Monoclonal antibodies targeted against the C-terminal domain of dystrophin or utrophin.

The structure-function relationships of dystrophin, a protein which is absent or defective in patients with Duchenne or Becker muscular dystrophies, and utrophin can only be compared if specific antibodies are produced. We expressed C-terminal parts of dystrophin and utrophin in expression vectors. Mice were immunized with recombinant proteins and 26 monoclonal antibodies were produced and analyzed. Their respective epitopes were determined using other overlapping recombinant products. We observed antibody specificity towards 400 kDa dystrophin and/or utrophin protein bands, either by Western blot analysis or immunodetection in human skeletal (quadriceps) and smooth (uterus) muscles. These antibodies have been used to compare the relative abundance of both dystrophin and utrophin relative to the structures analyzed.

Expression of myosin heavy chain isoforms in Duchenne muscular dystrophy patients and carriers.

The expression of MHC isoforms in the skeletal muscles of nine patients with Duchenne muscular dystrophy (DMD) (from 2.5 to 15 yr of age) and three DMD carriers was studied using different specific anti-MHC MAbs. We also analyzed muscle fiber size and fiber reactivity with acridine orange and/or with a surface antigen marker. One-quarter of all fibers of DMD patients, or less with age, were of normal size and contained only adult slow MHC. Half of the muscle fibers contained adult and developmental MHCs. Only half of these fibers were representative of an active regenerative process. MHC co-expression also altered the proportion of normal fast or slow fibers. Adult fast MHCs were expressed as unique MHC only in small and very small fibers in the oldest DMD patients. In DMD carrier muscles, the greatest alterations in MHC expression were observed in patients with the most reduced dystrophin expression. However, MHC changes in dystrophin-positive fibers were similar to those observed in dystrophin-free fibers. In conclusion, disruptions or delays in the switching of all genes coding for adult fast and slow MHC and developmental MHC coincided with dystrophin deletion and with perturbations in its expression.

Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system.

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining. These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.

Anti-myosin heavy chain monoclonal antibodies reveal two IIB (fast) fiber subtypes.

Indirect immunofluorescence analysis of different rat skeletal muscles using anti-myosin heavy chain (MHC) monoclonal antibodies (MAb) revealed the presence of two immunologically distinct kinds of fibers within the IIB fibers, histochemically identified by myosin ATPase staining. Some IIB fibers (designated here as IIB1) were unreactive with one anti-fast MHC MAb, whereas they did react with another anti-fast MHC MAb; other IIB fibers (designated here as IIB2) reacted with both anti-fast MAbs. Neither of the two IIB fiber subtypes was significantly reactive with a neonatal MHC MAb. The number of each IIB fiber subtype was age-dependent, at least in the plantaris muscle. IIB1 fibers were observed only in the superficial portion of the plantaris and gastrocnemius muscle. The ratio of IIB1:IIB2 fibers was about the same throughout the extensor digitorum longus and extraocular muscles. Therefore, the two kinds of IIB fibers here observed have a different myosin heavy chain content. On the basis of their specific immunoreactivities, we suggest that IIB1 fibers contain the previously described MHCB. IIB2 fibers contain either a unique new MHC isoform or a mixture of at least two MHC, possibly composed of the MHCB and either the previously described MHCA or a new MHC isoform.

A homologue of dystrophin is expressed at the blood vessel membrane of DMD and BMD patients: immunological evidence.

Muscles from Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) patients were analysed using monoclonal and polyclonal antibodies raised against different regions of the dystrophin molecule. On blot, two of the antibodies detected a protein of Mr 400K in muscle extracts from all patients, including a BMD patient with a deletion which spanned more than 40% of the central rod domain of the Xp21 encoded dystrophin. Immunocytochemical labelling of tissue sections from the same patients showed that the same two antibodies labelled a protein at the surface membrane of smooth muscle fibers in blood vessels of both BMD and DMD muscles. Thus we have demonstrated a 400K blood vessel-associated protein, which is immunologically homologous with dystrophin, for at least two epitopes from the carboxy terminal and the central rod domains must be encoded by another gene than the dystrophin gene.

Isolation of L-forms from the spleens of Brucella suis-infected, penicillin-treated mice.

Previous attempts to obtain in vitro wall-deficient stable L-forms of various strains of Brucella have failed because the obtained spheroplasts revert quickly to bacterial form. Here, we report the isolation of L-forms from mice infected with a B. suis strain type 1 and treated with penicillin. In defined experimental conditions, L-type microcolonies associated with tissue debris were observed in primary spleen cultures, even on antibiotic free media. After several transfers on penicillin-containing medium. typical, tissue-free L colonies were obtained. At first, when cultivated on antibiotic-free medium, these colonies reverted to the bacterial form (identified as B suis, biotype 1). Later, after approximately fifteen transfers on penicillin-supplemented medium, they no longer reverted even after several subcultures on antibiotic-free medium. The L-forms' ultrastructural features included many giant empty bodies, considerable variation in size, shape and density of the wall-deficient cells, and many multilayered membranes. The stabilized L-forms were propagated in vitro and inoculated into mice, and then recovered from their spleens as tissue associated L-microcolonies. An occasional in vivo revertant was identified as B. suis, biotype 1. These data provide one possible explanation for earlier failures to detect the presence of atypical bacteria in clinical or experimental Brucella infections.

