Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma.

Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas.

Ultrastructure of Endogenous Stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in Experimentally Infected Goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 105 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

An activation-independent role of transcription factors in insulator function.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.

Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C.

The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1.

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorphogenesis. The ubiquitin E3 ligase COP1 (constitutively photomorphogenic 1) regulates the stability of several light-signaling components. HFR1 (long hypocotyl in far-red light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes. HFR1 is closely related to PIF1, PIF3, and PIF4 (phytochrome interacting factor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling , as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response.

The nuclear matrix: a critical appraisal.

It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently described to occur in tight association with the nuclear matrix.

Sperm Ultrastructure of the Spider Crab Maja brachydactyla (Decapoda: Brachyura)

This study describes the morphology of the sperm cell of Maja brachydactyla, with emphasis on localizing actin and tubulin. The spermatozoon of M. brachydactyla is similar in appearance and organization to other brachyuran spermatozoa. The spermatozoon is a globular cell composed of a central acrosome, which is surrounded by a thin layer of cytoplasm and a cup-shaped nucleus with four radiating lateral arms. The acrosome is a subspheroidal vesicle composed of three concentric zones surrounded by a capsule. The acrosome is apically covered by an operculum. The perforatorium penetrates the center of the acrosome and has granular material partially composed of actin. The cytoplasm contains one centriole in the subacrosomal region. A cytoplasmic ring encircles the acrosome in the subapical region of the cell and contains the structures-organelles complex (SO-complex), which is composed of a membrane system, mitochondria with few cristae, and microtubules. In the nucleus, slightly condensed chromatin extends along the lateral arms, in which no microtubules have been observed. Chromatin fibers aggregate in certain areas and are often associated with the SO-complex. During the acrosomal reaction, the acrosome could provide support for the penetration of the sperm nucleus, the SO-complex could serve as an anchor point for chromatin, and the lateral arms could play an important role triggering the acrosomal reaction, while slightly decondensed chromatin may be necessary for the deformation of the nucleus.

Anatomia e Histologia das Glândulas Salivares nos Triatomíneos

Histological studies upon the salivary glands of ten species of triatomine bugs were performed looking for their number and structural organization in different genera. It was possible to evaluate the celular epithelium type of each gland, as well as the merocrine and apocrine secretions of the glands. Secretion run until the hilo and after to salivary pump and hypofaringe. The glandular components, D1, D2 and D3 are always present in the Triatoma, Panstrongylus and Diptelogaster but in Rhodnius there are only the first two pairs of glands. The salivary channels and the hilo are analyzed by histology. The whole pair D3 has a clear valve that regularizes the exit of the secretions to the hilo. According to the genus the valves appear in different locations. They have low and dense epithelium, and their nucleus are rich in chromatin. The secondary channels leaving these valves, are very different, with clear chitinous ringer, low level of chromatin in the nucleus and homogeneous cytoplasm.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed. We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo. Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.