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.     

Molecular characterization and functional expression of the human cardiac gap junction channel

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.     

Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.     

Intercellular calcium signaling via gap junctions in glioma cells

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.     

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.     

Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive

Hydropathy analysis of the Mr 27,000 rat liver gap junction protein sequence deduced from a cDNA clone has suggested the presence of four transmembrane segments (Paul, D. L. (1986) J. Cell Biol. 103, 123-134). In the present report, several features of the molecular topology of the protein were investigated by microsequence analysis of peptides generated by treatment of isolated gap junctions with a variety of proteases. Under the experimental conditions used, the proteases had access only to the portion of the Mr 27,000 protein that was originally (in vivo) the cytoplasmic surface of the gap junction. Microsequencing of the peptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the amino terminus of the protein is disposed at or near the cytoplasmic surface of the gap junction, and that this surface also contains a protease-hypersensitive hydrophilic sequence between residues 109 and 123, presumably connecting the second and third transmembrane segments. Immunocytological localization of binding of monoclonal antipeptide antibodies demonstrates that the carboxyl terminus of the protein is also localized to the cytoplasmic surface of the gap junction. No protease sensitivity was found in the hydrophilic sequences thought to connect either the first and second transmembrane segments or the third and fourth segments, supporting the model's prediction that these sequences face the narrow intercellular gap which cannot be penetrated by proteases.     

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved. Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons. In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays. By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling. These experiments demonstrate that, contrary to the conclusions of some earlier studies (Baldwin, K. M., 1979, J. Cell Biol., 82:66-75; Peracchia, C., and L. L. Peracchia, 1980, J. Cell Biol., 87:708-718), the arrangement of gap junction connexons, in cardiac tissue at least, cannot be used as a reliable guide to the functional state of the junctions.     

Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.     

A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae.

The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence. In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M. T. Fuller, J. H. Caulton, J. A. Hutchens, T. C. Kaufman, and E. C. Raff, J. Cell Biol. 104:385-394, 1987, and J. E. Rudolph, M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff, Mol. Cell. Biol. 7:2231-2242, 1987). Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.     

Books

Book reviewed in this article: Brown , B. T., Carotheks , S. W., & Johnson , R. R. 1987. Grand Canyon Birds. Brown , D. E. 1985. Arizona Wetlands and Waterfowl. Cheng , T.-H. (Zheng , Z.). 1986. Birds of the World: Latin, Chinese, and English. Feige , K.-D. 1986. Der Pirol. Pp. 216, 92 figures (many black-and-white photographs), 24 tables. Die Neue Brehm-Biicherei 578. Wittenberg Lutherstadt: A. Ziemsen Verlag. Fraga , R. & Narosky , S. 1985. Nidificacion de las Aves Argentinas (Formicariidae a Cinclidae). GWINNER, E. 1986. Circannual Rhythms: Endogenous Annual Clocks in the Organization of Seasonal Processes. Harwood , M. (ed.) 1985. Proceedings of Hawk Migration Conference IV. Pp. 393, various tables and figures. Hawk Migration Association of North America. Hilty , S. L. & Brown , W. L. 1986. A Guide to the Birds of Colombia. Ito , J., Brown , L. & Kikkawa , J. 1987. Animal Societies: Theories and Facts. Kavanau , J. L. 1987. Lovebirds, Cockatiels, Budgerigars: Behavior and Evolution. Knystautas , A. J. V. & Sibnev , J. B. 1987. Die Vogelwelt Ussuriens. Lane , B. A. 1987. Shorebirds in Australia. MEDMARAVIS and Monbailliu X. (eds). 1986. Mediterranean Marine Avifauna: Population Studies and Conservation. Narosky , S., Fraga , R. &de la Pe～a , M. 1983. Nidificacion de las Aves Argentinas (Dendrocolaptidae y Furnariidae) Fraga , R. & Narosky , S. 1985. Nidificacion de las Aves Argentinas (Formicariidae a Cinclidae) Noval , A. 1986. Guia de las Aves de Asturias. O'Connor , R. J. & Shrubb , M. 1986. Farming and Birds. Paz , U. 1986. The Plants and Animals of the Land of Israel. Perrins , C. M. 1987. Collins New Generation Guide to the Birds of Britain and Europe. Perry , K. 1987. The Irish Dipper. Ridley , M. (ed.) 1986. Proceedings of the Third International Symposium on Pheasants in Asia, Chiang Mai, Thailand. 26–28 January 1986. Rubenstein , D. I. & Wrangham , R. W. (eds) 1986. Ecological Aspects of Social Evolution: Birds and Mammals. Skutch , A. F. 1987. Helpers at Birds' Nests: a Worldwide Survey of Cooperative Breeding and Related Behavior. Solomonov , N. G. (ed.) 1987. Red Data Book of the Yakut ASSR: Rare and Endangered Species of Animals. (Russian.) Ulrich , T. J. 1984. Birds of the Northern Rockies. Wink , M. 1987. Die Vogel des Rheinlandes, III. Atlas zur Brutvogelverbreitung.     

