FTDP-17 missense mutations site-specifically inhibit as well as promote dephosphorylation of microtubule-associated protein tau by protein phosphatases of HEK-293 cell extract

FTDP-17 missense tau mutations: G272V, P301L, V337M and R406W promote tau phosphorylation in human and transgenic mice brains by interfering with the tau phosphorylation/dephosphorylation balance. The effect of FTDP-17 mutations on tau phosphorylation by different kinases has been studied previously. However, it is not known how various FTDP-17 mutations affect tau dephosphorylation by phosphoprotein phosphatases. In this study we have observed that when transfected into HEK-293 cells, tau is phosphorylated on various sites that are also phosphorylated in diseased human brains. When transfected cells are lysed and incubated, endogenously phosphorylated tau is dephosphorylated by cellular protein phosphatase 1 (PP1), phosphatase 2A (PP2A) and phosphatase 2B (PP2B), which are also present in the lysate. By using this assay and specific inhibitors of PP1, PP2A and PP2B, we have observed that the G272V mutation promotes tau dephosphorylation by PP2A at Ser(396/404), Ser(235), Thr(231), Ser(202/205) and Ser(214) and by PP2B at Ser(214) but inhibits dephosphorylation by PP2B at Ser(396/404). The P301L mutation promotes tau dephosphorylation at Thr(231) by PP1 and at Ser(396/404), Thr(231), Ser(235) and Ser(202/205) by PP2A but inhibits dephosphorylation at Ser(214) by PP2B. The V337M mutation promotes tau dephosphorylation at Ser(235), Thr(231) and Ser(202/205) by PP2A and at Ser(202/205) by PP2B whereas the R406W mutation promotes tau dephosphorylation at Ser(396/404) by PP1, PP2A and PP2B but inhibits dephosphorylation at Ser(202/205) and Ser(235) by PP1 and PP2A, respectively. Our results indicate that each FTDP-17 tau mutation not only site-specifically inhibits tau dephosphorylation on some sites but also promotes dephosphorylation by phosphatases on other sites.     

Selective isolation,evaluation and characterization of antagonistic actinomycetes against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The baiting bag method was found to be useful for isolating antagonistic actinomycetes from terrestrial habitat. Out of total 110 actinomycetes isolated from rhizospheric and non-rhizospheric soil of Indo Gangetic Plains (IGP) of India, 9 isolates exhibited aggressive antagonism against Rhizoctonia solani, screened through dual culture, well diffusion and sealed plate technique. Maximum growth inhibition was recorded up to 50% under well diffusion (S. toxytricini vh22) and 52.6% in a direct confrontation (Actinomycetales bacterium vh41). Whereas maximum disease suppression (53.33%) under green house condition was achieved on seedling treated with S. tricolor vh85. Scanning electron microscopy of antagonists and pathogen interaction exhibited pore formation and hyphal degradation of test pathogen. Physiological and molecular characterization of selected isolates showed wide diversity and uncommon species has been encountered through the selective isolation technique.     

A Phosphate Transporter from the Root Endophytic Fungus Piriformospora indica Plays a Role in Phosphate Transport to the Host Plant

Because pure cultures and a stable transformation system are not available for arbuscular mycorrhizal fungi, the role of their phosphate transporters for the symbiotic interaction with the plant up till now could not be studied. Here we report the cloning and the functional analysis of a gene encoding a phosphate transporter (PiPT) from the root endophytic fungus Piriformospora indica, which can be grown axenically. The PiPT polypeptide belongs to the major facilitator superfamily. Homology modeling reveals that PiPT exhibits twelve transmembrane helices divided into two halves connected by a large hydrophilic loop in the middle. The function of the protein encoded by PiPT was confirmed by complementation of a yeast phosphate transporter mutant. The kinetic analysis of PiPT (K_m 25 μm) reveals that it belongs to the high affinity phosphate transporter family (Pht1). Expression of PiPT was localized to the external hyphae of P. indica colonized with maize plant root, which suggests that external hyphae are the initial site of phosphate uptake from the soil. To understand the physiological role of PiPT, knockdown transformants of the gene were prepared using electroporation and RNA interference. Knockdown transformants transported a significantly lower amount of phosphate to the host plant than wild-type P. indica. Higher amounts of phosphate were found in plants colonized with wild-type P. indica than that of non-colonized and plants colonized with knockdown PiPT P. indica. These observations suggest that PiPT is actively involved in the phosphate transportation and, in turn, P. indica helps improve the nutritional status of the host plant.     

