Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Genetic characterization of staphopain genes in Staphylococcus aureus

Staphylococcus aureus , a leading cause of bacterial infections in humans, is endowed with a wealth of virulence factors that contribute to the disease process. Several extracellular proteolytic enzymes, including cysteine proteinases referred to as the staphopains (staphopain A, encoded by the scpA gene, and staphopain B, encoded by sspB ), have proposed roles for staphylococcal virulence. Here we present data regarding the distribution, copy number and genetic variability of the genes encoding the staphopains in a large number of S. aureus strains. The polymorphism of the scpA and sspB genes in three laboratory strains and 126 clinical isolates was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Both genes were detected in all isolates by PCR amplification and, based on the PCR-RFLP patterns, classified as four types for scpA and six types for sspB . Those with the most divergent patterns were subjected to DNA sequencing and compared with genomic sequence data for the seven available strains of S. aureus . Southern blot analysis of the scpA and sspB sequences indicates that they are strongly conserved as single-copy genes in the genome of each S. aureus strain investigated. Taken together, these data suggest that the staphopains have important housekeeping and/or virulence functions, and therefore may constitute an interesting target for the development of therapeutic inhibitors for the treatment of staphylococcal diseases.     

The Drosophila transmembrane protein Fear-of-intimacy controls glial cell migration

Development of complex organs depends on intensive cell-cell interactions, which help coordinate movements of many cell types. In a genetic screen aimed to identify genes controlling midline glia migration in the Drosophila nervous system, we have identified mutations in the gene kastchen. Here we show that during embryogenesis kastchen is also required for the normal migration of longitudinal and peripheral glial cells. During larval development, kastchen non-cell autonomously affects the migration of the subretinal glia into the eye disc. During embryonic development, kastchen not only affects glial cell migration but also controls the migration of muscle cells toward their attachment sites. In all cases, kastchen apparently functions in terminating or restricting cell migration. We identified the molecular nature of the gene by performing transgenic rescue experiments and by sequence analysis of mutant alleles. Kastchen corresponds to the recently described gene fear-of-intimacy (foi) that was identified in screen for genes affecting germ cell migration, suggesting that Foi-Kastchen is more generally involved in regulating cell migration. It encodes a member of an eight-transmembrane domain protein family of putative Zinc transporters or proteases. We determined the topology of the Foi protein by using antisera against luminal and intracellular domains of the protein and provide evidence that it does not act as a Zinc transporter. Genetic evidence suggests that one of the functions of foi may be associated with hedgehog signaling.     

Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749)

Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a cellulose-binding module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/).     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Actin cytoskeleton in the extra-ovular embryo sac of Utricularia nelumbifolia (Lentibulariaceae)

The actin cytoskeleton in the mature female gametophyte of angiosperms has been examined in only a few dicot and monocot species. The main purposes of this study were to identify how the actin cytoskeleton is arranged in the mature extra-ovular embryo sac in Utricularia nelumbifolia (Lentibulariaceae). We found that the extra-ovular part of the central cell has a well-developed actin cytoskeleton: actin microfilaments formed of long strands which run longitudinally or transversally to the long axis of the embryo sac. The exerted part of the central cell, which is exposed to the environment of the ovary chamber, is highly vacuolated and in the thin peripheral cytoplasm possesses a complicated network of actin microfilaments. The epidermal cells of the placenta that are in contact with the extra-ovular part of the embryo sac are crushed. The ultrastructure data of these cells are presented. We detected the accumulation of the actin cytoskeleton between the micropylar parts of the synergids and the extra-ovular part of central cell. This actin accumulation is unusual because in typical angiosperms the micropylar parts of the synergids form the apex of the female gametophyte.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

A high-density screen for linkage in multiple sclerosis

To provide a definitive linkage map for multiple sclerosis, we have genotyped the Illumina BeadArray linkage mapping panel (version 4) in a data set of 730 multiplex families of Northern European descent. After the application of stringent quality thresholds, data from 4,506 markers in 2,692 individuals were included in the analysis. Multipoint nonparametric linkage analysis revealed highly significant linkage in the major histocompatibility complex (MHC) on chromosome 6p21 (maximum LOD score [MLS] 11.66) and suggestive linkage on chromosomes 17q23 (MLS 2.45) and 5q33 (MLS 2.18). This set of markers achieved a mean information extraction of 79.3% across the genome, with a Mendelian inconsistency rate of only 0.002%. Stratification based on carriage of the multiple sclerosis–associated DRB1*1501 allele failed to identify any other region of linkage with genomewide significance. However, ordered-subset analysis suggested that there may be an additional locus on chromosome 19p13 that acts independent of the main MHC locus. These data illustrate the substantial increase in power that can be achieved with use of the latest tools emerging from the Human Genome Project and indicate that future attempts to systematically identify susceptibility genes for multiple sclerosis will have to involve large sample sizes and an association-based methodology.     

Relative warp analysis of cranial and upper molar shape in rock miceApodemus mystacinus sensu lato

We studied phenotypic relationships among 13 samples of two rock mice species:Apodemus mystacinus (Danford and Alston, 1877) from Anatolia (n = 38) andA. epimelas (Nehring, 1902) from the Balkans (n = 71). Cartesian coordinates of landmarks were collected on the skull and on the occlusal projection of the upper molars (18 landmarks). Centroid size (a measure of overall size) suggested that molars vary independently of overall skull size in both species. Discriminant function analysis on relative warp scores classified >80% of specimens into the correct species, with the best results obtained for the ventral aspect of the skull and for molars. Projection of the 1st discriminant function scores against centroid size provided good separation between the two species. Analysis of vector displacements associated with extremes of variation suggested considerable phenetic differences on the ventral side of the skull and in the molar shape of the two species. The great majority of shifts in landmarks were in a longitudinal direction and the rearrangements of molar cusps were more complex than was the case with the cranium. A bivariate plot of the posterior hard palate length against the incisive foramen length separatedA. mystacinus andA. epimelas well.