A 130K protein from chicken gizzard: Its localization at the termini of microfilament bundles in cultured chicken cells

A protein with a molecular weight of 130,000 (130K protein) was extracted from chicken gizzard and purified to homogeneity by ammonium sulfate fractionation and ion-exchange chromatography. Antibodies prepared against the pure protein were used for its immunochemical characterization and immunofluorescent visualization in cultured chicken cells. Both peptide mapping and immunochemical analysis indicated that the 130K protein is not related either structurally or antigenically to other mechanochemical proteins, including α-actinin, actin, myosin, tropomyosin, filamin and tubulin. Immunofluorescent labeling of different cultured embryonic chicken cells (from skin, heart and gizzard) indicated that the label was predominantly organized in intracellular plaques at the bottom of the cells and in some areas of cell-cell contact. Immunoprecipitation of the 130K protein from biosynthetically ³⁵S-methionine-labeled cultured cells, using the pure antibodies and Staphylococcus aureus, resulted in the specific isolation of a single labeled electrophoretic band indistinguishable from the chicken gizzard 130K protein. The 130K protein-rich plaques were found, by interference-reflection microscopy, to coincide with cell substrate adhesion plaques. Double immunofluorescent labeling for the 130K protein and other cytoskeletal proteins (actin, α-actinin and tropomyosin) indicated that the 130K protein-rich areas are localized at the termini of stress fibers. α-Actinin was found in close association with the 130K protein, while tropomyosin was usually excluded from those areas.     

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.     

A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites

Recently we developed an isolation procedure for the cell-to-cell adherens junctions (AJ; cadherin-based junctions) from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41). In this study, using the isolated AJ, we have obtained two mAbs specific to the 220-kD undercoat-constitutive protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this 220-kD protein was highly concentrated at the undercoat of cell-to-cell AJ in various types of tissues and that this protein was located in the immediate vicinity of the plasma membrane in the undercoat of AJ. In the cells lacking typical cell-to-cell AJ, such as fibroblasts, the 220-kD protein was immunofluorescently shown to be coconcentrated with cadherin molecules at cell-cell adhesion sites. These localization analyses appeared to indicate the possible direct or indirect association of the 220-kD protein with cadherin molecules. Furthermore, it was revealed that the 220-kD protein and alpha-spectrin were coimmunoprecipitated with the above mAbs in both the isolated AJ and the brain. The affinity-purified 220-kD protein molecule looked like a spherical particle, and its binding site on the spectrin molecule was shown to be in the position approximately 10-20 nm from the midpoint of spectrin tetramer by low-angle rotary-shadowing electron microscopy. Taking all these results together with biochemical and immunological comparisons, we are persuaded to speculate that the 220-kD protein is a novel member of the ankyrin family. However, the possibility cannot be excluded that the 220-kD protein is an isoform of beta-spectrin. The possible roles of this 220-kD protein in the association of cadherin molecules with the spectrin-based membrane skeletons at the cadherin-based cell-cell adhesion sites are discussed.     

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin–catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin–catenin system and may control cadherin-mediated cell–cell adhesion.     

A new 82-kD barbed end-capping protein (radixin) localized in the cell- to-cell adherens junction: purification and characterization

An 82-kD protein has been purified from the undercoat of the adherens junction isolated from the rat liver. The purification scheme includes low salt extraction followed by DEAE-cellulose ion exchange, DNase I-actin affinity, and carboxyl methyl-cellulose ion exchange chromatographies. The purified 82-kD protein was essentially free of contaminants as judged by SDS-PAGE combined with silver staining. The substoichiometric 82-kD protein largely inhibited the actin filament assembly; when the molar ratio of the 82-kD protein to G-actin was 1:1,000, the viscosity was reduced to 28% of the control value. Direct electron microscopic studies revealed that the 82-kD protein selectively inhibited monomer addition at the barbed ends of actin filaments. By use of the antibody raised against the 82-kD protein, this protein was shown by immunofluorescence microscopy to be localized at the cell-to-cell adherens junction in various types of cells. In contrast, the 82-kD protein was not concentrated at the cell-to-substrate adherens junctions (focal contacts). These findings have led us to conclude that the 82-kD protein is a barbed end-capping protein which is associated with the undercoat of the cell-to-cell adherens junction. Hence, we have tentatively designated the 82-kD protein as radixin (from the Latin word radix meaning root).     