Differential partitioning of Gαi1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells

Background

Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results

Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Gαi1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the α-subunit of the G protein have been identified and the Gαi (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Gαi1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Gαi1 in these cells. Moreover, Gαi1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Gαi1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion

Based on the opposing effects of taxol and the Gαi1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Gαi1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.     

Regional admixture mapping and structured association testing: conceptual unification and an extensible general linear model

Individual genetic admixture estimates, determined both across the genome and at specific genomic regions, have been proposed for use in identifying specific genomic regions harboring loci influencing phenotypes in regional admixture mapping (RAM). Estimates of individual ancestry can be used in structured association tests (SAT) to reduce confounding induced by various forms of population substructure. Although presented as two distinct approaches, we provide a conceptual framework in which both RAM and SAT are special cases of a more general linear model. We clarify which variables are sufficient to condition upon in order to prevent spurious associations and also provide a simple closed form “semiparametric” method of evaluating the reliability of individual admixture estimates. An estimate of the reliability of individual admixture estimates is required to make an inherent errors-in-variables problem tractable. Casting RAM and SAT methods as a general linear model offers enormous flexibility enabling application to a rich set of phenotypes, populations, covariates, and situations, including interaction terms and multilocus models. This approach should allow far wider use of RAM and SAT, often using standard software, in addressing admixture as either a confounder of association studies or a tool for finding loci influencing complex phenotypes in species as diverse as plants, humans, and nonhuman animals.     

Comparative study of fine needle aspiration and fine needle capillary sampling of thyroid lesions

OBJECTIVE: To investigate whether fine needle capillary (FNC) sampling gives quantitatively and qualitatively superior cytologic material as compared to the conventional technique of fine needle aspiration (FNA) when performed by a single aspirator. STUDY DESIGN: Cross-sectional diagnostic test evaluation study. FNA and FNC were performed by a single operator on 200 diffuse and nodular thyroid lesions. RESULTS: A statistically significant difference in favor of FNC was observed for the parameter amount of cellular material. For the rest of the parameters--background blood or clot, degree of cellular degeneration, degree of cellular trauma and retention of architecture--the average score favored FNC but was not statistically significant--i.e., smears prepared from FNC displayed cellular material that was more concentrated, less damaged and less likely to be obscured by blood. CONCLUSION: Although FNC sampling was diagnostic in a greater number of cases than FNA sampling, this study did not prove a clear superiority of FNC over FNA. Until greater experience shows clear sampling superiority of FNC alone, rather than performing only FNA in diffuse or nodular thyroid lesions, incorporating FNC into the second puncture will definitely improve the quality and quantity of material at the patient's first visit.     

Down-regulation of the tumor suppressor gene C-terminal Src kinase: an early event during premalignant colonic epithelial hyperproliferation

Hyperproliferation of the premalignant epithelium is critical for colonic carcinogenesis; however the mechanisms remain largely unexplored. We report herein that prior to occurrence of neoplastic lesions in the azoxymethane-rat model of colon carcinogenesis; the tumor suppressor gene C-terminal Src kinase (Csk) was down-regulated with a concomitant increase in Src activity. Furthermore, pharmacological or genetic (RNA interference) inhibition of Csk resulted in increased proliferation in colon cancer cell lines through the mitogen-activated protein kinase dependent pathway. Thus, we demonstrate, for the first time, that Csk suppression is an important early event in colorectal cancer pathogenesis.