Inhibition of ChoK is an efficient antitumor strategy for Harvey-, Kirsten-, and N-ras-transformed cells

An increasing amount of evidence suggests that elevated PCho levels are related to the transforming properties of the H-Ras oncoprotein. Based on these observations, we have designed an antitumor strategy using choline kinase, the enzyme responsible of PCho production, as a novel target for drug discovery. However, little relationship between this lipid-related pathway and the other two Ras members, N- and K-ras, has been established. Since N- and K-ras are the most frequently mutated ras genes in human tumors, we have analyzed the PC-PLD/ChoK pathway and the sensitivity to ChoK inhibition of all three ras-transformed cells. Here we demonstrate that transformation by the three Ras oncoproteins results in increased levels of PCho to a similar extent, resulting from a similar constitutive increase of ChoK activity. As well, sensitivity to choline kinase inhibitors as antiproliferative drugs is similar in cell lines transformed by each of the three ras oncogenes, being in all cases higher than parental, nontransformed cells. In addition, H, K and N-ras-induced alterations in PC metabolism is discussed. These results indicate that ChoK can be used as a general target for anticancer drug design against Ras-dependent tumorigenesis.     

Periodic maternal deprivation and lesion of anterodorsal thalami nuclei induce alteration on hypophyso adrenal system activity in adult rats

The hypothalamic-pituitary-adrenal (HPA) axis is normally regulated by extrahypothalamic limbic structures, among these, the anterodorsal thalami nuclei (ADTN), which exert an inhibitory influence on HPA, in basal and acute stress conditions in rats. In the present work we have investigated whether neonatal maternal deprivation (MD) produces long-term changes in the ADTN regulation of HPA activity. Maternal deprivation, in female rats, for 4.5 hs daily, during the first 3 weeks of life, produced at 3 months old, a significant decrease in plasma ACTH concentration (p<0.001) and an increase in plasma corticosterone (C) (p<0.001), compared to control non-deprived rats (NMD). Also MD showed higher plasma epinephrine (E) and norepinephrine (NE) levels than NMD rats. The increase of NE (66.6% p<0.001) was higher than that observed in E (19%). After 30 days of ADTN lesion, plasma ACTH values were higher than in sham lesioned rats, in both NMD and MD animals. ACTH response was greater in MD rats. Plasma C, in NMD, was higher, whereas in MD lesioned animals, it was significantly lower than in sham lesioned. In MD rats, lesion produced a significant increase in plasma E and NE (p<0.001), and again, NE increase was higher than E increase. The more accentuated increase of NE than E, suggests sympathetic nervous system hyperactivity. In summary, neonatal maternal deprivation induces long-term alterations on HPA axis sensitivity and medullo adrenal secretion; enhanced sympathetic nervous system activity and, therefore affected the ADTN inhibitory influence on ACTH and adrenal glands secretion.     

Methyl 5-O-benzoyl-2,3-oxazole-D-ribofuranoside: a useful intermediate for the synthesis of conformationally restrained nucleosides

The synthesis of methyl 5-O-benzoyl-2,3-oxazole-D-ribofuranoside, a tetrahydrofuro [3,4-d]oxazole is described. The key step involves the reaction of methyl 3-amino-3-deoxy-5-O-benzoyl-D-ribofuranoside with N,N-dimethylformamide dimethyl acetal with cyclisation to the 2,3-oxazole via a prototropic rearrangement-elimination reaction.     

Molecular association of lectin and beta-glucosidase in corn coleoptile

Corn coleoptile lectin is present with beta-glucosidase (EC. 3.2.1.2.1) in a single tightly bound molecular association complex (88.7 kDa). SDS-PAGE of the molecular complex dissociates into two main components. Of these, at a concentration of 75%, the corn coleoptile beta-glucosidase (60 kDa) is identified by enzymatic activity, with two 16-amino acid tryptic peptides displaying close homology with the primary structure of the enzyme. In separate experiments, we isolated homogenous monomeric enzyme of corn coleoptile. This allowed us to conclude that lectin properties like erythrocyte agglutination, found in the (88.7 kDa) molecular complex, is not due to the beta-glucosidase bound in it. Another protein (30 kDa) dissociated from the same SDS-PAGE gels rendered several tryptic peptides, including a 20-amino acid sequence V(L)GP(Q)W(A)GGSGGSPVDITAEPQR closely homologous to the putative beta-glucosidase aggregating factor (BGAF) precursor described recently. Tryptic peptide SAFTE(A)WN(V)ELK(V) was also present in the BGAF precursor. KFHEQR peptide was not present in BGAF precursor or any other protein sequence examined. Tryptic peptide TYGPFGA showed good homology with the BGAF precursor protein, FEGLYLFHTPLGSGAN peptide displayed identity with the BGAF precursor sequence. Thus, the 30 kDa protein does not appear to be identical to BGAF, but is rather a similar molecule which could be endowed with the lectin properties of the 88.7 kDa molecular complex.     

