THE FINE LOCALIZATION OF NUCLEOSIDE TRIPHOSPHATASE ACTIVITY IN THE RETINA OF THE FROG

Nucleoside triphosphatase (NTPase) activity was demonstrated at the submicroscopic level in the frog retina by the Wachstein-Meisel method utilizing various purine and pyrimidine nucleosides. Under the electron microscope magnesium-activated NTPase was localized in the outer and inner segments, and in the plexiform layers. NTPase active sites in the cones were localized diffusely in the 70 to 80 A interspaces between the double membranes of the stacked lamellae and in the investing cytoplasm. In the rods, on the other hand, sites of activity were observed at the periphery of the stacked lamellae as discrete electron opaque deposits measuring 1000 to 1500 A which interdigitated between the lamellae for short distances. Deposits of reaction product appeared more numerous in rods of dark-adapted frogs stimulated with monochromatic light with a wave length of 510 mµ. Enzyme activity was also observed in mitochondria of the rod and cone ellipsoids. In the outer and inner plexiform layers NTPase active sites were present on and between the membranes of axons and the plasma membranes of some of the neurons.     

Copulatory mechanics in the wolf spider Agalenocosa pirity reveals a hidden diversity of locking systems in Lycosidae (Araneae)

Genital traits are among the fastest to evolve, and the processes that drive their evolution are intensively studied. Spiders are characterized by complex genitalia, but the functional role of the different structures during genital coupling is largely unknown. Members of one of the largest spider groups, the retrolateral tibial apophysis (RTA) clade, are characterized by a RTA on the male palp, which is thought to play a crucial role during genital coupling. However, the RTA was lost in several families including the species-rich wolf spiders (Lycosidae) leading to the hypothesis that the genital coupling is achieved by alternative mechanisms. Here, we investigate the genital interactions during copulation in the wolf spider Agalenocosa pirity (Zoicinae) on cryofixed mating pairs using electron, optical and X-ray microscopy and compare our findings with other lycosids and entelegyne spiders. We found an unprecedented coupling mechanism for lycosid spiders involving the palea and a membranous cuticle folding adjacent to the epigynal plate. Additionally, we show an uncommon coupling between the median apophysis and the contralateral genital opening, and confirmed that the terminal apophysis acts as functional conductor, as previously hypothesized for males of Zoicinae. Phylogenetic mapping of RTA indicated that the basal tibial process found in Agalenocosa is a secondary acquisition rather than a modified RTA.     

Periconceptional alcohol consumption-induced changes in embryonic prostaglandin E levels in mouse organogenesis: modulation by nitric oxide

The mechanisms of the teratogenic effects of maternal alcohol consumption remain unclear. The aim of the present work was to study the organogenic PGE(2) levels and the modulation of PGE(2) levels by NO after periconceptional alcohol ingestion. Female mice were intoxicated with a 10% ethanol in drinking water before pregnancy and up to day 10 of gestation. The PGE(2) released from organogenic embryos was measured by radio immunoassay following incubation with or without the addition of either a NO donor or a NO synthase (NOS) inhibitor. In the ethanol-treated females, we found increased percentages of retarded embryos, associated with a significantly elevated resorption rate (p<0.05), very high quantities of morphologically abnormal E.10 embryos (p<0.001) and significantly increased PGE(2) release, as compared to the embryo parameters of control females. While in the control-derived E.10 embryos the NO donor produced significantly increased PGE(2) release, in the ethanol-derived embryos decreased quantities of PGE(2) were observed. L-NMMA inhibited PGE(2) release in both control and ethanol-derived embryos at different concentrations, whereas it decreased PGE(2) content in controls but not in ethanol-derived embryos. The periconceptional alcohol ingestion produced excessive PGE(2) release, decreased PGE(2) content and disruption of the regulatory NO-PGE(2) pathways. These PGs alterations may be related to delayed organogenesis and abnormal neural tube development after chronic periconceptional consumption of alcohol.     

Altered Functional Connectivity and Small-World in Mesial Temporal Lobe Epilepsy

Background

The functional architecture of the human brain has been extensively described in terms of functional connectivity networks, detected from the low–frequency coherent neuronal fluctuations that can be observed in a resting state condition. Little is known, so far, about the changes in functional connectivity and in the topological properties of functional networks, associated with different brain diseases.