Books

Andrews , J.R.H. 1987. The Southern Ark. Zoological Discovery in New Zealand 1769–1900. Andru?aitis , G. (ed.) 1985. Red Data Book of the Latvian SSR: Rare and Endangered Species of Animals and Plants. (Latvian and Russian.) Bakeman , R. & Gottman , J. 1986. Observing Interaction: An Introduction to Sequential Analysis. Bergmann , H.-H. 1987. Die Biologie des Vogels. Boag , D. & Alexander , M. 1986. The Atlantic Puffin. Boehme , R.L., Zhordania , R.G. & Kuznetsov , A.A. 1987. The Birds of Georgia. Bond , J. 198S. Birds of the West Indies. Buden , D.W. 1987. The Birds of the Southern Bahamas. Clews , B., Heryet , A. & Trodd , P. 1987. Where to Watch Birds in Bedfordshire, Berkshire, Buckinghamshire, Hertfordshire & Oxfordshire. Croxall , J.P. (ed.) 1987. Seabirds: Feeding Ecology and Role in Marine Ecosystems. Dubois , P.J. &Mahéo , R. 1986. Limicoles Nicheurs de France. Eriksson , M.O.G. (ed.) 1987. Proceedings of the Fifth Nordic Ornithological Congress, 1985. Onsala, Sweden, 5–9 August, 1985. Gillham , E. 1987. Tufted Ducks in a Royal Park. Hails , C. 1987. Birds of Singapore. Illustrated by Frank Jarvis. Hill , D.J. (ed.) 1987. Breeding and Management in Birds of Prey. Hill , M. & Langsbury , G. 1987. A Field Guide to Photographing Birds in Britain and Western Europe. Hudson , P.J. & Lovel , T.W.I, (eds). 1986. 3rd International Grouse Symposium, York, 1984. Huntingford , F.A. & Turner , A.K. 1987. Animal Conflict. Isler , M.L. & Isler , P.R. 1987. The Tanagers: Natural History, Distribution, and Identification. Johnsgard , P.A. 1986. Birds of the Rocky Mountains. Johnston , R.F. (ed.) 1986. Current Ornithology Kenward , R.E. 1987. Wildlife Radio Tagging: Equipment, Field Techniques and Data Analysis. Lobkov , E.G. 1986. The Breeding Birds of Kamchatka. Peklo , A.M. 1987. The Flycatchers of the USSR. Pratt , H.D., Bruner , P.L. & Berrett , G. 1987. A Field Guide to the Birds of Hawaii and the Tropical Pacific. Princeton: Princeton University Press. Potapov , R.L. & Flint , V.E. (eds) 1987. The Birds of the USSR: Galliformes, Gruiformes. Orians , G. Blackbirds of the Americas. Ridley , M. (ed.) 1986. Proceedings of the Third International Symposium on Pheasants in Asia, Chiang Mai, Thailand, January 26–28 1986. Stephens , D.W. & Krebs , J.R. 1987. Foraging Theory. Taylor , D., Wheatly , J. & Prater , T. 1987. Where to Watch Birds in Kent, Surrey and Sussex. Whitmore , T.C. & Prance , G.T. (eds.) 1987. Biogeography and Quaternary History of Tropical America. Wink , M. 1987. Die Vögel des Rheinlandes, III. Atlas zur Brutvogelverbreitung.     