Glitazones differentially regulate primary astrocyte and glioma cell survival. Involvement of reactive oxygen species and peroxisome proliferator-activated receptor-gamma

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.     

Ependymal Cell Differentiation and GLUT1 Expression is a Synchronous Process in the Ventricular Wall

Ependymal cells appear to be totally differentiated during the first 3 weeks in the mouse brain. Early during postnatal development ependymal cells differentiate and undergo metabolic activation, which is accompanied by increased glucose uptake. We propose that ependymal cells induce an overexpression of the glucose transporter, GLUT1, during the first 2 weeks after delivery in order to maintain the early metabolic activation. During the first postnatal day, GLUT1 is strongly induced in the upper region of the third ventricle and in the ventral area of the rostral cerebral aqueduct. During the next 4 days, GLUT1 is expressed in all differentiated ependymal cells of the third ventricle and in hypothalamic tanycytes. At the end of the first week, ependymal cell differentiation and GLUT1 overexpression is concentrated in the latero-ventral area of the aqueduct. We propose that ependymal cell differentiation and GLUT1 overexpression is a synchronous process in the ventricular wall.     

ERECTA receptor-like kinase and heterotrimeric G protein from Arabidopsis are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina

Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina is complex and depends on the ethylene, jasmonic acid and salicylic acid signaling pathways. A quantitative trait loci (QTL) analysis of resistance to this fungus was performed using two populations of recombinant inbred lines. Three loci QRP1-QRP3 (for Quantitative Resistance to Plectosphaerella) were identified and mapped on chromosome 2 (QRP1 and QRP2) and 5 (QRP3). QRP1, the locus showing the strongest effect, was found to correspond to the ERECTA (ER) gene that encodes a receptor-like-kinase (RLK), which has been previously implicated in plant development, and resistance to the bacterium Ralstonia solanacearum. The leucine-rich repeat and the kinase domains of ERECTA were specifically required for resistance to P. cucumerina, as er mutant alleles impaired in any of these domains showed enhanced susceptibility to this fungus, but not to other virulent pathogens. The involvement of the ER-signaling pathway in resistance to P. cucumerina was supported by the fact that three mutants defective in this pathway, elk2, elk5 and elk4 (agb1-1), which encodes the beta-subunit of Arabidopsis heterotrimeric G protein, were also impaired in their resistance to this fungus. The putative function of the Arabidopsis heterotrimeric G protein in resistance to P. cucumerina suggested by the enhanced susceptibility of agb1-1 was corroborated by the demonstration that a null allele (gpa1-4) of the G protein alpha-subunit showed enhanced resistance to this pathogen. Deposition of beta-1,3-glucan callose at infection sites was specifically impaired in er-1 and agb1-1 mutants upon P. cucumerina inoculation. Taken together, these data suggest a putative function of ERECTA and heterotrimeric G protein in P. cucumerina perception.     

Patterns of vegetative growth and gene flow in Rhizopogon vinicolor and R. vesiculosus (Boletales, Basidiomycota)

We have collected sporocarps and tuberculate ectomycorrhizae of both Rhizopogon vinicolor and Rhizopogon vesiculosus from three 50 x 100 m plots located at Mary's Peak in the Oregon Coast Range (USA); linear map distances between plots ranged from c. 1 km to c. 5.5 km. Six and seven previously developed microsatellite markers were used to map the approximate size and distribution of R. vinicolor and R. vesiculosus genets, respectively. Genetic structure within plots was analysed using spatial autocorrelation analyses. No significant clustering of similar genotypes was detected in either species when redundant samples from the same genets were culled from the data sets. In contrast, strong clustering was detected in R. vesiculosus when all samples were analysed, but not in R. vinicolor. These results demonstrate that isolation by distance does not occur in either species at the intraplot sampling scale and that clonal propagation (vegetative growth) is significantly more prevalent in R. vesiculosus than in R. vinicolor. Significant genetic differentiation was detected between some of the plots and appeared greater in the more clonal species R. vesiculosus with Phi(ST) values ranging from 0.010 to 0.078*** than in R. vinicolor with Phi(ST) values ranging from -0.002 to 0.022** (*P < 0.05, **P < 0.01, ***P < 0.001). When tested against the null hypothesis of no relationship between individuals, parentage analysis detected seven likely parent/offspring pairs in R. vinicolor and four in R. vesiculosus (alpha = 0.001). Of these 11 possible parent/offspring pairs, only two R. vinicolor pairs were still supported as parent/offspring when tested against the alternative hypothesis of being full siblings (alpha = 0.05). In the latter two cases, parent and offspring were located at approximately 45 m and 28 m from each other. Challenges to parentage analysis in ectomycorrhizal fungi are discussed.