Methodology/Principal Findings

In this study, we investigated alterations related to mesial temporal lobe epilepsy (mTLE), using resting state functional magnetic resonance imaging on 18 mTLE patients and 27 healthy controls. Functional connectivity among 90 cortical and subcortical regions was measured by temporal correlation. The related values were analyzed to construct a set of undirected graphs. Compared to controls, mTLE patients showed significantly increased connectivity within the medial temporal lobes, but also significantly decreased connectivity within the frontal and parietal lobes, and between frontal and parietal lobes. Our findings demonstrated that a large number of areas in the default-mode network of mTLE patients showed a significantly decreased number of connections to other regions. Furthermore, we observed altered small-world properties in patients, along with smaller degree of connectivity, increased n-to-1 connectivity, smaller absolute clustering coefficients and shorter absolute path length.

Conclusions/Significance

We suggest that the mTLE alterations observed in functional connectivity and topological properties may be used to define tentative disease markers.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.     

Use of the D-R model to define trends in the emergence of Ceftazidime-resistant Escherichia coli in China

Objective

To assess the efficacy of the D-R model for defining trends in the appearance of Ceftazidime-resistant Escherichia coli.

Methods

Actual data related to the manifestation of Ceftazidime-resistant E. coli spanning years 1996–2009 were collected from the China National Knowledge Internet. These data originated from 430 publications encompassing 1004 citations of resistance. The GM(1,1) and the novel D-R models were used to fit current data and from this, predict trends in the appearance of the drug-resistant phenotype. The results were evaluated by Relative Standard Error (RSE), Mean Absolute Deviation (MAD) and Mean Absolute Error (MAE).

Results

Results from the D-R model showed a rapid increase in the appearance of Ceftazidime-resistant E. coli in this region of the world. These results were considered accurate based upon the minor values calculated for RSE, MAD and MAE, and were equivalent to or better than those generated by the GM(1,1) model.

Conclusion

The D-R model which was originally created to define trends in the transmission of swine viral diseases can be adapted to evaluating trends in the appearance of Ceftazidime-resistant E. coli. Using only a limited amount of data to initiate the study, our predictions closely mirrored the changes in drug resistance rates which showed a steady increase through 2005, a decrease between 2005 and 2008, and a dramatic inflection point and abrupt increase beginning in 2008. This is consistent with a resistance profile where changes in drug intervention temporarily delayed the upward trend in the appearance of the resistant phenotype; however, resistance quickly resumed its upward momentum in 2008 and this change was better predicted using the D-R model. Additional work is needed to determine if this pattern of “increase-control-increase” is indicative of Ceftazidime-resistant E. coli or can be generally ascribed to bacteria acquiring resistance to drugs in the absence of alternative intervention.     

The insertion of the antimicrobial peptide dicynthaurin monomer in model membranes: thermodynamics and structural characterization

This paper is focused on the thermodynamics and the structural investigation of the interaction of the antimicrobial peptide dicynthaurin monomer with model lipid membranes composed of mixtures of 1-palmitoyl-2-oleyl-glycerophosphocholine and -glycerophosphoglycerol. The thermodynamic binding parameters as obtained by isothermal titration calorimetry reveal strong binding toward the lipid model system dominated by large chemical binding constants which exceeds the electrostatic binding effects and thus suggests insertion of the amphipathic alpha-helical peptide into the hydrophobic membrane core. Circular dichroism study shows that the peptide exhibits trans-membrane alpha-helix secondary structure. Neutron diffraction measurements using partially deuterated sequences were successfully applied to determine the orientation of the peptide thus proving insertion into the hydrophobic membrane core. This insertion and the formation of higher order porelike aggregates is assumed to be the most relevant event in microbial membrane perturbation that in vivo finally leads to bacterial cell death on a fast time scale.     

Localization of coenzyme Q10 in the center of a deuterated lipid membrane by neutron diffraction

Quinones (e.g., coenzyme Q, CoQ10) are best known as carriers of electrons and protons during oxidative phosphorylation and photosynthesis. A myriad of mostly more indirect physical methods, including fluorescence spectroscopy, electron-spin resonance, and nuclear magnetic resonance, has been used to localize CoQ10 within lipid membranes. They have yielded equivocal and sometimes contradictory results. Seeking unambiguous evidence for the localization of ubiquinone within lipid bilayers, we have employed neutron diffraction. CoQ10 was incorporated into stacked bilayers of perdeuterated dimyristoyl phosphatidyl choline doped with dimyristoyl phosphatidyl serine containing perdeuterated chains in the natural fluid-crystalline state. Our data show CoQ10 at the center of the hydrophobic core parallel to the membrane plane and not, as might be expected, parallel to the lipid chains. This localization is of importance for its function as a redox shuttle between the respiratory complexes and, taken together with our recent result that squalane is in the bilayer center, may be interpreted to show that all natural polyisoprene chains lie in the bilayer center. Thus ubiquinone, in addition to its free radical scavenging and its well-known role in oxidative phosphorylation as a carrier of electrons and protons, might also act as an inhibitor of transmembrane proton leaks.