The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.     

The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.     

Prevalence and upper limit of cardiac hypertrophy in professional cyclists

The term athlete's heart refers to an increased left ventricular mass. Few studies have assessed the prevalence and normal upper limit of cardiac hypertrophy in highly trained cyclists and this was the aim of this study. A group of 40 professional road cyclists [mean age 26 (SD 3) years] who had participated in European competitions for 3–10 years, were evaluated at the beginning of the 1992–93 season. Evaluation included a clinical history and physical examination, one and two-dimensional echocardiography, 12-lead resting electrocardiogram and a graded exercise test. Determination of the left ventricular mass index (LVMI) was performed using Devereux's formula with correction for the body surface area. Systolic and diastolic blood pressure were measured at rest and at peak exercise. Of the group 23 cyclists (58%) presented a LVMI greater than 130 g · m^–2, 21 cyclists presented a diastolic ventricular thickness equal to or greater than 13 mm, with a superior limit of 19 mm; 3 cyclists presented asymmetrical septum hypertrophy; and the relationship between posterior wall and left ventricular diastolic radius was equal to or greater than 0.45 in 14 cases (35%). Electrocardiographic abnormalities of ST-T segment were seen in only 1 subject. No correlation was found between the degree of ventricular hypertrophy and arterial blood pressure. We concluded that these professional cyclists showed a high prevalence of cardiac hypertrophy (58%). The distribution of this hypertrophy was concentric in 20/33 and asymmetric in 3/23 of the subjects with left ventricular hypertrophy. The electrocardiograms were normal in 98% of the subjects.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Production of hydrogen peroxide by Lactobacillus hilgardii and its effect on Leuconostoc oenos growth

In mixed culture of Lactobacillus hilgardii X₁B and Leuconostoc oenos X₂L, isolated from Argentinian wines, an amensalistic growth response was observed: Leuconostoc oenos did not grow, and after 24 h of incubation at 30°C no viable cells were detected. In pure and mixed cultures, Lactobacillus hilgardii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. The values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the action of hydrogen peroxide on the growth of Leuconostoc oenos were: 4.08g ml^-1 and 17.00 g ml^-1 respectively.     

Tripeptides as selective inhibitors of src-SH2 phosphoprotein interactions

Summary This paper describes the synthesis of phosphorylated peptides of the general structural Ac-Tyr(PO₃H₂)-Glu-Xaa_NH₂, where Xaa represents a hydrophobic -amino acid of d-configuration. These peptides displayed activities in the micromolar range in inhibiting src-SH2 domain/epidermal growth factor receptor interactions.     

Purification of restriction endonuclease XcyI from Xanthomonas cyanopsidis

A new Type II restriction endonuclease XcyI, purified from Xanthomonas cyanopsidis 13D5, is an isoschizomer of SmaI and XmaI that cleaves at the nucleotide sequence 5'-C decreases CCGGG-3' of double-stranded DNA. The single restriction activity present in this strain permits rapid purification of 8000 units of cleavage activity from 10 g of freshly harvested cells. The resulting XcyI preparation is free of contaminating nuclease activities that interfere with in vitro manipulation of DNA.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

HLA antigens in a sample of the Spanish population: Common features among Spaniards,Basques, and Sardinians

Summary Frequencies of recently described HLA-A,-B (antigens or splits) and HLA-C and HLA-DR antigens are studied in a 450 normal Spaniards sample. The linkage disequilibria are also calculated. HLA-DR7 is more frequent than in any other population studied. Aw30-B18 and Aw33-B14 associations are common and specific Spanish, Basque, and Sardinian HLA features. A11-B27 association is found in our Spanish sample and also in Basques.HLA-Bw4,-Bw6 antigens are analyzed by family mating and segregation and also using unrelated individuals. It shows a good fit as a genetic system. HLA-B antigens are included either in Bw4 or Bw6 according to expectations from other Caucasoid population results. The possibility of a common and North African origen (Iberians) for Spaniards, Basques, and Sardinians is discussed on the basis of presently available HLA data.