Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .     

Effects of prepubertal castration on the spinal motor nucleus of the ischiocavernosus muscle of the rat

Summary The location, number and size of the motoneurons innervating the ischiocavernosus muscle, identified by means of horseradish-peroxidase (HRP) retrograde transport, were studied (1) in adult untreated male rats, (2) in adult male rats castrated before puberty, and (3) in adult male rats castrated before puberty and injected with testosterone from the day of castration. After injection of HRP into the ischiocavernosus muscle, labeled motoneurons were found in the dorsolateral and dorsomedial columns of the lamina IX, at the level of L6 and S1 segments of the spinal cord. Morphometric analysis demonstrated that prepubertal castration induces a statistically significant reduction in the somatic and nuclear areas (40% and 35%, respectively, if compared to those of the control rats) of both the dorsolateral and dorsomedial motoneurons, but does not affect their number. The effects of castration are prevented by exogenous testosterone.Preliminary results were presented at the International Conference on Hormones, Brain and Behaviour, Liège, Belgium, August, 1989     

Mechanics of the cellular actin cortex: From signalling to shape change

The actin cortex is a thin layer of actin, myosin and actin-binding proteins that underlies the membrane of most animal cells. It is highly dynamic and can undergo remodelling on timescales of tens of seconds, thanks to protein turnover and myosin-mediated contractions. The cortex enables cells to resist external mechanical stresses, controls cell shape and allows cells to exert forces on their neighbours. Thus, its mechanical properties are the key to its physiological function. Here, we give an overview of how cortex composition, structure and dynamics control cortex mechanics and cell shape. We use mitosis as an example to illustrate how global and local regulation of cortex mechanics gives rise to a complex series of cell shape changes.     

Secondary metabolite as therapeutic agent from endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis

Endophytic fungi, especially from mangrove plants, are rich source of secondary metabolites, which plays a major role in various pharmacological actions preferably in cancer and bacterial infections. To perceive its role in antidiabetic activity we isolated and tested the metabolites derived from a novel strain Alternaria longipes strain VITN14G obtained from mangrove plant Avicennia officinalis. The crude extract was analyzed for antidiabetic activity and subjected to column chromatography. The isolated fractions were screened in vitro for α-glucosidase and α-amylase inhibitory activities. The cytotoxicity of the isolated fractions was studied on L929 cell lines. Following which, the screened fraction 2 was allowed for structure elucidation using gas chromatography-mass spectrometry, one-dimensional, two-dimensional nuclear magnetic resonance spectroscopy, ultraviolet, and Fourier-transform infrared analysis. The binding energies of the isolated fraction 2 with glycolytic enzymes were calculated by molecular docking studies using AutoDock Vina. The isolated fraction 2 identified as 2,4,6-triphenylaniline, showed no significant difference in α-amylase inhibition rates and a significant difference of 10% in α-glucosidase inhibition rates than that of the standard drug acarbose. Further, the cytotoxicity assay of the isolated fraction 2 resulted in a cell viability of 73.96%. Supportingly, in silico studies showed 2,4,6-triphenylaniline to produce a stronger binding affinity toward the glycolytic enzyme targets. The compound 2,4,6-triphenylaniline isolated from A. longipes strain VITN14G exhibited satisfactory antidiabetic activity for type 2 diabetes in vitro, which will further be confirmed by in vivo studies. Successful outcome of the study will result in a natural substitute for existing synthetic antidiabetic drugs.     

Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC₅₀ values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC₅₀ > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.     

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48–50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.