Partial deafferentation of the developing rat spinal cord delays the spontaneous repression of GAD67 mRNAs in spinal cells.

Early and ubiquitous detection of GABA in the rat spinal cord before the occurrence of synaptogenesis has led to the concept of a neurotrophic role of GABA, in addition to a promoting effect on neurite extension and neurodevelopment. The aim of this study was to further establish, in vivo, evidence for a link between the maturation of spinal cord innervation and the regulation of several isoforms of the synthetic enzymes of GABA, the glutamic acid decarboxylases GAD65, GAD67, and EP10, the embryonic truncated form of GAD67. Neonatal capsaicin treatment was used to induce a specific loss of afferent fibers (unmyelinated C fibers, thin myelinated fibers A delta) to the dorsal horn. The regulation of various GAD mRNAs was investigated using sensitive techniques such as RT-PCR and in situ hybridization. The sensitivity of the methods was further enhanced by the use of a gaseous detector (beta-imager) to quantitate the mRNAs species. After neonatal capsaicin treatment, higher levels of GAD67 mRNA were detected transiently during the postnatal development of the rat spinal cord. A maximum two-fold increase of GAD67 mRNA was found on the day following the capsaicin injection and reached control values within 3 weeks. In contrast, GAD65 mRNA levels remained low and were unaffected by the treatment, and EP10 was not detected. In addition, we have found a similar upregulation, with the same time course, of the cytoskeletal protein beta-actin. The capsaicin-induction of mRNA synthesis was, however, two-fold greater for beta-actin than for GAD67. Moreover, since this upregulation of GAD67 mRNA coincides with the sprouting of unaffected afferent fibers and of 5HT axons, one can hypothesize that GAD67 participates in the structural plasticity occurring in reaction to the capsaicin-induced partial deafferentation.

Ultrastructural localization of dystrophin in chicken smooth muscle.

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.

Local changes in myosin types in diseased human atrial myocardium: a quantitative immunofluorescence study.

Two monoclonal antibody groups were prepared from adult human atrial and ventricular myosin heavy chains. Using these two groups, we were able to identify two myosin variants in human atria and to classify human atrial fibers into alpha-, mixed, or beta-fibers according to the reactivity of the two monoclonal antibody groups to alpha- and beta-myosin heavy chains of normal young and hypothyroid rat ventricles, respectively. The alpha-fiber percentage of the left atria in two normal human hearts was 15% higher than in the right atria. The auricles contained two to three times more alpha-fibers than beta-fibers, whereas the proportion was reversed in the crista terminalis. The mean fiber diameter of the alpha- and beta-fibers was 13.6 +/- 3 micron. A complete alpha- to beta-fiber transition was observed in all atrial regions of two hearts with severe ventricular myocardial infarction; a moderate alpha- to beta-fiber transition was observed only in the left atria of two hearts with primary congestive cardiomyopathy. The mean diameters of the two fiber types were significantly increased in all diseased hearts (19 +/- 3.8 micron). We hypothesize that pressure overload and increased wall tension successively induce an enlargement of the fiber diameter and an alpha- to beta-myosin transition.

Comparative distribution of GAD65 and GAD67 mRNAs and proteins in the rat spinal cord supports a differential regulation of these two glutamate decarboxylases in vivo.

Gamma-aminobutyric acid (GABA) synthesis can result from the action of at least two glutamic acid decarboxylase (GAD) isoforms, GAD65 and GAD67, possibly involved in distinct mechanisms. We have made the hypothesis that GAD65 may respond to short-term changes and is present in neurons with a phasic activity, while GAD67 may rather provide GABA for the metabolic pool and for supporting tonic levels of synaptic transmission (Erlander et al.: Neuron 7:91-100, 1991; Feldblum et al.: J Neurosci Res 34:689-706, 1993). In the present work we have tested this hypothesis in the rat spinal cord where both types of activities have been identified. The correlation of GABA immunodetection with the distribution of GAD65 and GAD67 mRNAs and proteins has evinced in the dorsal horn a differential regulation of the two isoforms. In situ hybridization has revealed, in the dorsal horn, relatively higher levels of GAD67 mRNA than of GAD65, while immunodetection of the proteins demonstrated numerous punctate profiles with both GAD antisera. Reverse transcription-polymerase chain reaction (RT-PCR) data confirmed the abundance of the GAD67 transcripts compared to GAD65 in the rat spinal cord. In contrast, within the ventral horn, there was a greter number of GAD67-immunoreactive (IR) profiles mostly located around motoneurons. The paucity of GAD65 immunoreactivity in the ventral horn cannot be related to a different accessibility of the antigens to the epitopes since on the same section a dense GAD65 staining was detected in the dorsal horn. Hence, a number of biochemical and electrophysiological data support the concept of the involvement of glycine as the major inhibitory system within the ventral horn which may explain the low levels of GAD transcription in this region. The paucity of GAD65 in the ventral horn may also reflect a functional difference, suggesting a predominance of GAD67 in neurons under tonic activity. In the dorsal horn, where neurons with phasic and tonic firing patterns have been disclosed, GAD65 may, in addition, provide GABA for responses to short-term changes.

