Alternate day fasting impacts the brain insulin-signaling pathway of young adult male C57BL/6 mice

Dietary restriction (DR) has recognized health benefits that may extend to brain. We examined how DR affects bioenergetics-relevant enzymes and signaling pathways in the brains of C57BL/6 mice. Five-month-old male mice were placed in ad libitum or one of two repeated fasting and refeeding (RFR) groups, an alternate day (intermittent fed; IF) or alternate day plus antioxidants (blueberry, pomegranate, and green tea extracts) (IF + AO) fed group. During the 24-h fast blood glucose levels initially fell but stabilized within 6 h of starting the fast, thus avoiding frank hypoglycemia. DR in general appeared to enhance insulin sensitivity. After six weeks brain AKT and glycogen synthase kinase 3 beta phosphorylation were lower in the RFR mice, suggesting RFR reduced brain insulin-signaling pathway activity. Pathways that mediate mitochondrial biogenesis were not activated; AMP kinase phosphorylation, silent information regulator 2 phosphorylation, peroxisomal proliferator-activated receptor-gamma coactivator 1 alpha levels, and cytochrome oxidase subunit 4 levels did not change. ATP levels also did not decline, which suggests the RFR protocols did not directly impact brain bioenergetics. Antioxidant supplementation did not affect the brain parameters we evaluated. Our data indicate in young adult male C57BL/6 mice, RFR primarily affects brain energy metabolism by reducing brain insulin signaling, which potentially results indirectly as a consequence of reduced peripheral insulin production.     

Micronuclei as biomarkers for evaluating the risk of malignant transformation in the uterine cervix

We evaluated micronucleus and apoptosis occurrence among women with normal smears and women with different kinds of cervical abnormalities, i.e., inflammatory processes and low- and high-grade squamous intraepithelial lesions (N = 12, N = 10 and N = 27, respectively). The sample included 59 women who were seen at a public medical service for cervical cancer prevention in Feira de Santana, Bahia, Brazil. The diagnosis was established by means of cytological, colposcopic, and histopathological examination. Cytogenetic analysis was performed on 2000 cells from each woman and included assessment of micronuclei and nuclear degenerative abnormalities indicative of apoptosis (karyorrhexis, pyknosis and condensed chromatin). Micronucleus frequency was significantly higher in the women with high-grade squamous intraepithelial lesions than in the women without cervical abnormalities or inflammatory processes (P< 0.001) or in the women with low-grade squamous intraepithelial lesions (P < 0.005). The frequency of apoptosis was similar in women without cervical abnormalities and women showing high-grade squamous intraepithelial lesions (P > 0.50), and significantly lower in women without cervical abnormalities and in women showing high-grade squamous intraepithelial lesions than in women showing inflammatory processes or low-grade squamous intraepithelial lesions (P < 0.0001). These results indicate that, in addition to Papanicolaou cervical cytological analysis, it would be useful to use micronucleus analysis to screen women who are at risk of developing cervical cancer. The assessment of nuclear degenerative abnormalities indicative of apoptosis increased the sensitivity of this test.     

Molecular sexing of unusually large numbers of Spheniscus magellanicus (Spheniscidae) washed ashore along the Brazilian coast in 2008

There have been few studies on Magellanic penguins (Spheniscus magellanicus). In 2008, these penguins washed ashore along the Brazilian coast in unusually high numbers, some reaching as far as northeast Brazil. As Magellanic penguins show little sexual dimorphism, sex determination by morphological features is not accurate. Here, we tested a molecular procedure for sexing specimens of S. magellanicus washed ashore along the coasts of Sergipe, Rio de Janeiro and Rio Grande do Sul in 2008, comparing the sex ratio between these localities. Tissue samples were collected from 135 dead, beached specimens. We carried out total genomic DNA extraction and CHD-Z/CHD-W gene amplification by PCR using P2 and P8 primers. Amplicons were separated by 12% acrylamide gel electrophoresis. We found a greater proportion of females (70%). Sex could be determined because females have two intronic regions of CHD gene of different size in the sex chromosomes, visualized as two bands on the gel (380 and 400 bp approximately), while males have only one (400 bp). Therefore, this method proved to be effective and sensitive for sex determination of S. magellanicus individuals. Data on sex ratios are useful for understanding the dynamics and ecology of Magellanic penguin populations.     

Perinatal Na(+) Overload Programs Raised Renal Proximal Na(+) Transport and Enalapril-Sensitive Alterations of Ang II Signaling Pathways during Adulthood

Background

High Na⁺ intake is a reality in nowadays and is frequently accompanied by renal and cardiovascular alterations. In this study, renal mechanisms underlying perinatal Na⁺ overload-programmed alterations in Na⁺ transporters and the renin/angiotensin system (RAS) were investigated, together with effects of short-term treatment with enalapril in terms of reprogramming molecular alterations in kidney.

