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Humans and mice with deficiency of the alpha2 subunit of the basement membrane protein laminin-2/merosin suffer from merosin-deficient congenital muscular dystrophy (MCMD). We have expressed a human laminin alpha2 chain transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restores the synthesis and localization of merosin in skeletal muscle, and greatly improves muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the nontransgenic dystrophic mice a progressive lameness of hind legs, suggestive of a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD. Future gene therapies of human MCMD, and perhaps of other forms of muscular dystrophy, may require restoration of the defective gene product in multiple tissues.

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis., (Copyright 1998 Academic Press.)

Background: Estrogen receptor (ER)-positive breast carcinomas possess a less aggressive phenotype than ER-negative breast carcinomas. We hypothesize that a set of genes exists that is expressed only in ER-negative breast carcinomas, which account for the more malignant phenotypic characteristics of these tumors., Methods: We have used a new technique of polymerase chain reaction select suppression subtractive hybridization to identify genes that are expressed only in ER-negative carcinomas., Results: Seventy-one cDNA clones generated by suppression subtractive hybridization were screened by Northern blot analysis with RNA from ER-positive MCF7 and ER-negative MDA-MB-231 breast carcinoma cell lines. Fifteen clones were differentially expressed in MDA-MB-231 cells. Five of these 15 clones were consistently found to be associated with the ER-negative phenotype in a panel of eight breast carcinoma cell lines. Sequence analysis demonstrated that three of these clones were derived from vimentin and two clones from moesin. Western blot analysis with antihuman moesin antibody confirmed that moesin protein was overexpressed in ER-negative breast carcinoma cell lines but absent from ER-positive breast carcinomas. Moesin mRNA was examined in a panel of 29 primary breast carcinomas with semi-quantitative reverse transcriptase-polymerase chain reaction. Moesin expression was found to be decreased significantly in ER-positive compared with ER-negative tumors (P < .01)., Conclusions: Vimentin and moesin are differentially expressed in association with the ER-negative breast cancer phenotype. Moesin is a membrane/actin filament protein involved in dynamic restructuring of the cell surface and filopodia, a cell structure needed for cell adhesion and motility. Moesin may play a role in the invasiveness and pattern of metastasis characteristic of ER-negative breast cancers.

Mutations in the gene coding for the alpha 2 chain of laminin-2 and -4 (merosin) cause a severe form of congenital muscular dystrophy in humans and mice. To establish a defined model for in vitro and in vivo studies of the role of laminin alpha 2/merosin in development and cell and tissue function, we generated several lines of mutant embryonic stem (ES) cell with disruption of the laminin alpha 2 chain gene. We find that homozygous mutant ES cells differentiate normally in vitro, giving rise to cardiomyocytes, myotubes, and smooth muscle cells in addition to many other cell types. However, the myotubes that are formed are unstable. They detach, collapse, and degenerate, a process which is initiated at the appearance of the mature, contractile phenotype of the cells. We propose that the detachment and death of contracting myotubes in vitro has its counterpart in vivo and that contraction-induced myofiber damage, along with the lack of survival cues provided by laminin alpha 2/merosin, is a significant contribution to muscle degeneration in merosin-deficient muscular dystrophy.

Differences in gene expression are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast cancer. We have identified differentially expressed cDNAs in the estrogen receptor (ER)-positive MCF7 breast carcinoma cell line compared with the ER-negative MDA-MB-231 breast carcinoma cell line. Differential screening isolated four differentially expressed genes: cytokeratin 8, cytokeratin 18, Hsp27 and GPCR -Br. To identify differentially expressed genes of lower abundance, suppression subtractive hybridization was utilized and 29 differentially expressed clones were isolated. Sequence analysis revealed that 11 clones were from previously described genes: HEK8, neuropeptide Y receptor Y1, p21 WAF-1, p55 PIK, cytokeratin 18 (cloned twice), fructose-1,6-biphosphatase, cytokeratin 8, TGFbeta1 binding protein, elongation factor 1alpha2 and pS2. The remaining 18 clones did not match sequences in the GenBank/EMBL database, indicating that they may be novel genes. Expression of pS2, neuropeptide Y receptor Y1 and three novel clones was induced by estradiol, indicating estrogen-responsiveness. The expression pattern of one novel gene, DEME -6, correlated with expression of ER and ERF -1/ AP -2gamma in a panel of breast carcinoma cell lines. A 2.6 kb cDNA of DEME -6 was sequenced and contains an open reading frame of 574 amino acids that demonstrates 62.4% similarity with a gene from Caenorhabditis elegans chromosome III. Expression of DEME -6 was also detected in primary breast carcinomas but not in normal breast tissue, as determined by RT-PCR. These findings support the hypothesis that a set of genes coordinately regulated with ER , but not necessarily estradiol-responsive, are characteristic of the hormone-responsive breast cancer phenotype.

