Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.     

CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease

CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.     

Effects of cadmium on the co-ordination of nitrogen and carbon metabolism in bean seedlings

The effect of cadmium (Cd) was investigated on the in vitro activities of leaf and root enzymes involved in carbon (C) and nitrogen (N) metabolism of bean (Phaseolus vulgaris L. cv. Morgane). Cd induced a high increase in maximal extractable activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). Cd promoted ammonium accumulation in leaves and roots, and a tight correlation was observed between ammonium amount and GDH activity. Changes in GDH activity appear to be mediated by the increase in ammonium levels by Cd treatment. Cd stress also enhanced the activities of phosphoenolypyruvate carboxylase (PEPC, EC 4.1.1.31) and NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH, EC 1.1.1.42) in leaves while they were inhibited in roots. Immuno-titration, the PEPC sensitivity to malate and PEPC response to pH indicated that the increase in PEPC activity by Cd was due to de novo synthesis of the enzyme polypeptide and also modification of the phosphorylation state of the enzyme. Cd may have modified, via a modulation of PEPC activity, the C flow towards the amino acid biosynthesis. In leaves, Cd treatments markedly modified specific amino acid contents. Glutamate and proline significantly accumulated compared to those of the control plants. This study suggests that Cd stress is a part of the syndrome of metal toxicity, and that a readjustment of the co-ordination between N and C metabolism via the modulation of GDH, PEPC and ICDH activities avoided the accumulation of toxic levels of ammonium.     

Increased expression of epidermal growth factor receptor induces sequestration of extracellular signal-related kinases and selective attenuation of specific epidermal growth factor-mediated signal transduction pathways

Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.     

Zooplankton distribution in four ponds of different salinity: a seasonal study in the solar salterns of Sfax (Tunisia)

The seasonal distribution of metazooplankton and large-sized ciliates was studied in four ponds of different salinity in the solar salterns of Sfax (Tunisia). Total zooplankton abundance varied from 1 × 10³ to 4.7 × 10⁶ ind m^–3. Salinity had a negative effect on the abundance of copepods and rotifers which were absent in the pond with the highest salt concentration (180) in which the number of taxa was low and Artemia or the ciliate Fabrea largely dominated the zooplankton community. Temperature and the presence of Dunaliella salina as prey appeared as key factors in controlling the abundance of Artemia, while organic detritus appeared as important in the diet of Fabrea. Change in zooplankton species composition along the hypersaline gradient (40–90) was primarily related to salinity. However, our data suggest the importance of both the abundance and composition of food in the spatial and temporal variations of some zooplankton species.     

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.     

Biosynthesis of fattiviracin FV-8, an antiviral agent

Streptomyces microflavus strain No. 2445 produces many derivatives of fattiviracin antibiotics. The major product of these derivatives is fattiviracin FV-8, which consists of four glucose and two trihydroxy fatty acid residues. We found that this strain has the ability to convert several sugars in the culture medium to glucose, and the glucose added to the medium is directly incorporated into the FV-8 molecule. Two trihydroxy fatty acid residues in the FV-8 molecule are derived from acetic acid, and production of FV-8 is inhibited by the addition of cerulenin, which is an inhibitor of fatty acid biosynthesis.     

The development of a potential single photon emission computed tomography (SPECT) imaging agent for the corticotropin-releasing hormone receptor type

A high-affinity radioligand for CRHR1 has been prepared that can serve as a template for the development of SPECT imaging agents. The 5-chloro-N-cyclopropylmethyl-N-(2,6-dichloro-4-iodophenyl)-2-methyl-N-propylpyrimidine-4,6-diamine (6b, Ki = 14 nM), and the corresponding 4-bromophenyl analogue (6a, Ki = 21 nM), were synthesized in four steps from compound 3.     

Quince (Cydonia oblonga Miller) peel polyphenols modulate LPS-induced inflammation in human THP-1-derived macrophages through NF-κB, p38MAPK and Akt inhibition

Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis and cancer. A wide range of anti-inflammatory chemicals have been used to treat such diseases while presenting high toxicity and numerous side effects. Here, we report the anti-inflammatory effect of a non-toxic, cost-effective natural agent, polyphenolic extract from the Tunisian quince Cydonia oblonga Miller. Lipopolysaccharide (LPS) treatment of human THP-1-derived macrophages induced the secretion of high levels of the pro-inflammatory cytokine TNF-α and the chemokine IL-8, which was inhibited by quince peel polyphenolic extract in a dose-dependent manner. Concomitantly, quince polyphenols enhanced the level of the anti-inflammatory cytokine IL-10 secreted by LPS-treated macrophages. We further demonstrated that the unexpected increase in IL-6 secretion that occurred when quince polyphenols were associated with LPS treatment was partially responsible for the polyphenols-mediated inhibition of TNF-α secretion. Biochemical analysis showed that quince polyphenols extract inhibited the LPS-mediated activation of three major cellular pro-inflammatory effectors, nuclear factor-kappa B (NF-κB), p38MAPK and Akt. Overall, our data indicate that quince peel polyphenolic extract induces a potent anti-inflammatory effect that may prove useful for the treatment of inflammatory diseases and that a quince-rich regimen may help to prevent and improve the treatment of such diseases.     

Interaction of a bacterial monorhamnolipid secreted by Pseudomonas aeruginosa MA01 with phosphatidylcholine model membranes

This work presents a biophysical study on the interactions of a monorhamnolipid (monoRL) produced by Pseudomonas aeruginosa MA01 with model phosphatidylcholine membranes. The molecular characterization of the biological activities, including the modulation of phospholipid membranes structure, of this monoRL biosurfactant is of importance for the validation of this particular Pseudomonas aeruginosa strain as a useful biosurfactant producer. The marked amphiphilic structure of monoRL is expected to result in strong interactions with the phospholipid constituents of membrane bilayers. Incorporation of monoRL into DMPC completely abolished the pretransition, and the main gel to liquid-crystalline phase transition was progressively broadened and shifted to lower temperatures, as observed by differential scanning calorimetry. Partial phase diagrams for DPPC and DSPC indicated near-ideal behavior. However, the DMPC diagram indicated fluid phase immiscibility. X-ray diffraction showed and apparent increase in d-value for DPPC containing monoRL, which might be the result of an effective increase in the bilayer thickness, or in the thickness of the hydration layer between bilayers. FTIR indicated that interaction of monoRL with the phospholipid acyl chains did not result in a large additional disordering of the acyl chain region of the fluid bilayer. Analysis of the CO stretching band of DPPC indicated an important effect of monoRL on the interfacial region of phosphatidylcholine bilayers, which might contribute to explain some of the biological activities of this glycolipid.