Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.     

Saturated free fatty acid release and intracellular ceramide generation during apoptosis induction are closely related processes

Apoptosis induced by cells from the immune system is frequently associated with an increase in the ceramide content of target cells, due to the activation of sphingomyelinases (SMase). Some studies have also reported the release of saturated and monounsaturated free fatty acids (FFA) from apoptotic cells. However, the possible relationship between these lipid biochemistry events has not been characterized. We have analysed for the first time the release of FFA triggered by tumor necrosis factor-alpha (TNF-alpha), Fas/CD95 or the perforin/granzyme system of cytotoxic T lymphocytes (CTL) and their relationship to intracellular ceramide generation. TNF-alpha- and Fas-induced apoptosis are associated with both intracellular ceramide generation from sphingomyelin (SM) and release of palmitic-derived FFA, with similar kinetics. Intracellular SMase activation and FFA release from target cells during Fas-induced apoptosis are much more rapid and efficient if Fas-based cytotoxicity is exerted by alloantigenic CTL. In the case of perforin/granzyme-based cytotoxicity exerted by CTL, intracellular ceramide generation and FFA release from target cells seem to depend on the type of lysis induction used. Importantly, the correlation between intracellular SMase activation and the release of palmitic acid-derived FFA from target cells has been observed in all types of cytotoxicity assayed. In addition, exogenous natural ceramide induces the rapid release of the same FFA, well before any apoptotic sign is detected, and FFA release during Fas-induced apoptosis is inhibited in SM-depleted cells by chronic fumonisin-B(1) treatment. These results demonstrate a novel connection between the release of palmitic acid-derived FFA and intracellular ceramide accumulation during apoptosis induction.     

Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.     

Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin

Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically characterized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of -175 mV was calculated for wild type Grx5. The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pKa. Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.     

Effectiveness of GF-120 fruit fly bait spray applied to border area plants for control of melon flies (Diptera: Tephritidae)

In a field study in Hawaii, color-marked protein-deprived and protein-fed female melon flies, Bactrocera cucurbitae Coquillett, were released within canopies of unsprayed sorghum plants (a nonhost of melon flies) outside of a border area of unsprayed or bait-sprayed sorghum plants or open space that surrounded cucumbers, a favored host of melon flies. Application of bait spray to sorghum or sugarcane surrounding host plants of melon flies is a common practice for melon fly control in Hawaii. GF-120 Fruit Fly Bait spray proved very effective in preventing protein-deprived females from alighting on cucumbers (23% of released females were observed dead on bait-sprayed sorghum; 0% were observed alive on cucumbers), but proved less effective in suppressing protein-fed females (14% of released females were observed dead on bait-sprayed sorghum; 11% were observed alive on cucumbers). No females were found dead on unsprayed sorghum. Compared with open space surrounding cucumbers, the presence of unsprayed sorghum as surrounding border area neither significantly enhanced nor significantly inhibited the ability of either type of female with respect to finding cucumbers. Greenhouse cage assays revealed that compared with droplets of water, droplets of GF-120 Fruit Fly Bait spray were highly attractive to protein-deprived females within 1 h of bait spray application to sorghum, but lost about half of their attractiveness within 5 h and all of it within 24 h under the dry greenhouse conditions used for maintaining baited-sprayed sorghum plants in these assays. Laboratory cup assays showed that bait spray droplets remained highly toxic to protein-deprived females 24 h after application, but lost nearly half of their toxicity within 4 d under laboratory exposure and nearly all of it after approximately 8 mm of rainfall. Combined findings suggest that application of GF-120 Fruit Fly Bait spray to nonhost plants for melon fly control either be made often enough to overcome loss of attractiveness of bait spray droplets to females or that bait spray be applied to nonhost plants that are themselves attractive to the females.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.     

Optimization and validation of an analytical procedure by high-performance liquid chromatography for the quantification of peroxisomicines and isoperoxisomicines

Peroxisomicine A1 is a potential antineoplastic substance extracted from plants of the genus Karwinskia. An RP-HPLC-DAD method was developed and validated for the separation and quantification of four isomers of this compound. These isomers coelute in the preparative procedure and are present at a proportion ranging between 3 and 5% in the peroxisomicine A1 purified in the laboratory. The desirability coefficient of the method described here was enhanced 140% with respect to the previously reported method.     

Monitoring the colonization of monuments by bacteria: cultivation versus molecular methods

Building materials commonly used in wall paintings and monuments (mortar, limestone and sandstone) were inoculated with an artificial consortium composed of 14 microorganisms and incubated for 6 months at 28 degrees C. The colonization of the different materials by the consortium was investigated. Culture-independent techniques revealed the presence of a diversity of bacteria, whereas culture-dependent techniques yielded mainly spore-forming bacteria. The data suggest that plating leads to an overestimation of the number of spore-forming bacteria with respect to quiescent vegetative forms; the latter are less easily cultured, but are readily detected by culture-independent techniques.     

Genesis and evolution of the <Emphasis Type="Italic">Evx</Emphasis> and <Emphasis Type="Italic">Mox</Emphasis>genes and the extended Hox and ParaHox gene clusters

Background

Hox and ParaHox gene clusters are thought to have resulted from the duplication of a ProtoHox gene cluster early in metazoan evolution. However, the origin and evolution of the other genes belonging to the extended Hox group of homeobox-containing genes, that is, Mox and Evx, remains obscure. We constructed phylogenetic trees with mouse, amphioxus and Drosophila extended Hox and other related Antennapedia-type homeobox gene sequences and analyzed the linkage data available for such genes.

Results

We claim that neither Mox nor Evx is a Hox or ParaHox gene. We propose a scenario that reconciles phylogeny with linkage data, in which an Evx/Mox ancestor gene linked to a ProtoHox cluster was involved in a segmental tandem duplication event that generated an array of all Hox-like genes, referred to as the 'coupled' cluster. A chromosomal breakage within this cluster explains the current composition of the extended Hox cluster (with Evx, Hox and Mox genes) and the ParaHox cluster.

Conclusions

Most studies dealing with the origin and evolution of Hox and ParaHox clusters have not included the Hox-related genes Mox and Evx. Our phylogenetic analyses and the available linkage data in mammalian genomes support an evolutionary scenario in which an ancestor of Evx and Mox was linked to the ProtoHox cluster, and that a tandem duplication of a large genomic region early in metazoan evolution generated the Hox and ParaHox clusters, plus the cluster-neighbors Evx and Mox. The large 'coupled' Hox-like cluster EvxHox/MoxParaHox was subsequently broken, thus grouping the Mox and Evx genes to the Hox clusters, and isolating the ParaHox cluster.