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].     

Proteolytic cleavage of the Fe-S subunit hinge region of Rhodobacter capsulatus bc(1) complex: effects of inhibitors and mutations

The three-dimensional structure of the mitochondrial bc(1) complex reveals that the extrinsic domain of the Fe-S subunit, which carries the redox-active [2Fe2S] cluster, is attached to its transmembrane anchor domain by a short flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatus). In various structures, this extrinsic domain is located in different positions, and the conformation of the hinge region is different. In addition, proteolysis of this region has been observed previously in a bc(1) complex mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokito, M., Zhang, Z., Berry E. A., and Daldal, F. (1998) Biochemistry 37, 8105-8114]. Thus, possible correlations between proteolysis, conformation of the hinge region, and position of the extrinsic domain of the Fe-S subunit within the bc(1) complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge region of the Fe-S subunit between its amino acid residues A46-M47 or D43-V44, respectively, to yield a protease resistant fragment with a M(r) of approximately 18 kDa. The cleavage was affected significantly by ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specific mutations located in the bc(1) complex. In particular, using either purified or detergent dispersed chromatophore-embedded R. capsulatus bc(1) complex, we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutations in various regions of the Fe-S subunit and cyt b subunit changed drastically proteolysis patterns, indicating that the structure of the hinge region of the Fe-S subunit was modified in these mutants. The overall findings establish that protease accessibility of the Fe-S subunit of the bc(1) complex is a useful biochemical assay for probing the conformation of its hinge region and for monitoring indirectly the position of its extrinsic [2Fe2S] cluster domain within the Q(o) pocket.     

Novel mutation in spectrin-like repeat 1 of dystrophin central domain causes protein misfolding and mild Becker muscular dystrophy

Mutations in the dystrophin gene without disruption of the reading frame often lead to Becker muscular dystrophy, but a genotype/phenotype correlation is difficult to establish. Amino acid substitutions may disrupt binding capacities of dystrophin and have a major impact on the functionality of this protein. We have identified two brothers (ages 8 and 10 years) with very mild proximal weakness, recurrent abdominal pain, and moderately elevated serum creatine kinase levels. Gene sequencing revealed a novel mutation in exon 11 of the dystrophin gene (c.1280T>C) leading to a L427P amino acid substitution in repeat 1 of the central rod domain. Immunostaining of skeletal muscle showed weak staining of the dystrophin region encoded by exons 7 and 8 corresponding to the end of the actin-binding domain 1 and the N-terminal part of hinge 1. Spectrofluorescence and circular dichroism analysis of the domain repeat 1-2 (R1-2) revealed partial misfolding of the L427P mutated protein as well as a reduced refolding rate after denaturation. Based on computational homology models of the wild-type and mutated R1-2, a molecular dynamics study showed an alteration in the flexibility of the structure, which also strongly affects the conformational space available in the N-terminal region of the fragment. Our results suggest that this missense mutation hinders the dynamic properties of the entire N-terminal region of dystrophin.     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

Embryonic axis induction by the armadillo repeat domain of beta- catenin: evidence for intracellular signaling

