Arabidopsis ssi2-conferred susceptibility to Botrytis cinerea is dependent on EDS5 and PAD4

Loss of a stearoyl-ACP desaturase activity in the Arabidopsis thaliana ssi2 mutant confers susceptibility to the necrotroph, Botrytis cinerea. In contrast, the ssi2 mutant exhibits enhanced resistance to Pseudomonas syringae, Peronospora parasitica, and Cucumber mosaic virus. The altered basal resistance to these pathogens in the ssi2 mutant plant is accompanied by the constitutive accumulation of elevated salicylic acid (SA) level and expression of the pathogenesis-related 1 (PR1) gene, the inability of jasmonic acid (JA) to activate expression of the defensin gene, PDF1.2, and the spontaneous death of cells. Here, we show that presence of the eds5 and pad4 mutant alleles compromises the ssi2-conferred resistance to Pseudomonas syringae pv. maculicola. In contrast, resistance to B. cinerea was restored in the ssi2 eds5 and ssi2 pad4 double-mutant plants. However, resistance to B. cinerea was not accompanied by the restoration of JA responsiveness in the ssi2 eds5 and ssi2 pad4 plants. The ssi2 eds5 and ssi2 pad4 plants retain the ssi2-conferred spontaneous cell death phenotype, suggesting that cell death is not a major factor that predisposes the ssi2 mutant to infection by B. cinerea. Furthermore, the high SA content of the ssi2 pad4 plant, combined with our previous observation that the SA-deficient ssi2 nahG plant succumbs to infection by B. cinerea, suggests that elevated SA level does not have a causal role in the ssi2-conferred susceptibility to B. cinerea. Our results suggest that interaction between an SSI2-dependent factor or factors and an EDS5- and PAD4-dependent mechanism or mechanisms modulates defense to B. cinerea.     

Open-channel block by the cytoplasmic tail of sodium channel beta4 as a mechanism for resurgent sodium current

Voltage-gated sodium channels with "resurgent" kinetics are specialized for high-frequency firing. The alpha subunits interact with a blocking protein that binds open channels upon depolarization and unbinds upon repolarization, producing resurgent sodium current. By limiting classical inactivation, the cycle of block and unblock shortens refractory periods. To characterize the blocker in Purkinje neurons, we briefly exposed inside-out patches to substrate-specific proteases. Trypsin and chymotrypsin each removed resurgent current, consistent with established roles for positively charged and hydrophobic/aromatic groups in blocking sodium channels. In Purkinje cells, the only known sodium channel-associated subunit that has a cytoplasmic sequence with several positive charges and clustered hydrophobic/aromatic residues is beta4 (KKLITFILKKTREK; beta4(154-167)). After enzymatic removal of block, beta4(154-167) fully reconstituted resurgent current, whereas scrambled or point-mutated peptides were ineffective. In CA3 pyramidal neurons, which lack beta4 and endogenous block, beta4(154-167) generated resurgent current. Thus, beta4 may be the endogenous open-channel blocker responsible for resurgent kinetics.     

A novel method of estimation of lipophilicity using distance-based topological indices: dominating role of equalized electronegativity

Attempt is made to propose yet another method of estimating lipophilicity of a heterogeneous set of 223 compounds. The method is based on the use of equalized electronegativity along with topological indices. It was observed that excellent results are obtained in multiparametric regression upon introduction of indicator parameters. The results are discussed critically on the basis various statistical parameters.     

Identification of domains of ataxia-telangiectasia mutated required for nuclear localization and chromatin association

Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin alpha1/beta1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP) x ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP x ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP x ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5-224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo.     

Distinct role of calmodulin and calmodulin-dependent protein kinase-II in lipopolysaccharide and tumor necrosis factor-alpha-mediated suppression of apoptosis and antiapoptotic c-IAP2 gene expression in human monocytic cells

Exposure of phagocytic cells to bacterial endotoxin (lipopolysaccharide; LPS) or inflammatory cytokines confers antiapoptotic survival signals; however, in the absence of the appropriate stimulus, monocytes are programmed to undergo apoptosis. Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. Herein, we demonstrate that LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF-alpha-induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF-alpha-stimulated cells through NF-kappaB activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF-alpha-mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF-alpha-induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-kappaB activation.     

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

Evaluation of protective potentials of a potentized homeopathic drug, Chelidonium majus, during azo dye induced hepatocarcinogenesis in mice

Several cytogenetical and enzymatic protocols were used to test if two microdoses of Chelidonium majus, namely Chelidonium-30 (Ch-30) and Chelidonium-200 (Ch-200), used as homeopathic drugs, showed anti-tumor activity and also favorably modulated genotoxic damages produced by an azo dye in mice at several intervals of fixation. Different sets of healthy mice were fed: (i) hepatocarcinogen, p-dimethylaminoazobenzene (p-DAB, initiator) + phenobarbital (PB, promoter), (ii) only p-DAB, (iii) only PB, and (iv) neither p-DAB nor PB (normal control). Mice fed with p-DAB + PB were divided into different sets that were also fed either Ch-30 (v) or Ch-200 (vi) or diluted alcohol (vii), the "vehicle" of the microdoses of Chelidonium. All mice of group (i), a few of group (ii) and group (vii) and none of groups (iii) and (iv) developed tumors in liver at the longer intervals of fixation. The frequencies of chromosome aberrations (CA), micronucleated erythrocytes (MN), mitotic index (MI) and sperm head abnormality (SHA) were much higher in groups (i) and (vii) mice than in groups (ii), (iii) and (iv) mice at all fixation intervals. However, in mice of both groups (v) and (vi), the frequencies of CA, MN, SHA were strikingly less than those of groups (i) and (vii), and moderately less than those of groups (ii) and (iii). Both Ch-30 and Ch-200 also modulated favourably some toxicity marker enzymes like acid and alkaline phosphatases, peroxidases, glutamate oxaloacetate and glutamate pyruvate transaminases in liver, kidney and spleen tissues of the carcinogen fed mice. The microdoses of Chelidonium having no visible ill effects of their own, may be strong candidates for use in delaying/protecting liver cancer.     

Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis <Emphasis Type="Italic">NPR1</Emphasis>

Cotton is an economically important crop worldwide that suffers severe losses due to a wide range of fungal/bacterial pathogens and nematodes. Given its susceptibility to various pathogens, it is important to obtain a broad-spectrum resistance in cotton. Resistance to several fungal and bacterial diseases has been obtained by overexpressing the Non-expressor of Pathogenesis-Related genes-1 (NPR1) in various plant species with apparently minimal or no pleiotropic effects. We examined the efficacy of this approach in cotton by constitutive expression of the Arabidopsis (Arabidopsis thaliana) NPR1 gene. The results show that NPR1-expressing lines exhibited significant resistance to Verticillium dahliae isolate TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternata. Interestingly, the transformants also showed significant resistance to reniform nematodes. Analysis of defense-related, biochemical and molecular responses suggest that when challenged with pathogens or certain systemic acquired resistance-inducing chemicals, the transgenic lines respond to a greater degree compared to the wild-type plants. Importantly, the basal activities of the defense-related genes and enzymes in uninduced transformants were no different than those in their non-transgenic counterparts. The results provide additional evidence supporting the role of NPR1 as an important part of the plant defense system and suggest a means to achieve broad-spectrum resistance to pathogens via genetic engineering.