Low-frequency dielectric behavior of membrane/electrolyte systems was studied with artificial membranes made from thin plastic films and NaCl electrolyte of various concentrations. A low-frequency dielectric dispersion or relaxation, which is associated with membrane pores and membrane permeability, was observed and investigated. A qualitative explanation is proposed. Membrane structure characteristics, such as thickness, pore size, and number, were found to affect significantly the low-frequency dielectric dispersion, which indicates some potential applications for low-frequency dielectric measurements in membrane- or tissue-related areas. Copyright 1997 Academic Press. Copyright 1997Academic Press

Adhalin, or alpha-sarcoglycan, is a 50-kDa glycoprotein that was originally characterized as a muscle membrane protein. The importance of adhalin is suggested by the diseases associated with its absence, notably the limb-girdle muscular dystrophies. However, the function of adhalin is unknown. To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation in vitro. The proper localization of adhalin to the muscle cell membrane was observed only in late stages of myotube maturation, coincident with the re-distribution of caveolin-3 and dystrophin. These data suggest that adhalin is highly specific for striated muscle and that it is linked with the formation of a fully functional muscle fiber.

Cardiotrophin-1 (CT-1) is a new member of the interleukin-6 cytokine family that was identified from a mouse embryoid body cDNA library by expression cloning. Mouse CT-1 induces features of hypertrophy in neonatal rat cardiac myocytes and binds to and activates the leukaemia inhibitory factor/gp130 receptor complex. In this work we report the isolation and characterization of cDNA and genomic clones encoding human CT-1. These clones encode a 201 amino acid protein that is 80% identical to the mouse protein. Human CT-1 produced by transfection of the cDNA clones into mammalian cells induces the hypertrophy of neonatal rat cardiac myocytes. Human and mouse CT-1 bind to the leukaemia inhibitory factor receptor on both human and mouse cell lines indicating a lack of species specificity. No binding to the human oncostatin M specific receptor was detected. A 1.7 kb CT-1 mRNA is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung. The coding region of CT-1 is contained on three exons and is located on human chromosome 16p11.1-16p11.2.

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q27-28.

Physiological platelet synthesis is thought to require the humoral activities of meg-CSF and thrombopoietin, which respectively promote proliferation and maturation of megakaryocytic cells. A meg-CSF/thrombopoietin-like protein that is present in plasma of irradiated pigs has been purified and cloned. This protein binds to and activates the c-mpl protein, a member of the cytokine receptor superfamily. The isolated Mpl ligand shares homology with erythropoietin and stimulates both megakaryocytopoiesis and thrombopoiesis.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

CDC42, a member of the Rho family of small GTP-binding proteins, regulates cytoskeletal rearrangements required for cell division. Activating mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-binding proteins, CDC42 is activated by a guanosine exchange factor and inactivated by a GTPase-activating protein (GAP). An unidentified 25-kDa platelet protein has been shown to function as a specific CDC42GAP. Here we report the cloning of a cDNA encoding this GAP from a human platelet-precursor cell line. Sequence analysis reveals the presence of three consensus box regions characteristic of rhoGAPs. A glutathione S-transferase fusion protein containing the three boxes derived from the new clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA clone encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85 alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.

Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR. A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor. We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini. Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein. Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR. Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts. These results demonstrate that guanylin is an endogenous activator of STaR.

IL-8 is a proinflammatory cytokine that functions as a chemoattractant for neutrophils. Recently, cDNA clones encoding the human neutrophil IL-8R were isolated by an expression cloning strategy. The amino acid sequence of the human IL-8R was sufficiently similar to a published sequence for an isoform of the rabbit FMLP receptor that we considered the possibility that the rabbit sequence might bind IL-8 as well. In order to establish its ligand specificity, we have isolated and characterized cDNA clones encoding the rabbit receptor. These cDNA clones, when expressed in mammalian cells, confer high affinity IL-8 binding (Kd = 3.6 nM), lack detectable binding of FMLP, and produce a transient increase in the intracellular Ca2+ concentration in response to IL-8 but not to FMLP. These data demonstrate that the reported rabbit FMLP receptor is the rabbit IL-8R, not an isoform of the FMLP receptor. In addition, the amino acid sequence of the rabbit IL-8R encoded by these cDNA clones differs at 23 amino acids (of 355) from that previously published.

Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.

The sequence of 20,114 bp of DNA including the human glucose-6-phosphate dehydrogenase (G6PD) gene was determined. The region included a prominent CpG island, starting about 680 nucleotides upstream of the transcription start site, extending about 1050 nucleotides downstream of the start site, and ending just at the start of the first intron. The transcribed region from the start site to the poly(A) addition site covers 15,860 bp. The sequence of the 13 exons agreed with published cDNA sequence and for the 11 exons tested, with the corresponding sequence in a yeast artificial chromosome (YAC). The latter confirms YAC cloning fidelity at the DNA sequence level. Sixteen Alu sequences constitute 24% of the total sequence tract. Four were outside the borders of the mRNA transcript of the gene; all the others were found in a large (9858 bp) intron between exons 2 and 3. Two Alu clusters each contain Alus lying between the monomers of another.

Structural features of v-kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto-oncogene coding for a receptor tyrosine kinase confirmed this possibility: c-kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF-1) and the receptor for platelet-derived growth factor. The c-kit gene is widely expressed as a single, 5-kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c-kit peptide antibody permitted the identification of a 145,000 dalton c-kit gene product that is inserted in the cellular plasma membrane and is capable of self-phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c-kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy- and amino-terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v-kit.

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells, respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins, in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF.

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.

The human albumin gene spans 16,961 nucleotides from the putative "Cap" site to the first poly(A) addition site. It is split into 15 exons by 14 intervening sequences which are symmetrically placed within the three domains of albumin. The 5' region is highly conserved up to position -250 and contains the putative TATA (-32) and CAT (-88) boxes. A consensus 5' splice sequence reads /GTAGAGT while the 3' splice sequence is pyrimidine rich and contains CTAG/ at the splice junction. The gene contains three polyadenylation signals, and this 3' region presumably arose by triplication of a shorter fragment prior to mammalian radiation. The albumin gene exhibits a high degree of DNA polymorphism and appears to have been recently invaded by Alu repetitive sequences.

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.

Protein F1 is a neuron-specific, synaptic-enriched, membrane-bound substrate of protein kinase C (PKC) whose phosphorylation is related to synaptic plasticity in the adult. The sequence of 26 N-terminal amino acids was determined from purified rat protein F1. A 78-mer synthetic oligonucleotide designed from the partial N-terminal sequence enabled identification of protein F1 cDNA clones in a rat brain library. F1 protein is a 226 amino acid protein encoded by a 1.5 kb brain-specific, developmentally-regulated mRNA. Transcripts for protein F1 can be detected at birth, and their level declines after maturation. A full-length cDNA clone was transcribed and translated in vitro. Translation products could be immunoprecipitated with anti-F1 antibodies. In situ hybridization analysis revealed protein F1 transcripts in hippocampal pyramidal cells, but not in granule cells. In cerebellum, granule cells contained protein F1 mRNA, while Purkinje cells did not. Co-localization of protein F1 with protein kinase C-II [PKC-II (beta)], rather than PKC-I (gamma) suggests that PKC-II may phosphorylate protein F1.

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.

Lipoprotein(a) is an LDL-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) which is disulphide-linked to apolipoprotein B-100. Sequencing of cloned human apolipoprotein(a) complementary DNA shows that it is very similar to human plasminogen. It contains a serine protease domain and two types of plasminogen-like kringle domains, one of which is present in 37 copies.

The primary structure of human uromodulin, a 616-amino acid, 85-kilodalton glycoprotein with in vitro immunosuppressive properties, was determined through isolation and characterization of complementary DNA and genomic clones. The amino acid sequence encoded by one of the exons of the uromodulin gene has homology to the low-density-lipoprotein receptor and the epidermal growth factor precursor. Northern hybridization analyses demonstrate that uromodulin is synthesized by the kidney. Evidence is provided that uromodulin is identical to the previously characterized Tamm-Horsfall glycoprotein, the most abundant protein in normal human urine.