Synergistic effect of coronatine and sorbitol on artemisinin production in cell suspension culture of Artemisia annua L. cv. Anamed

Plant Cell, Tissue and Organ Culture (PCTOC) - Artemisinin is an efficient anti-malarial drug and it possesses biological activity against a wide range of cancers. The combined application of two...     

Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin

G17 has growth promoting and antiapoptotic effects on the AR4-2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4-2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4-2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4-2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4-2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4-2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.     

Tetrathiomolybdate therapy protects against concanavalin a and carbon tetrachloride hepatic damage in mice

Tetrathiomolybdate, an anticopper drug, has been shown to protect mice against pulmonary fibrosis from bleomycin. Our hypothesis is that it does so by inhibiting fibrosis-inducing cytokines. Indeed, we have good evidence, not yet published, that tetrathiomolybdate inhibits pulmonary levels of transforming growth factor-beta and tumor necrosis factor-alpha expression in these bleomycin experiments. Herein, we evaluate tetrathiomolybdate's effectiveness in mitigating hepatitis and fibrosis in mice from the hepatotoxins, concanavalin A and carbon tetrachloride, and its inhibition of cytokines as a possible mechanism. In short-term experiments, concanavalin A elevated serum amino leucine transferase levels several fold, and tetrathiomolybdate completely prevented this increase. In additional experiments, tetrathiomolybdate therapy reversed the elevated serum transaminase levels despite continued concanavalin A injections, with nearly significant serum interleukin-1beta inhibition. Concanavalin A given for 12 weeks produced mild fibrosis, whereas concomitant tetrathiomolybdate treatment resulted in normal histology. Carbon tetrachloride given for 12 weeks resulted in very high serum amino leucine transferase levels, high serum transforming growth factor-beta levels, cirrhosis as seen histologically, and increase in liver hydroxyproline, a measure of fibrosis. Concomitant tetrathiomolybdate partially and significantly protected against increases in amino leucine transferase and transforming growth factor-beta, fully protected against the increase in hydroxyproline, and resulted in normal histology. In conclusion, tetrathiomolybdate protects against the hepatitis and fibrosis produced by these hepatotoxins, probably by inhibiting the excessive increase in inflammatory and fibrotic cytokines.     

Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.     

Application of an antibody biochip for p53 detection and cancer diagnosis

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immuno-biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein-labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.     

Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.     

A product inhibition study on adenosine deaminase by spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the inosine product on the activity of adenosine deaminase in a 50 mM sodium phosphate buffer, pH 7.5, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for inosine as a product of the enzymatic reaction. A graphical-fitting method was used for determination of the binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 140 microM by the microcalorimetry method, which agrees well with the value of 143 microM for the inhibition constant that was obtained from the spectroscopy method     

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-kappaB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IkappaB, which expresses a repressor of NF-kappaB. Addition of SB-203580 with Ad.dom.neg.IkappaB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE(2) release by enzyme-linked immunoassay. Carbachol induced PGE(2) release. Ad.dom.neg.IkappaB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE(2) production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE(2) release without affecting basal PGE(2) production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE(2) release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca(2+), PKC, p38 kinase, and activation of NF-kappaB. The functional significance of these effects seems to be stimulation of PGE(2) release.     

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens