Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.     

Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21-26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.     

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation

Background

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.

Methods

Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli''s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.

Results

We now demonstrate that Angeli''s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli''s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli''s salt were mimicked by BNP. We also demonstrate that the effects of Angeli''s salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).

Conclusions

Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Translation inhibitors induce cell death by multiple mechanisms and Mcl-1 reduction is only a minor contributor

There is significant interest in treating cancers by blocking protein synthesis, to which hematological malignancies seem particularly sensitive. The translation elongation inhibitor homoharringtonine (Omacetaxine mepesuccinate) is undergoing clinical trials for chronic myeloid leukemia, whereas the translation initiation inhibitor silvestrol has shown promise in mouse models of cancer. Precisely how these compounds induce cell death is unclear, but reduction in Mcl-1, a labile pro-survival Bcl-2 family member, has been proposed to constitute the critical event. Moreover, the contribution of translation inhibitors to neutropenia and lymphopenia has not been precisely defined. Herein, we demonstrate that primary B cells and neutrophils are highly sensitive to translation inhibitors, which trigger the Bax/Bak-mediated apoptotic pathway. However, contrary to expectations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal role and cautions that strong correlations do not always signify causality. On the other hand, the killing of T lymphocytes was less dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via alternative mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis altogether. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells.     

Suppression of Na+K+ -ATPase activity by reversible phosphorylation over the winter in a freeze-tolerant insect

Larvae of the gall fly, Eurosta solidaginis, use the cold hardiness strategy of freeze tolerance as well as entry into a hypometabolic state (diapause) to survive the winter. Cold hardiness strategies have been extensively explored in this species, but the metabolic features of winter hypometabolism have received little attention. A primary consumer of energy in cells is the ATP-dependent sodium-potassium ion pump (Na(+)K(+)-ATPase) so inhibitory controls over transmembrane ion movements could contribute substantially to energy savings over the winter months. Na(+)K(+)-ATPase activity was quantified in larvae sampled between October and April. Activity was high in October (0.56+/-0.13nmol/min/mg) but fell by 85% in November, remained low through midwinter, and then increased strongly in April. To determine whether the seasonal change in Na(+)K(+)-ATPase activity was linked with posttranslational modification of the enzyme, extracts from 15 degrees C-acclimated larvae were incubated under conditions that stimulated protein kinases A, G, or C. The action of all three kinases suppressed Na(+)K(+)-ATPase activity to levels just 3-8% of control values whereas the opposite treatment with alkaline phosphatase had no effect. Hence, the seasonal suppression of Na(+)K(+)-ATPase activity may be linked to enzyme phosphorylation. Furthermore, acute cold (3 degrees C) or hypoxia exposures of 15 degrees C-acclimated larvae did not alter enzyme activity, and freezing at -16 degrees C increased activity, so environmental factors do not appear to directly influence enzyme activity. Rather, it appears that winter suppression of ion motive ATPase activity may be part of a program of winter metabolic suppression.