A new member of the LIM protein family binds to filamin B and localizes at stress fibers

Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats. They also interact with a number of other cellular proteins. All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin. In a yeast two-hybrid search of a human placental library, using as bait repeats 10-18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus. We term this protein filamin-binding LIM protein-1, FBLP-1. Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10-13. FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells. We also have identified two FBLP-1 variants. Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain. Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells. The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.     

Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions

We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.     

Immunolocalization of the intermediate filament-associated protein plectin at focal contacts and actin stress fibers.

The distribution of plectin in the cytoplasm of Rat1 and glioma C6 cells was examined using a combination of double and triple immunofluorescence microscopy and interference reflection microscopy. In cells examined shortly after subcultivation (less than 48 h), filamentous networks of plectin structures, resembling and partially colocalizing with vimentin filaments, were observed as reported in previous studies. In cells kept attached to the substrate without growth for periods of 72 h to 8 days (stationary cultures), thick fibrillary plectin structures were observed. These structures were located at the end of actin filament bundles and showed co-distribution with adhesion plaques (focal contacts), vinculin, and vimentin. Only relatively large adhesion plaques (dash-like contacts) were decorated by antibodies to plectin, smaller dot-like contacts at the cell edges remained undecorated. Moreover, in stationary Rat1 cells plectin structures were found to be predominantly colocalized with actin stress fibers. However, after treatment of such cells with colcemid, plectin's distribution changed dramatically. The protein was no longer associated with actin structures, but was distributed diffusely throughout the cytoplasm. After a similar treatment with cytochalasin B, plectin's association with stress fibers again was completely abolished, although stress fibers were still present. The association of plectin with focal contact-associated intermediate filaments was demonstrated also by immunogold electron microscopy of quick-frozen, deep-etched replicas of rat embryo fibroblasts. These data confirm previous reports suggesting a relationship between intermediate filaments on the one hand, and actin stress fibers and their associated plasma membrane junctional complexes, on the other. Furthermore, the data establish plectin as a novel component of focal contact complexes and suggest that plectin plays a role as mediator between intermediate filaments and actin filaments.     

Central root cap cells are depleted of endoplasmic microtubules and actin microfilament bundles: implications for their role as gravity-sensing statocytes

Summary Indirect immunofluorescence, using monoclonal antibodies to actin and tubulin, applied to sections of root tips ofLepidium, Lycopersicon, Phleum, andZea, revealed features of the cytoskeleton that were unique to the statocytes of their root caps. Although the cortical microtubules (CMTs) lay in dense arrays against the periphery of the statocytes, these same cells showed depleted complements of endoplasmic microtubules (EMTs) and of actin microfilament (AMF) bundles, both of which are characteristic of the cytoskeleton of other post-mitotic cells in the proximal portion of the root apex. The scarcity of the usual cytoskeletal components within the statocytes is considered responsible for the exclusion of the larger organelles (e.g., nucleus, plastids, ER elements) from the interior of the cell and for the absence of cytoplasmic streaming. Furthermore, the depletion of dense EMT networks and AMF bundles in statocyte cytoplasm is suggested as being closely related to the elevated cytoplasmic calcium content of these cells which, in turn, may also favour the formation of the large sedimentable amyloplasts by not permitting plastid divisions. These latter organelles are proposed to act as statoliths due to their dynamic interactions with very fine and highly unstable AMFs which enmesh the statoliths and merge into peripheral AMFs-CMTs-ER-plasma membrane complexes. Rather indirect evidence for these interactions was provided by showing enhanced rates of statolith sedimentation after chemically-induced disintegration of CMTs. All these unique properties of the root cap statocytes are supposed to effectively enhance the gravity-perceptive function of these highly specialized cells.Dedicated to Prof. Dr. Benno Parthier on the occasion of his retirement     

A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin- catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway.     

Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.     

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full- length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.     

Interferon action: nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster

The interferons are cytokines with antiviral, cell growth regulatory, and immunomodulatory activities. These activities are mediated by the proteins induced by the interferons. Earlier we described a gene cluster (the 200 cluster) consisting of at least six adjacent, interferon-activatable genes located next to the erythroid alpha-spectrin locus on murine chromosome 1. The genes of the cluster arose by repeated gene duplication and they specify proteins with pronounced sequence similarity. We have now raised polyclonal antibodies against a segment from one of these proteins (the 204 protein of 72 kD). Using these, we established that the 204 protein is a phosphoprotein whose level in cells from various murine lines can be increased up to 75-fold upon treatment with alpha interferon. Experiments involving fractionation of cell lysates and indirect immunofluorescence microscopy of control and interferon-treated cells revealed that the 204 protein is nucleolar and nucleoplasmic. This conclusion was confirmed by co-localization with B23, a known nucleolar protein. The 204 protein is the first interferon-induced protein found to be located in the nucleoli, the subcellular organelles of ribosomal RNA production and ribosome assembly. It remains to be seen whether the 204 protein affects any of these processes. Studies on 204 protein function should be facilitated by the availability of complete cDNA clones and the finding of cell lines in which the expression of this protein is impaired.     

Voltammetry of tobacco mosaic virus and its isolated protein at the graphite electrode

The electrochemical behaviour of tobacco mosaic virus (TMV) and its isolated protein was studied using differential pulse (DP) voltammetry at a graphite electrode and by direct current (DC) polarography in Brdicka solution. TMV and its isolated protein were found to be electrooxidized at the graphite electrode in the adsorbed state. Both species yielded two oxidation peaks on DP voltammograms. The first, more negative peak, corresponded to electrooxidation of tyrosine residues, whereas the other, more positive, peak corresponded to electrooxidation of tryptophan residues. DC polarography was used to detect degradation of TMV and denaturation of TMV-protein induced by an increased pH and by the addition of urea, respectively. These structural transformations resulted in increased DP voltammetric oxidation currents as recorded using a graphite working electrode. It has been suggested that the higher oxidation currents were due to an increase in the number of tyrosine and tryptophan residues accessible to the reaction at the graphite electrode. The results of these electrochemical investigations were in a good agreement with the estimation of the accessibility of tyrosine and tryptophan residues based on the well-explored three-dimensional structure of TMV and its isolated protein.     

Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development

Cellular localization of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in vascular bundles of leaf blades of rice (Oryza sativa L.), at the stage at which leaf blades 6 (the lowest position) to 10 were fully expanded, was investigated immunocytologically with an affinity-purified anti-GS1 immunoglobulin G. Strong signals for GS1 protein were detected in companion cells of large vascular bundles when blades 6–8 were tested. Signals for GS1 were also observed in vascular-parenchyma cells of both large and small vascular bundles. The results further support our hypothesis that GS1 is important for the export of leaf nitrogen from senescing leaves. The signals in companion cells were less striking in the younger green leaves and were hardly detected in the non-green portion of the 11th blade. In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylemparenchyma cells. When total GS extracts prepared from the 6th,10th, and the non-green 11th blades were subjected to anion-exchange chromatography, the activity of GS1 was clearly separated from that of chloroplastic GS, indicating that GS1 proteins detected in the vascular tissues were able to synthesize glutamine. The function of GS1 detected in the developing leaves is discussed.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GS1 cytosolic glutamine synthetase - GS2 plastidic glutamine synthetase - IgG immunoglobulin G     

Cdc42 and its BORG2 and BORG3 effectors control the subcellular localization of septins between actin stress fibers and microtubules

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A new mechanism of dominance in hypophosphatasia: the mutated protein can disturb the cell localization of the wild-type protein

The dominant negative effect of mutations is rare in metabolic diseases and its mechanism has not been studied much. Hypophosphatasia, a bone inherited metabolic disorder, is a good model because the disease can be dominantly transmitted. The gene product activity depends on a homodimeric configuration and many mutations have been reported in the ALPL gene responsible for the disease. Using CFP/YFP-tagged-TNSALP plasmids, transfections in COS cells and confocal fluorescence analyses, we studied the point mutation G232V (c.746G>T). We showed that the G232V protein sequestrates some of the wild-type protein into the cells and prevents it from reaching the membrane where it plays its physiological role. A. S. Lia-Baldini and I. Brun-Heath equally contributed to this work.     

Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border

The extracellular compartment where bone resorption occurs, between the osteoclast and bone matrix, is shown in this report to be actively acidified. The weak base acridine orange accumulates within this compartment but dissipates after incubation with ammonium chloride. Upon removal of ammonium chloride, the cells are able to rapidly reacidify this compartment. The highly convoluted plasma membrane of the osteoclast facing this acidic compartment (ruffled border) is shown to contain a 100-kD integral membrane protein otherwise present in limiting membranes of lysosomes and other related acidified organelles (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526; Tougard, C., D. Louvard, R. Picart, and A. Tixier-Vidal, 1985, J. Cell Biol. 100:786-793). Antibodies recognizing this 100-kD lysosomal membrane protein cross-react with a proton-pump ATPase from pig gastric mucosae (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526), therefore raising the possibility that it plays a role in the acidification of both intracellular organelles and extracellular compartments. Lysosomal enzymes are also directionally secreted by the osteoclast into the acidified extracellular compartment which can therefore be considered as the functional equivalent of a secondary lysosome with a low pH, acid hydrolases, the substrate, and a limiting membrane containing the 100-kD antigen.     

分离自蚊幼虫的腐霉一新种及其rDNA的ITS区段分析

在一次室外蚊虫生物防治实验中,从被病菌感染的蚊幼虫体中分离到一株卵菌。根据其形态特征及其rDNA的ITS区段的碱基系列,鉴定为腐霉属一新种,命名为贵阳腐霉Pythiumguiyangense。对新种作了拉丁文描述。对新种与其近似种卡地腐霉P.carolinianum、奇雄腐霉P.middletonii和多卵腐霉P.multisporum的区别进行了讨论。模式标本(干培养物)保存在中国科学院微生物研究所菌物标本馆(HMAS),活培养物保存在贵阳医学院。     

Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA

While a number of different gap junction proteins have now been identified, hepatic gap junctions are unique in being the first demonstrated case where two homologous, but distinct, proteins (28,000 and 21,000 Mr) are found within a single gap junctional plaque (Nicholson, B. J., R. Dermietzel, D. Teplow, O. Traub, K. Willecke, and J.-P. Revel. 1987. Nature [Lond.]. 329:732-734). The cDNA for the major 28,000-Mr component has been cloned (Paul, D. L. 1986. J. Cell Biol. 103:123-134) (Kumar, N. M., and N. B. Gilula. 1986. J. Cell Biol. 103:767-776) and, based on its deduced formula weight of 32,007, has been designated connexin 32 (or Cx32 as used here). We now report the selection and characterization of clones for the second 21,000-Mr protein using an oligonucleotide derived from the amino-terminal protein sequence. Together the cDNAs represent 2.4 kb of the single 2.5- kb message detected in Northern blots. An open reading frame of 678 bp coding for a protein with a calculated molecular mass of 26,453 D was identified. Overall sequence homology with Cx32 and Cx43 (64 and 51% amino acid identities, respectively) and a similar predicted tertiary structure confirm that this protein forms part of the connexin family and is consequently referred to as Cx26. Consistent with observations on Cx43 (Beyer, E. C., D. L. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629) the most marked divergence between Cx26 and other members of the family lies in the sequence of the cytoplasmic domains. The Cx26 gene is present as a single copy per haploid genome in rat and, based on Southern blots, appears to contain at least one intron outside the open reading frame. Northern blots indicate that Cx32 and Cx26 are typically coexpressed, messages for both having been identified in liver, kidney, intestine, lung, spleen, stomach, testes, and brain, but not heart and adult skeletal muscle. This raises the interesting prospect of having differential modes of regulating intercellular channels within a given tissue and, at least in the case of liver, a given cell.