beta-catenin was identified as a cytoplasmic cadherin-associated protein required for cadherin adhesive function (Nagafuchi, A., and M. Takeichi. 1989. Cell Regul. 1:37-44; Ozawa, M., H. Baribault, and R. Kemler. 1989. EMBO [Eur. Mol. Biol. Organ.] J. 8:1711-1717). Subsequently, it was found to be the vertebrate homologue of the Drosophila segment polarity gene product Armadillo (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science [Wash. DC]. 254:1359-1361; Peifer, M., and E. Wieschaus. 1990. Cell. 63:1167-1178). Also, antibody perturbation experiments implicated beta-catenin in axial patterning of the early Xenopus embryo (McCrea, P. D., W. M. Brieher, and B. M. Gumbiner. 1993. J. Cell Biol. 123:477-484). Here we report that overexpression of beta-catenin in the ventral side of the early Xenopus embryo, by injection of synthetic beta-catenin mRNA, induces the formation of a complete secondary body axis. Furthermore, an analysis of beta-catenin deletion constructs demonstrates that the internal armadillo repeat region is both necessary and sufficient to induce axis duplication. This region interacts with C-cadherin and with the APC tumor suppressor protein, but not with alpha-catenin, that requires the amino-terminal region of beta-catenin to bind to the complex. Since alpha-catenin is required for cadherin-mediated adhesion, the armadillo repeat region alone probably cannot promote cell adhesion, making it unlikely that beta-catenin induces axis duplication by increasing cell adhesion. We propose, rather, that beta-catenin acts in this circumstance as an intracellular signaling molecule. Subcellular fractionation demonstrated that all of the beta-catenin constructs that contain the armadillo repeat domain were present in both the soluble cytosolic and the membrane fraction. Immunofluorescence staining confirmed the plasma membrane and cytoplasmic localization of the constructs containing the armadillo repeat region, but revealed that they also accumulate in the nucleus, especially the construct containing only the armadillo repeat domain. These findings and the beta-catenin protein interaction data offer several intriguing possibilities for the site of action or the protein targets of beta- catenin signaling activity.     

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.     

DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes

Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.     

Proteolytic susceptibility of the central domain in chicken gizzard and skeletal muscle dystrophins.

We investigated proteolytic susceptibility of the central domain in dystrophin molecules from chicken smooth and skeletal muscles. Dystrophin-enriched preparations from both muscles were made as described in Pons et al. (Proc. Natl. Acad. Sci. USA (1990) 87, 7851-7855). These preparations contained other protein components in addition to dystrophin. Three enzymes (Staphylococcus aureus proteinase, chymotrypsin and trypsin) having different proteolytic specificities were used. Time-courses of proteinase degradation were examined by the Western immunoblot technique using a specific polyclonal serum directed against a fragment (residues 1173-1728) of the dystrophin central domain. We observed accumulation of some major proteinase-resistant fragments, in the 110-160 kDa range originating from that central region of the molecule. Cleavage patterns of the smooth and skeletal muscle preparations were quite similar, but molecular weights of the breakdown products differed slightly. Interpretation of the results was based on two predictive structural models of the dystrophin central domain (Koenig and Kunkel (1990) J. Biol. Chem. 265, 4560-4566 and Cross et al. (1990) FEBS Lett. 262, 87-90). Skip residues at the end of repeat 13 (around the 1740th residue of the dystrophin amino acid sequence), as hypothesized in the Cross model, constitute probably the most sensitive site within the dystrophin central domain for any exogenous (or even endogenous) proteinase. Variations observed between dystrophins from skeletal and smooth muscles also suggest that the structures of both dystrophins differ slightly even within the dystrophin central domain. This precise identification of proteinase-resistant dystrophin fragments of variable lengths is a first step towards further physicochemical studies on the very large and rare dystrophin molecule.     

Molecular extensibility of mini-dystrophins and a dystrophin rod construct

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.     

Modeling of the human intercellular adhesion molecule-1, the human rhinovirus major group receptor

A model has been built of the amino-terminal domain of the intercellular adhesion molecule-1 (ICAM-1), the receptor for most human rhinovirus serotypes. The model was based on sequence and presumed structural homology to immunoglobulin constant domains. It fits well into the putative receptor attachment site, the canyon, on the human rhinovirus-14 (HRV14) surface in a manner consistent with most of the mutational data for ICAM-1 (Staunton, D. E., Dustin, M. L., Erickson, H. P., Springer, T. A. Cell, in press, 1989) and HRV14 (Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M. E., Murcko, M. A. Proc. Natl. Acad. Sci. U.S.A. 85: 5449-5453, 1988).     

The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes.

Previous studies have shown that nuclear lamin B binds specifically to the C-terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B-agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles.     

Utrophin lacks the rod domain actin binding activity of dystrophin

We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.     

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).     

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D)

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.     