Recent advances in the molecular analysis of inherited disease

Many important human genes have been cloned during the last ten years. In some cases, using reverse genetic techniques [Orkin, S. H. (1986) Cell 47, 845-850], disease-causing genes have been isolated whose product was previously unknown. Important examples include the dystrophin protein which, when mutated, gives rise to either Duchenne or Becker muscular dystrophy [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Cell 50, 509-517; Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S. & Kunkel, L. M. (1988) Genomics 2, 90-95; Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228] and the cystic fibrosis transmembrane conductance regulator (CFTR) [Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989) Science 245, 1066-1073]. Recently the technology for systematically detecting single base-pair changes by chemical methods, enzymatic methods or direct DNA sequencing has greatly expanded and simplified. In addition to providing structural information about these clinically important genes and information on disease-causing mutations, these studies have led to an increased understanding of mechanisms of mutation, to the discovery of novel genetic mechanisms and to important clinical applications of carrier detection and pre-natal diagnosis. The recent rapid progress has been made possible by the development of DNA amplification using the polymerase chain reaction (pcr) invented by Saiki and colleagues [Saiki, R. K., Chang, C-A., Levenson, C. H., Warren, T. C., Boehm, C. D., Kazazian, H. H. & Ehrlich, H. A. (1988) N. Engl. J. Med. 319, 537-541].     

Versican modulates gap junction intercellular communication

Versican is a large chondroitin sulfate proteoglycan and belongs to the family of lecticans. Versican possesses two globular domains, G1 and G3 domain, separated by a CS-attachment region. The CS-attachment region present in the middle region is divided into two spliced domains named CSalpha and beta. Alternative splicing of versican generates at least four versican isoforms named V0, V1, V2, and V3. We have successfully cloned the full-length cDNA of chick versican isoforms V1 and V2 and found that versican isoform V1 induced mesenchymal-epithelial transition in NIH3T3 cells. Mesenchymal-epithelial transition induced by V1 in NIH3T3 cells is characterized by expression of E-cadherin and occludin, two epithelial markers, and reduced expression of fibroblastic marker vimentin (Sheng et al., 2006, Mol Biol Cell. 17, 2009-2020). In the present studies, we found that versican V1 isoform not only induced cell transition, but also increased intercellular communication via gap junction channels composed of connexin proteins. Our results showed that V1 induces plasma membrane localization of connexin 43, resulting in increased cell communication. This was further confirmed by blocking assays. Gap junctions mediated the transfer of small cytoplasmic molecules and the diffusion of second messenger molecules between adjacent cells. The ability of versican in regulating gap junction implied a potential role of versican in coordinating functions.     

Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.     

Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin

X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 A) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P2(1), with unit cell dimensions a = 90.3 A, b = 82.9 A, c = 73.4 A, beta = 122.5 degrees for the native Fab and a = 63.9 A, b = 73.0 A, c = 49.1 A, beta = 120.6 degrees for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.     

Membrane binding characteristics of two forms of transforming growth factor-beta

Cartilage-inducing factors A and B (CIF-A and CIF-B) from bovine bone have recently been identified as transforming growth factor-beta (TGF-beta) (Seyedin, S.M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem., 261, 5693-5695) and a unique protein homologous to TGF-beta (Seyedin S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987) J. Biol. Chem., 262, 1946-1949), respectively. Although the biological activities of TGF-beta and CIF-B are similar, the divergence of CIF-B from the highly conserved amino acid sequence of TGF-beta prompted an investigation of its receptor binding properties. Three classes of cell surface binding components were identified. Class A has exclusive affinity for TGF-beta; class B has greater affinity for CIF-B; and class C has equal affinity for both proteins. A high molecular weight component, the predominant binding species, was further characterized and shown to consist of two components that are either class B or class C. The differential binding properties of TGF-beta and CIF-B to cell surface components suggest that there are biological activities unique to each of the proteins.     

Expression of gap junction genes during postnatal neural development

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.