Identification of a novel antiapoptotic human protein kinase C delta isoform, PKCdeltaVIII in NT2 cells

Protein kinase C (PKC) delta plays an important role in cellular proliferation and apoptosis where it is involved in the caspase-3 mediated apoptotic pathway. Cleavage of PKCdeltaI by caspase-3 releases a catalytically active C-terminal fragment that is sufficient to induce apoptosis. In this paper, we identified a novel human PKCdelta isozyme, PKCdeltaVIII (Genbank accession number DQ516383) in human teratocarcinoma (NT2) cells that differentiate into hNT neurons upon retinoic acid (RA) treatment. Expression of PKCdeltaVIII was confirmed by real-time RT-PCR analysis, and we observed that after an initial peak at 24 h following RA treatment, its expression gradually declined with prolonged RA treatment. PKCdeltaVIII is generated via the utilization of an alternative 5' splice site, and this results in an insertion of 31 amino acids in the caspase-3 recognition sequence DMQD. The function of PKCdeltaVIII was examined by subcloning it into an expression vector and raising an antibody specific to PKCdeltaVIII. Using in vivo and in vitro assays, we demonstrated that PKCdeltaVIII is resistant to caspase-3 cleavage. Next, we sought to determine the role of PKCdeltaVIII in apoptosis in NT2 cells. Overexpression of PKCdeltaVIII and knockdown using PKCdeltaVIII siRNA suggest an antiapoptotic function for the PKCdeltaVIII isozyme. We demonstrate that antisense oligonucleotides (ASO) directed toward the 5' splice site I promote the expression of the PKCdeltaVIII isozyme. Our results indicated that ASO mediated PKCdeltaVIII expression rescued NT2 cells from etoposide-induced apoptosis. We conclude that the novel human PKCdeltaVIII splice variant functions as an antiapoptotic protein in NT2 cells.     

Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major

10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP⁺-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF ( K _i 105 nM), as was Leishmania growth (EC₅₀ 1.1 μM). Previous studies showed null ftl ^- mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1 ^- null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania , based on segregational loss of episomal complementing genes rather than transfection; analysis of ∼1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1 ^- null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major , and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.     

Relationship between multidrug resistance,hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells.

We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines. We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers. Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.     

Are all data types and connectivity models created equal? Validating common connectivity approaches with dispersal data

Aim

There is enormous interest in applying connectivity modelling to resistance surfaces for identifying corridors for conservation action. However, the multiple analytical approaches used to estimate resistance surfaces and predict connectivity across resistance surfaces have not been rigorously compared, and it is unclear what methods provide the best inferences about population connectivity. Using a large empirical data set on puma (Puma concolor), we are the first to compare several of the most common approaches for estimating resistance and modelling connectivity and validate them with dispersal data.

Location

Southern California, USA.

Methods

We estimate resistance using presence‐only data, GPS telemetry data from puma home ranges and genetic data using a variety of analytical methods. We model connectivity with cost distance and circuit theory algorithms. We then measure the ability of each data type and connectivity algorithm to capture GPS telemetry points of dispersing pumas.

Results

We found that resource selection functions based on GPS telemetry points and paths outperformed species distribution models when applied using cost distance connectivity algorithms. Point and path selection functions were not statistically different in their performance, but point selection functions were more sensitive to the transformation used to convert relative probability of use to resistance. Point and path selection functions and landscape genetics outperformed other methods when applied with cost distance; no methods outperformed one another with circuit theory.

Main conclusions

We conclude that path or point selection functions, or landscape genetic models, should be used to estimate landscape resistance for wildlife. In cases where resource limitations prohibit the collection of GPS collar or genetic data, our results suggest that species distribution models, while weaker, may still be sufficient for resistance estimation. We recommend the use of cost distance‐based approaches, such as least‐cost corridors and resistant kernels, for estimating connectivity and identifying functional corridors for terrestrial wildlife.