Fiber size and sensory endings of the middle cardiac nerve of the domestic fowl (Gallus domesticus)

The middle cardiac nerve, a branch of the vagus, innervates the ventricles of the avian heart. Of 533 myelinated sensory fibers, the size range was 2 micron. The ratio of myelinated to unmyelinated sensory fibers ranged from 2.17 to 3.48. Sensory endings resembled a network pattern with no distinct receptor-like endings. Frequency of nerve population increased from apex to base of the heart.     

Forest Loss and the Biodiversity Threshold: An Evaluation Considering Species Habitat Requirements and the Use of Matrix Habitats

Habitat loss is the main driver of the current biodiversity crisis, a landscape-scale process that affects the survival of spatially-structured populations. Although it is well-established that species responses to habitat loss can be abrupt, the existence of a biodiversity threshold is still the cause of much controversy in the literature and would require that most species respond similarly to the loss of native vegetation. Here we test the existence of a biodiversity threshold, i.e. an abrupt decline in species richness, with habitat loss. We draw on a spatially-replicated dataset on Atlantic forest small mammals, consisting of 16 sampling sites divided between forests and matrix habitats in each of five 3600-ha landscapes (varying from 5% to 45% forest cover), and on an a priori classification of species into habitat requirement categories (forest specialists, habitat generalists and open-area specialists). Forest specialists declined abruptly below 30% of forest cover, and spillover to the matrix occurred only in more forested landscapes. Generalists responded positively to landscape heterogeneity, peaking at intermediary levels of forest cover. Open area specialists dominated the matrix and did not spillover to forests. As a result of these distinct responses, we observed a biodiversity threshold for the small mammal community below 30% forest cover, and a peak in species richness just above this threshold. Our results highlight that cross habitat spillover may be asymmetrical and contingent on landscape context, occurring mainly from forests to the matrix and only in more forested landscapes. Moreover, they indicate the potential for biodiversity thresholds in human-modified landscapes, and the importance of landscape heterogeneity to biodiversity. Since forest loss affected not only the conservation value of forest patches, but also the potential for biodiversity-mediated services in anthropogenic habitats, our work indicates the importance of proactive measures to avoid human-modified landscapes to cross this threshold.     

Circadian variations in self-monitoring,a component of executive functions

The objective of this study was to identify circadian rhythms in self-monitoring, a component of executive functions. Participants were 10 undergraduate students, age: 18.5 ± 2.68 years, two male and eight female. They were recorded on a 30-h constant routine protocol; rectal temperature was recorded every minute and performance on a tracking task was assessed every 100 min. Self-monitoring indicators were adjustments of responses to random changes of speed and trajectory of a circle moving on the computer screen. Participants showed better accuracy during the afternoon, with decreases in the morning (06:20 and 08:00 h). These variations showed a phase delay of 2:29 ± 2:19 h with respect to the circadian rhythm of body temperature. In conclusion, there are circadian variations in self-monitoring. The decline in this component of executive functions could cause serious accidents among people working or studying during a morning shift, as well as commuting to and from work or school.     

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.     

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.     

Expression of the receptor tyrosine kinase Tie2 in neoplastic glial cells is associated with integrin beta1-dependent adhesion to the extracellular matrix

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.     

Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction

The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.     

DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Using tunable diode laser spectroscopy to measure carbon isotope discrimination and mesophyll conductance to CO₂ diffusion dynamically at different CO₂ concentrations

In C₃ leaves, the mesophyll conductance to CO₂ diffusion, g_m, determines the drawdown in CO₂ concentration from intercellular airspace to the chloroplast stroma. Both g_m and stomatal conductance limit photosynthetic rate and vary in response to the environment. We investigated the response of g_m to changes in CO₂ in two Arabidopsis genotypes (including a mutant with open stomata, ost1), tobacco and wheat. We combined measurements of gas exchange with carbon isotope discrimination using tunable diode laser absorption spectroscopy with a CO₂ calibration system specially designed for a range of CO₂ and O₂ concentrations. CO₂ was initially increased from 200 to 1000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm, or the sequence was reversed. In 2% O₂ a step increase from 200 to 1000 ppm significantly decreased g_m by 26–40% in all three species, whereas following a step decrease from 1000 to 200 ppm, the 26–38% increase in g_m was not statistically significant. The response of g_m to CO₂ was less in 21% O₂. Comparing wild type against the ost1 revealed that mesophyll and stomatal conductance varied independently in response to CO₂. We discuss the effects of isotope fractionation factors on estimating g_m.