Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions.

In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37 degrees C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.     

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa^®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.     

Detection of Terminal Mismatches on DNA Duplexes in Homogeneous Assays or with Immobilized Probes

We recently reported the design of new fluorescent oligo-2′-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.     

Mitochondrial protein import in plants – Signals,Sorting, Targeting,Processing and Regulation

Plant Molecular Biology - Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and...     

Characterization and risk assessment of the invasive papaya mealybug,Paracoccus marginatus,in Kenya under changing climate

The present study was conducted to characterize the newly invasive papaya mealybug Paracoccus marginatus Williams and Granara de Willink in Kenya using molecular techniques and to establish the potential risk of spread of the pest. Although abundant literature of P. marginatus outbreaks exists in other parts of the world, studies from Africa are rare. Our results revealed significant similarity between Kenyan samples with GenBank accession number KP692333.1 of P. marginatus. Phylogenetic analyses generated a tree that was paraphyletic with two clusters showing low genetic distance values for the P. marginatus sequences, which diverged from that of Planococcus citri. Our models displayed an optimal performance with mean area under the curve value of 0.82 and 0.98 for Genetic Algorithm for Rule-Set Production (GARP) and maximum entropy modelling (MaxEnt), respectively. Isothermality was the most influential variable in determining the potential distribution of P. marginatus with a 69% contribution to the models. Other variables included temperature mean diurnal range temperature seasonality, temperature annual range and annual precipitation in decreasing order of contribution. Current prediction by both models exceeded the existing range of P. marginatus, exacerbating the potential threat of the pest. GARP was more conservative in predicting suitable areas for P. marginatus, while MaxEnt showed further expansion by the year 2050. Our findings provide important information to guide biosecurity agencies in decision-making to prevent the spread and enhance control efforts of P. marginatus.     

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization

Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.     

Predation selectively culls medium-sized species from island mammal faunas

Globally, elevated extinction risk in mammals is strongly associated with large body size. However, in regions where introduced predators exert strong top-down pressure on mammal populations, the selectivity of extinctions may be skewed towards species of intermediate body size, leading to a hump-shaped relationship between size and extinction risk. The existence of this kind of extinction pattern, and its link to predation, has been contentious and difficult to demonstrate. Here, we test the hypothesis of a hump-shaped body size–extinction relationship, using a database of 927 island mammal populations. We show that the size-selectivity of extinctions on many islands has exceeded that expected under null models. On islands with introduced predators, extinctions are biased towards intermediate body sizes, but this bias does not occur on islands without predators. Hence, on islands with a large-bodied mammal fauna, predators are selectively culling species from the lower end of the size distribution, and on islands with a small-bodied fauna they are culling species from the upper end. These findings suggest that it will be difficult to use predictable generalizations about extinction patterns, such as a positive body size–extinction risk association, to anticipate future species declines and plan conservation strategies accordingly.     

Life cycle analysis on the design of induction motors

Purpose  

Herein is reported an application of life cycle analysis (LCA), using the Methodology for the Ecodesign of Energy Using Products (MEEUP), in order to assess the influence of some design parameters in the environmental impact of three-phase induction motors. A motor design procedure to minimize the total environmental impact, based on data obtained from commercial motors, is presented. This procedure is specially intended for the low power range due to the greater potential for energy savings in motors having an output power of 0.75 to 4 kW.