An efficient solubilization buffer for plant proteins focused in immobilized pH gradients

The solubilization of a large array of proteins before electrophoresis itself is a very critical point for proteomic analyses. We compared the efficiency of several different solubilization buffers. From this work, we defined a very efficient solubilization buffer, including two chaotropes, two reducing agents (R2), two detergents (D2), and two kinds of carrier ampholytes in combination. This so-called R2D2 buffer (5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris(2-carboxyethyl) phosphine, 0.5% carrier ampholytes 4-6.5, 0.25% carrier ampholytes 3-10) proved to be very efficient for a large range of different samples and allowed us to obtain two-dimensional gels of high resolution and quality.     

The composition,structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon

The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.     

Membrane protein import in yeast mitochondria

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn(2+)-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous intermembrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.     

Styryl- and dihydrostyryl-2-pyrones derivatives from Polygala sabulosa

Two dihydrostyrylpyrones and a styrylpyrone were isolated from Polygala sabulosa, together with five known styrylpyrones. Their structures were established on the basis of spectral evidence as 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone, 4-methoxy-6-(11,12-methylenedioxy-10,14-dimethoxydihydrostyryl)-2-pyrone, and 4-methoxy-6-(11,12-methylenedioxy-14-methoxystyryl)-2-pyrone.     

Reproducibility and complications in gene searches: linkage on chromosome 6, heterogeneity, association, and maternal inheritance in juvenile myoclonic epilepsy

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.     

Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa nalB mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in lasI expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of lasI and the virulence genes.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Phylogenetic relationships in Orchis and some related genera: an approach using chloroplast DNA

A chloroplast DNA restriction fragment length polymorphism analysis has been carried out on representatives species of Orchis (Orchidaceae) and of the allied genera Aceras, Dactylorhiza , and Anacamptis . One species of Cephalanthera and one of Serapias were used as outgroups. The consensus tree from a cladistic analysis showed that Orchis , as presently defined, is paraphyletic, as it contains also Aceras anthropophorum and Dactylorhiza saccifera . The genus Orchis is divided in two clades: one including O. laxiflora, O. papilionacea, O. coriophora , and O. morio in a ladderized sequence, the other showing D. saccifera at the base, followed by a clade in which a collapse of O. mascula, O. pauciflora, O. quadripunctata is sister group to a clade composed by O. italica, O. simia , and A. anthropophorum . These results, which agree to a great extent with literature evidence on chromosomes and isozymes, have been compared with various traditional systematic hypotheses for the genus.