Thyrotropin-releasing hormone (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.     

Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse

Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc) (Partridge, T. A., J. E. Morgan, G. R. Coulton, E. P. Hoffman, and L. M. Kunkel. 1989. Nature (Lond.). 337:176-179). However, it is difficult to determine whether this biochemical "rescue" results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration (Coulton, G. R., N. A. Curtin, J. E. Morgan, and T. A. Partridge. 1988. Neuropathol. Appl. Neurobiol. 14:299- 314). By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily (Wakeford, S., D. J. Watt, and T. A. Patridge. 1990. Muscle & Nerve.) Here, we have implanted normal mpc, obtained from newborn mice, into such preirradiated mdx muscles, finding that it is far more extensively permeated and replaced by implanted mpc than is nonirradiated mdx muscle; this is evident both from analysis of glucose-6-phosphate isomerase isoenzyme markers and from immunoblots and immunostaining of dystrophin in the treated muscles. Incorporation of normal mpc markedly reduces the loss of muscle fibers and the deterioration of muscle structure which otherwise occurs in irradiated mdx muscles. Surprisingly, the regenerated fibers are largely peripherally nucleated, whereas regenerated mouse skeletal muscle fibers are normally centrally nucleated. We attribute this regeneration of apparently normal muscle to the tendency of newborn mouse mpc to recapitulate their neonatal ontogeny, even when grafted into 3-wk-old degenerating muscle.     

Isolation and sequence of a tropomyosin-binding fragment of turkey gizzard calponin

Limited chymotryptic cleavage of turkey gizzard calponin yields a 13 kDa fragment which could be purified by its ability to bind to Sepharose-immobilized tropomyosin. This 13 kD polypeptide is shown to be derived from a 22 kDa fragment. Complete amino acid sequence analysis of the 13 kD and 22 kD fragments reveals high homology with the formerly characterized smooth muscle-specific protein SM22 alpha (Pearlstone, J.R., Weber, M., Lees-Miller, J.P., Carpenter, M.R. and Smillie L.B., 1987, J. Biol. Chem. 262, 5985-5991) and the product of gene mp20 of Drosophila (Ayme-Southqate, A., Lasko, P., French, C, and Pardue, M.L. [(1989) J. Cell Biol. 108, 521-531]. Futhermore we recognize sequence elements of a putative actin-binding domain of alpha-actinin, the calpactin I or p 36 sequence, and a consensus motif present in the repeats of the gene product of the candidate unc-87 gene of C. elegans (S.D. Goetinck and R.H. Waterston, personal communication).     

T- and B-lymphocyte responses to human immunodeficiency virus (HIV) type 1 in macaques immunized with hybrid HIV/hepatitis B surface antigen particles.

Recombinant human immunodeficiency virus (HIV)/hepatitis B surface antigen (HBsAg) subviral particles of dual antigenicity and immunogenicity were obtained by fusing 84 amino acids of the HIV type 1 external envelope glycoprotein within the pre-S2 part of the hepatitis B middle protein (M.-L. Michel, M. Mancini, E. Sobczak, V. Favier, D. Guétard, E.-M. Bahraoui, and P. Tiollais, Proc. Natl. Acad. Sci. USA 85:7957-7961, 1988). We now describe the humoral and cellular immune response of rhesus monkeys immunized with these hybrid particles. Macaque antisera raised by subcutaneous injections of the HIV/HBsAg particles were shown to be specific for HIV in peptide-binding assays. Moreover, we were able to generate in these vaccinated animals a T-cell-proliferative response to both parts of the hybrid particle, i.e., HIV and HBsAg. These results establish the presence of a T-cell epitope in this HIV segment, which has been shown previously (L.A. Lasky, G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon, Cell 50:975-985, 1987) to be an important domain involved in the binding of the virus to its cellular receptor, the CD4 molecule. This work demonstrates the feasibility of using the HBsAg subviral particle as a carrier protein for the presentation of foreign immunogenic epitopes to the immune system.