Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.

The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.

The ketogenic diet revisited.

The ketogenic diet is a high-fat diet that maintains the body's starvation mechanism, with exogenous fat provided for metabolism in lieu of stored fat. Mild dehydration is important to prevent dilution of the level of ketones in circulation at any given time. It is not known why or how ketosis affects seizure activity, so the principles behind the therapy have been developed from years of clinical experience and theoretical assumptions. Dietitians are essential providers of ketosis therapy, but the dietitian must work with a physician who understands the theories behind the therapy and is an active member of the ketosis therapy team.

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrin-radixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant conformation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.

Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis.

Ezrin is a component of the microvillus cytoskeleton of a variety of polarized epithelial cells and is believed to function as a membrane-cytoskeletal linker. In this study, we isolated microvilli from human placental syncytiotrophoblast as a model system for biochemical analysis of ezrin function. In contrast to intestinal microvilli, ezrin is a major protein component of placental microvilli, comprising approximately 5% of the total protein mass and present at about one quarter of the molar abundance of actin. Gel filtration and chemical cross-linking studies demonstrated that ezrin exists mainly in the form of noncovalent dimers and higher order oligomers in extracts of placental microvilli. A novel form of ezrin, apparently representing covalently cross-linked adducts, was present as a relatively minor constituent of placental microvilli. Both oligomers and adducts remained associated with the detergent-insoluble cytoskeleton, indicating a tight interaction with actin filaments. Moreover, stimulation of human A431 carcinoma cells with EGF induces the rapid formation of ezrin oligomers in vivo, thus identifying a signal transduction pathway involving ezrin oligomerization coincident with microvillus assembly. In addition to time course studies, experiments with tyrosine kinase and tyrosine phosphatase inhibitors revealed a correlation between the phosphorylation of ezrin on tyrosine and the onset of oligomer formation, consistent with the possibility that phosphorylation of ezrin might be required for the generation of stable oligomers. Based on these observations, a model for the assembly of cell surface structures is proposed.

Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains.

Previous work has indicated that ezrin, a membrane-microfilament linking protein, exists largely as a monomeric protein in solution. Here we purify from human placenta two cytosolic ezrin species that chromatography differently on gel filtration, anion, and cation exchange resins. Both species contain only the ezrin polypeptide, yet they do not readily interconvert in vitro as determined by gel filtration analysis. Determination of the physical properties of the two species indicates that one represents the conventional monomer, whereas the other represents highly asymmetric dimers. Chemical cross-linking data support this conclusion. Purified ezrin monomers normally have a masked C-terminal domain (termed a C-ERMAD) that, upon exposure, can associate with an N-terminal domain (termed N-ERMAD) of another ezrin molecule. Here we show that purified ezrin dimers also have masked C-ERMADs. On the basis of these results, we suggest a working model for the molecular organization of ezrin monomers and dimers and propose a hypothesis that explains the stable coexistence of ezrin monomers and dimers in placenta. Since radixin and moesin, the two other members of the closely related ERM protein family, both contain N- and C-ERMADs, the results we have documented and models proposed for ezrin are likely to apply to radixin and moesin as well.

Beta 2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat.

Maternal IgG crosses the proximal small intestine of the suckling rat by receptor-mediated endocytosis and transepithelial transport. The Fc receptor resembles the major histocompatibility complex class I antigens in that it consists of two subunits: a transmembrane glycoprotein (gp50) in association with beta 2-microglobulin. We used immunofluorescence microscopy and quantitative immunogold cytochemistry to study the subcellular distribution of the two subunits. In mature absorptive cells both subunits were colocalized in each of the membrane compartments that mediate transcytosis of IgG. IgG administered in situ apparently caused both subunits to concentrate within endocytic pits of the apical plasma membrane, suggesting that ligand causes redistribution of receptors at this site. These results support a model for transport in which IgG is transferred across the cell as a complex with both subunits. During absorptive cell differentiation, gp50 and beta 2-microglobulin showed nearly identical patterns of increased expression that accompanied the development of the apical endocytic apparatus and terminal web. However, absorptive cells in weanling rats expressed no detectable gp50 and only low levels of beta 2-microglobulin in the Golgi region and on the basolateral plasma membrane where class I antigens would likely reside. Thus, beta 2-microglobulin has a novel distribution unrelated to its function as a subunit of the class I antigens. The co-expression of the two receptor subunits is restricted to neonatal epithelial cells engaged in IgG transport and is coordinately regulated during absorptive cell differentiation and during postnatal intestinal development.

Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells.

Ezrin and moesin are two cytoskeletal proteins originally purified from human placenta that are 74% identical in overall protein sequence. They are believed to be membrane-cytoskeletal linking proteins because they share sequence homology with erythrocyte band 4.1 and colocalize with actin specifically in microvilli and membrane ruffles in cultured cells. To determine if ezrin and moesin share similar distributions in vivo, we studied their localizations with respect to F-actin in tissue sections. Surprisingly, ezrin and moesin exhibited very different cellular distributions. Ezrin was highly concentrated and colocalized with actin on the apical surface of many epithelial cell types. During enterocyte differentiation, the pattern of expression and redistribution of ezrin was consistent with it performing a role in microvillus assembly. Immunoelectron microscopy in differentiated cells revealed that ezrin was restricted mainly to the plasma membrane of microvilli and other actin-rich surface projections. Moesin was found in endothelial cells and was also enriched in the apical microvilli of a restricted set of epithelial cells. All polarized cell types with abundant microvilli contained one or both proteins, suggesting that ezrin and moesin perform related functions. However, the differential expression of ezrin and moesin indicates that they have distinct properties, which are uniquely adapted to specific cell types.

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.

Letter memory loads change more than visual-field advantage: interhemispheric coupling effects.

A derivation from Denenberg's (Denenberg, 1980. American Journal of Physiology, 238, R3-R13) model of brain organization is that significant correlations between left-hemisphere (LH) and right-hemisphere (RH) measures indicate that the hemispheres are coupled together in a general system. Two formally similar visual-field (VF) reaction-time (RT) tasks, one a LH-right (R) VF and the other a RH-left (L) VF advantage task, were performed by 30 female and 30 male right-handers under single-task (zero load) and dual task (three or six consonants in memory) conditions. Expected letter load effects upon VF performances were obtained. Particularly for RT variability, correlations of measures lateralized to different hemispheres (i.e., LVF-RVF, LH and RH tasks) increased linearly as load increased, while correlations of measures lateralized to the same hemisphere (i.e., two LH tasks) did not. Bihemispheric cortical activation may play a role in this general system coupling caused by complexity or load. Females demonstrated greater interhemispheric coupling.

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.

Components of dental fear in adults?

The functional relatedness between dental fear and multiple other fears was studied in a normative sample of 285 undergraduates. Rachman and Lopatka's work on the inter-dependence of multiple fears within individuals, as well as Lang's bioinformational theory of emotion, provided a theoretical background for this investigation. Fears about social contact, pain, mutilation (e.g. injury, blood, disfigurement), and being closed-in were assessed within the realm of verbal report; they were studied as possible components and/or concomitants of the dental fear construct. Multiple regression analyses with these variables utilized the Dental Fear Survey total score as a criterion variable. Fear of pain was found to be the most significant predictor of dental fear in both males and females. For females only, mutilation fear was the next strongest determinant. Fear of being closed-in was an additional significant dental fear predictor for both sexes. The possible role of social fears in the manifestation of dental fear was not confirmed and awaits further investigation. Results were consistent with the idea that there may often be a moderate degree of functional dependence between dental fear and the other fears identified here.