies the possible influence of EDRF/NO and nicotinic ganglionar receptors on the Ach-induced relaxation in an effort to elucidate the probable mechanisms involved in ACh effect. It was observed that the relaxation of the lizard intestine elicited by ACh (10(-7) - 4 x 10(-4) M) was not affected by hexametonium (5 x 10(-4) M) or tetrodotoxin (10(-6) M). Nicotine (10(-7) to 10(-4) M) induced relaxation was significantly antagonized by hexametonium; however, it was not influenced by tetrodotoxin. These results allow us to discard a neuronal pathway in cholinergic-induced relaxation, suggesting a more direct cholinergic effect on the smooth muscle, perhaps mediated by an unknown substance released by some specialized tissue. N-nitro-L-arginine, used to block NO-synthase and NO production, induced no changes in ACh-induced relaxation. Methylene blue, a soluble guanylate cyclase inhibitor, induced no changes in ACh-induced relaxation. These results allow us to discard a probable role of EDRF/nitric oxide in the ACh-induced relaxation of lizard small intestine, providing evidence that this mechanism could be different from that reported in other species.     

Increased estrogen receptor alpha immunoreactivity in the forebrain of sexually satiated rats

Estrogen receptor alpha (ERalpha) participates in the neuroendocrine regulation of male sexual behavior, primarily in brain areas located in the limbic system. Males of many species present a long-term inhibition of sexual behavior after several ejaculations, known as sexual satiety. It has been shown that androgen receptor density is reduced 24 h after a single ejaculation or mating to satiety, in the medial preoptic area, nucleus accumbens and ventromedial hypothalamus. The aim of this study was to analyze if the density of ERalpha was also modified 24 h after a single ejaculation or mating to satiety. Sexual satiety was associated with an increased ERalpha density in the anteromedial bed nucleus of the stria terminalis (BSTMA), ventrolateral septum (LSV), posterodorsal medial amygdala (MePD), medial preoptic area (MPA) and nucleus accumbens core (NAc). A single ejaculation was related to an increase in ERalpha density in the BSTMA and MePD. ERalpha density in the arcuate (Arc) and ventromedial hypothalamic nuclei (VMN), and serum estradiol levels remained unchanged 24 h after one ejaculation or mating to satiety. These data suggest a relationship between sexual activity and an increase in the expression of ERalpha in specific brain areas, independently of estradiol levels in systemic circulation.