Regulation of telomere length by fatty acid elongase 3 in yeast. Involvement of inositol phosphate metabolism and Ku70/80 function

In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Delta cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Delta cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Delta cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Delta cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku.     

Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer

Myofibroblasts, also known as activated fibroblasts, constitute an important niche for tumor development through the promotion of angiogenesis. However, the mechanism of stromal fibroblast activation in tumor tissues has not been fully understood. A gastric cancer mouse model (Gan mice) was recently constructed by simultaneous activation of prostaglandin (PG) E2 and Wnt signaling in the gastric mucosa. Because both the PGE2 and Wnt pathways play a role in human gastric tumorigenesis, the Gan mouse model therefore recapitulates the molecular etiology of human gastric cancer. Microvessel density increased significantly in Gan mouse tumors. Moreover, the expression of vascular endothelial growth factor A (VEGFA) was predominantly induced in the stromal cells of gastric tumors. Immunohistochemistry suggested that VEGFA-expressing cells in the stroma were alpha-smooth muscle actin-positive myofibroblasts. Bone marrow transplantation experiments indicated that a subset of gastric myofibroblasts is derived from bone marrow. Importantly, the alpha-smooth muscle actin index in cultured fibroblasts increased significantly when stimulated with the conditioned medium of Gan mouse tumor cells, indicating that gastric tumor cells activate stromal fibroblasts. Furthermore, conditioned medium of Gan mouse tumor cells induced VEGFA expression both in embryonic and gastric fibroblasts, which further accelerated the tube formation of human umbilical vein endothelial cells in vitro. Notably, stimulation of fibroblasts with PGE2 and/or Wnt1 did not induce VEGFA expression, thus suggesting that factors secondarily induced by PGE2 and Wnt signaling in the tumor cells are responsible for activation of stromal fibroblasts. Such tumor cell-derived factors may therefore be an effective target for chemoprevention against gastric cancer.     

Human calpain 7/PalBH associates with a subset of ESCRT-III-related proteins in its N-terminal region and partly localizes to endocytic membrane compartments

Calpain 7 (also known as PalBH) is a mammalian homologue of the Aspergillus, atypical calpain PalB. Knowledge of the biochemical properties of calpain 7 is limited and its function is not yet known. In this study, we investigated the interactions of calpain 7 with all 11 ESCRT-III-related proteins, named charged multivesicular body proteins (CHMPs), and the subcellular localization of calpain 7. Pulldown assays using stable HEK293T transfectants of Strep-tagged calpain 7 revealed interactions of calpain 7 with a subset of FLAG-tagged CHMPs, among which CHMP1B was selected for further analyses. The N-terminal region containing a tandem repeat of MIT domains of calpain 7 was found to be necessary and sufficient for interaction with CHMP1B. Direct interaction was confirmed by a pulldown assay using recombinant proteins. Fluorescence microscopic analysis using HeLa cells revealed that overexpression of GFP-fused CHMPs or a dominant-negative construct of SKD1/Vps4B caused accumulation of epitope-tagged calpain 7 in a punctate pattern in the perinuclear area. Subcellular fractionation revealed that the most of endogenous calpain 7 is present in the cytosol but a small portion is present in particulate fractions. Punctate fluorescence signals of monomeric GFP-fused calpain 7 partly merged with those of endocytosed tetramethylrhodamine-labelled EGF. These results suggest that calpain 7 plays roles in the endosomal pathway by interacting with a subset of ESCRT-III-related proteins.     

Charge stabilization in reaction center protein investigated by optical heterodyne detected transient grating spectroscopy

Photosynthetic reaction center (RC) pigment protein complex converts the free energy of light into chemical potential of charge pairs with extremely high efficiency. A transient phase in the absorption spectrum in the sub-millisecond time scale is expected to be especially important to examine the conformational gating model of the Q (A) (-) Q(B) to Q(A)Q (B) (-) (here Q(A )and Q(B) are the primary and secondary quinone type electron acceptors, respectively) electron transport. Essential kinetic components at few tens of microseconds scale and at around 200 mus have been suggested. We investigated the conformation change of RCs using heterodyne detection of the laser-induced transient grating method. An about 25 mus dynamics was observed, which coincides with the one described by the conformational gating model and possibly related to the nonadiabatic intrinsic Q (A) (-) Q(B) to Q(A)Q (B) (-) electron transport. The relative intensity of this component decreased with increasing quinone concentration indicating an initial (P(+)Q (A) (-) )(1) or a relaxed (P(+)Q (A) (-) )(2 )conformational substate. We did not find the decay component at few hundreds of microseconds time scale indicating that there is no large displacement in the RC structure if Q(B) is present. The diffusion coefficient of the RC/LDAO detergent micelles calculated from the kinetic component was D = 3.8 x 10(-11 )m(2)/s that agrees fairly well with the number estimated from the Einstein-Stokes relationship, and relates to a hydrodynamic diameter of 11.4 nm of the RC in LDAO micellar solution.     

Regulation of radiation-induced protein kinase Cdelta activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines

Protein kinase Cdelta (PKCdelta) has an important role in radiation-induced apoptosis. The expression and function of PKCdelta in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCdelta-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCdelta activation correlated with the degradation of PKCdelta, indicating that PKCdelta activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCdelta activation was lower than that in radiosensitive 3SBH5. Cytosol PKCdelta levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCdelta levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCdelta activation compared to that of 3SBH5. On the other hand, Atm(-/-) mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm(-/-) cells, had decreased PKCdelta levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCdelta degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCdelta activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species

The domestication and improvement of fruit-bearing crops resulted in a large diversity of fruit form. To facilitate consistent terminology pertaining to shape, a controlled vocabulary focusing specifically on fruit shape traits was developed. Mathematical equations were established for the attributes so that objective, quantitative measurements of fruit shape could be conducted. The controlled vocabulary and equations were integrated into a newly developed software application, Tomato Analyzer, which conducts semiautomatic phenotypic measurements. To demonstrate the utility of Tomato Analyzer in the detection of shape variation, fruit from two F2 populations of tomato (Solanum spp.) were analyzed. Principal components analysis was used to identify the traits that best described shape variation within as well as between the two populations. The three principal components were analyzed as traits, and several significant quantitative trait loci (QTL) were identified in both populations. The usefulness and flexibility of the software was further demonstrated by analyzing the distal fruit end angle of fruit at various user-defined settings. Results of the QTL analyses indicated that significance levels of detected QTL were greatly improved by selecting the setting that maximized phenotypic variation in a given population. Tomato Analyzer was also applied to conduct phenotypic analyses of fruit from several other species, demonstrating that many of the algorithms developed for tomato could be readily applied to other plants. The controlled vocabulary, algorithms, and software application presented herein will provide plant scientists with novel tools to consistently, accurately, and efficiently describe two-dimensional fruit shapes.