Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Topology of the membrane-associated hepatitis C virus protein NS4B

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in FLAVIVIRIDAE:     

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.     

Dopamine receptors for every species: Gene duplications and functional diversification in Craniates

The neuromodulatory effects of dopamine on the central nervous system of craniates are mediated by two classes of G protein-coupled receptors (D1 and D2), each comprising several subtypes. A systematic isolation and characterization of the D1 and D2-like receptors was carried out in most of the Craniate groups. It revealed that two events of gene duplications took place during vertebrate evolution, before or simultaneously to the emergence of Gnathostomes. It led to the conservation of two-to-four paralogous receptors (subtypes), depending on the species. Additional duplication of dopamine receptor gene occurred independently in the teleost fish lineage. Duplicated genes were maintained in most of the vertebrate groups, certainly by the acquisition of a few functional characters, specific of each subtypes, as well as by discrete changes in their expression territories in the brain. The evolutionary scenario elaborated from these data suggests that receptor gene duplications were the necessary conditions for the expansion of vertebrate forebrain to occur, allowing dopamine systems to exert their fundamental role as modulator of the adaptive capabilities acquired by vertebrate species.     

Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.     

Development of TaqMan assays for the quantitative detection of Fusarium avenaceum/Fusarium tricinctum and Fusarium poae esyn1 genotypes from cereal grain

Fungi of the genus Fusarium are important plant pathogens and contaminants of cereal grains producing different types of mycotoxins. Enniatins are a group of mycotoxins with ionophoric properties frequently detected in North European grains. Within the Fusarium complex responsible for grain infection, Fusarium avenaceum, Fusarium poae and Fusarium tricinctum are the most potential enniatins producers. This study presents the development of two quantitative TaqMan MGB (Minor Groove Binder) assays for the specific quantification of F. avenaceum/F. tricinctum and F. poae esyn1 genotypes, respectively. Two sets of genotype-specific primers/probes were designed on the basis of esyn1 gene homologues encoding multifunctional enzyme enniatin synthetase. The specificity of the assays developed has been tested successfully on 111 Fusarium isolates from different geographical origins. The detection limits for F. avenaceum/F. tricinctum esyn1 genotype and F. poae genotype were 19 and 0.3?pg, respectively. The application of the assays developed on asymptomatic wheat grain samples revealed significant positive correlations between the enniatins levels and the amount of F. avenaceum/F. tricinctum esyn1 genotype (R=0.61) and F. poae esyn1 genotype (R=0.42).     

Female genital system of the folding-trapdoor spider Antrodiaetus unicolor (Hentz, 1842) (Antrodiaetidae, Araneae): ultrastructural study of form and function with notes on reproductive biology of spiders

The genitalia of the female folding-trapdoor spider Antrodiaetus unicolor are characterized by two pairs of spermathecae that are arranged in a single row and connected to the roof of the bursa copulatrix. Each single spermatheca is divided into three main parts: stalk, bowl, and bulb, which are surrounded by the spermathecal gland. The epithelium of the spermathecal gland is underlain by a muscle meshwork and consists of different types of cells partly belonging to glandular cell units (Class 3 gland cells) that extend into pores in the cuticle of the stalk and bowl. Interestingly, the bulb lacks glandular pores and is characterized by a weakly sclerotized cuticle. This peculiarly structured bulb probably plays an important role in the discharge of the sperm mass. It is suggested that by contraction of the muscle layer the sperm mass may be squeezed out, when the bulb invaginates and expands into the spermathecal lumen, pushing the sperm to the uterus lumen. Each glandular unit consists of usually one or two central secretory cells that are for the most part surrounded by a connecting cell that again is surrounded by a canal cell. The canal cell, finally, is separated from the other epithelial cells (intercalary cells) located between the glandular units by several thin sheath cells that form the outer enveloping layer of the unit. The secretions are released through a cuticular duct that originates proximally between the apical part of the connecting cell and the apical microvilli of the secretory cells and runs into a pore of the spermathecal cuticle. The glandular products of the Class 3 gland cells likely contribute to the conditions allowing long-term storage of the spermatozoa in this species. Details regarding the ovary, the uterus internus, and the uterus externus are reported. Most of the secretion that composes the chorion of the egg is produced in the ovary. Glandular cell units observed in the uterus externus differ structurally from those in the spermathecae and likely play a different role. Finally, we briefly discuss our results on the female genitalia of A. unicolor in the light of knowledge about the reproductive biology of spiders.