Probing functional regions in cardiac isomyosins with monoclonal antibodies.

Seven Mabs prepared against subfragment 1 (S1) of either bovine cardiac beta-specific or rabbit fast skeletal muscle myosin were used to identify functional regions in cardiac isomyosin heavy chains. This approach was designed to improve the understanding of structure-function relationships within the myosin molecule and between alpha and beta myosin heavy chains (MHCs). We used bacterial expression of human beta myosin fragments and determined that the seven antibodies were localized within four different MHC subdomains: amino acid residues 33-37 (one beta-specific antibody), 67-84 (one alpha/beta-specific antibody), 85-106 (four alpha/beta-specific antibodies) and 215-248 (one alpha/beta-specific antibody). All epitopes were accessible on myosin and actomyosin with the same affinities. Therefore, none of these MHC epitopes were located on the interfaces between the myosin head and actin. Three antibodies reacting at three out of the four investigated epitopes enhanced acto-S1 ATPase activities but not myosin, S1, or actomyosin activities. One antibody, which was strictly beta-specific and bound to five amino acid residues near the most N-terminal MHC end, substantially inhibited all myosin or S1 ATPase activities measured with or without actin. The epitope of this antibody coincides with one difference cluster observed between both cardiac MHC isoforms [McNally et al (1989) J. Mol. Biol. 210, 665-671], suggesting that this small variable MHC area could be one of the structural bases to explain observed functional differences in cardiac alpha and beta myosin isoforms.

Probing conformational changes within LC2 domains of cardiac myosin.

Nine monoclonal antibodies were used to test calcium and EDTA effects on the molecular conformation of ventricular VLC2 within myosin. Antibody epitopes were located in six domains of VLC2 using recombinant proteins. The apparent association constants of these antibodies were measured in solution in the presence of calcium or EDTA. An immunofluorescence study was performed to establish whether the observed effects would occur in more integrated systems, as compared to isolated proteins in solution. Our results showed (1) a slight effect of calcium on isolated VLC2, located in the aa 29-45 domain, (2) a clear-cut effect of calcium on VLC2 within myosin, only in the aa 45-59 domain, and (3) in the presence of EDTA, antibody affinities for VLC2 within myosin similar to the affinities for isolated VLC2. These results are discussed in terms of spatial arrangements and binding mechanisms between HC and VLC2. They suggest that there are two processes for stabilizing HC/VLC2 complex formation: one binding via calcium chelation and another involving hydrophobic interactions.

Probing myosin light chain 1 structure with monoclonal antibodies.

Five monoclonal antibodies that react with different regions of myosin light chain 1 from human ventricular myocardial muscle were used to obtain information on interactions between the light chain 1 and heavy chains and generally on the tertiary structure of the light chain 1 within the myosin head. We performed Western blot assays of the five antibodies with myosins from different cardiac and skeletal muscles, with different proteolytic fragments of bovine ventricular myosin light chain 1 (LC1) and to different recombinant fragments of human ventricular LC1 and rat fast skeletal light chain LC1/LC3. The five antibodies were mapped in three different regions of the light chain 1: two antibodies mapped within the first eight amino-terminal residues, two between residues 71 and 74, and one between residues 129 and 134. The apparent dissociation constants of the last three antibodies, determined by antibody-antigen equilibria in solution, were lower than when isolated light chains were used as antigens. It is probable that the corresponding amino acids involved in the antibody epitopes were either involved in interactions between the light and heavy myosin subunits, or somehow hindered by the myosin heavy chain bulk. In contrast, the apparent dissociation constants measured for both other antibodies were higher when myosin, rather than isolated light chains, was used as antigen. Thus LC1 fixation to heavy chains within the myosin molecule induced conformation changes at the amino-terminal end of the light chain 1. No difference in the accessibility of this mobile LC1 segment was detected in the presence of actin. Finally, observed differences in epitope accessibility on the light chain LC1 in myosin, as compared with chymotryptic subfragment 1 (SF1), indicated conformational differences between native myosin and extensively studied SF1 molecules.