Methodology/Principal Findings

Male adult Wistar rats were obtained from dams maintained throughout pregnancy and lactation on a standard diet and drinking water (control) or 0.17 M NaCl (saline group). Enalapril (100 mg/l), an angiotensin converting enzyme inhibitor, was administered for three weeks after weaning. Ninety day old offspring from dams that drank saline presented with proximal tubules exhibiting increased (Na⁺+K⁺)ATPase expression and activity. Ouabain-insensitive Na⁺-ATPase activity remained unchanged but its response to angiotensin II (Ang II) was lost. PKC, PKA, renal thiobarbituric acid reactive substances (TBARS), macrophage infiltration and collagen deposition markedly increased, and AT₂ receptor expression decreased while AT₁ expression was unaltered. Early treatment with enalapril reduced expression and activity of (Na⁺+K⁺)ATPase, partially recovered the response of Na⁺-ATPase to Ang II, and reduced PKC and PKA activities independently of whether offspring were exposed to high perinatal Na⁺ or not. In addition, treatment with enalapril per se reduced AT₂ receptor expression, and increased TBARS, macrophage infiltration and collagen deposition. The perinatally Na⁺-overloaded offspring presented high numbers of Ang II-positive cortical cells, and significantly lower circulating Ang I, indicating that programming/reprogramming impacted systemic and local RAS.

Conclusions/Significance

Maternal Na⁺ overload programmed alterations in renal Na⁺ transporters and in its regulation, as well as severe structural lesions in adult offspring. Enalapril was beneficial predominantly through its influence on Na⁺ pumping activities in adult offspring. However, side effects including down-regulation of PKA, PKC and AT₂ receptors and increased TBARS could impair renal function in later life.     

Potentiation of dietary restriction-induced lifespan extension by polyphenols

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF+PAO). Both IF and IF+PAO groups outlived the control group and the IF+PAO group outlived the IF group (all p<0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.     

Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars

Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple (Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant–pathogen relationship and establishing strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed. The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective cicatrization response compared to the susceptible cultivar.     

Epigenetic Variation in Mangrove Plants Occurring in Contrasting Natural Environment

Background

Epigenetic modifications, such as cytosine methylation, are inherited in plant species and may occur in response to biotic or abiotic stress, affecting gene expression without changing genome sequence. Laguncularia racemosa, a mangrove species, occurs in naturally contrasting habitats where it is subjected daily to salinity and nutrient variations leading to morphological differences. This work aims at unraveling how CpG-methylation variation is distributed among individuals from two nearby habitats, at a riverside (RS) or near a salt marsh (SM), with different environmental pressures and how this variation is correlated with the observed morphological variation.

Principal Findings

Significant differences were observed in morphological traits such as tree height, tree diameter, leaf width and leaf area between plants from RS and SM locations, resulting in smaller plants and smaller leaf size in SM plants. Methyl-Sensitive Amplified Polymorphism (MSAP) was used to assess genetic and epigenetic (CpG-methylation) variation in L. racemosa genomes from these populations. SM plants were hypomethylated (14.6% of loci had methylated samples) in comparison to RS (32.1% of loci had methylated samples). Within-population diversity was significantly greater for epigenetic than genetic data in both locations, but SM also had less epigenetic diversity than RS. Frequency-based (G_ST) and multivariate (β_ST) methods that estimate population structure showed significantly greater differentiation among locations for epigenetic than genetic data. Co-Inertia analysis, exploring jointly the genetic and epigenetic data, showed that individuals with similar genetic profiles presented divergent epigenetic profiles that were characteristic of the population in a particular environment, suggesting that CpG-methylation changes may be associated with environmental heterogeneity.

Conclusions

In spite of significant morphological dissimilarities, individuals of L. racemosa from salt marsh and riverside presented little genetic but abundant DNA methylation differentiation, suggesting that epigenetic variation in natural plant populations has an important role in helping individuals to cope with different environments.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

High levels of genetic structuring as a result of population fragmentation in the tropical tree species <Emphasis Type="Italic">Caesalpinia echinata</Emphasis> Lam.

We have investigated levels of genetic diversity within and among seven remnant populations of Caesalpinia echinata Lam., an endangered species found as fragmented populations in three major areas around the coastal regions of Brazil. Using amplified fragment length polymorphism (AFLP) genetic markers, we detected levels of within-population genetic diversity ranging from 0.092 to 0.163, with the lowest values generally being found in the smallest populations. Estimates of between-population genetic differentiation were strongly correlated with geographical distance (r = 0.884, p < 0.001), which,along with a neighbour-joining phylogenetic analysis, strongly suggested high levels of genetic isolation by distance. Over half (62%) of the total genetic diversity was partitioned between populations, further highlighting the genetic distinctness of individual populations. Taken together, these results suggest that fragmentation has led to an increase in population differentiation between fragments of C. echinata. These formations will be of great value in the development of conservation plans for species exhibiting high levels of genetic differentiation due to fragmentation, such as indication of conservation unit size, which populations should be chosen as priority in conservation plans and which samples should be introduced in areas with a low number of individuals of brazilwood.