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Biomedical subjects

J A Rafael

Publications and source records attributed to J A Rafael.

16 recordsLinked to original sources
PubMed1998

mdx muscle pathology is independent of nNOS perturbation.

In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS-dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.

Animals↗
PubMed1997

Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.

Genetic defects in a number of components of the dystrophin-glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy2J/dy2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin alpha2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.

Animals↗
PubMed1997

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Animals↗
PubMed1997

Contractile properties of diaphragm muscle segments from old mdx and old transgenic mdx mice.

Diaphragm muscles of young (4- to 6-mo-old) mdx mice show severe fiber necrosis and have normalized forces and powers 60 and 46% of the values for control C57BL/10 mice. In contrast, microinjection of mdx mouse embryos with a truncated dystrophin minigene has produced young transgenic mdx (tg-mdx) mice with a level of dystrophin expression and structural and functional properties of diaphragm muscle strips measured in vitro not different from those of control mice. Whether dystrophin expression and functional corrections persist for the life span of these animals is not know. We tested the null hypothesis that, in old (24 mo) tg-mdx mice, dystrophin expression is adequate and diaphragm muscle strips have forces and powers not different from values for diaphragm muscle strips from young tg-mdx mice or control mice. Compared with control values, diaphragm muscle strips from old mdx mice had normalized forces and powers of 48 and 31%, respectively. Expression of dystrophin persisted in diaphragm muscles of old tg-mdx mice, and functional properties were not different from diaphragm muscles of young tg-mdx or young or old control mice. These results suggest that, with a transgenic animal approach, dystrophin expression and functional corrections persist for the life span of the animals.

Aging↗
PubMed1997

Interactions between dystrophin and the sarcolemma membrane.

Dystrophin serves as a link between the subsarcolemmal cytoskeleton and the extracellular matrix. The NH2 terminus attaches to the cytoskeleton, while the COOH terminus attaches to the dystrophin associated protein (DAP) complex, which can be separated into the dystroglycan, sarcoglycan, and syntrophin subcomplexes. While the function of each DAP is not known, the dystroglycan complex binds laminin in the extracellular matrix, and binds the dystrophin COOH terminus in vitro. The syntrophins also bind the dystrophin COOH terminus in vitro, but no evidence has been reported for an interaction between dystrophin and the sarcoglycans. Human mutations have been found in dystrophin, the sarcoglycans and laminin, all of which lead to various types of muscular dystrophy. We have been studying the dystrophin domains necessary for formation of a functional complex by generating transgenic mdx (dystrophin minus) mice expressing internally truncated dystrophins. These mice provide in vivo models to study the localization of truncated dystrophin isoforms, the association of the truncated proteins with the DAP complex, and the functional capacity of the assembled DAP complexes. Expression of a dystrophin deleted for most of the NH2-terminal domain in mdx mice leads to only a mild dystrophy, indicating that dystrophin can attach to the cytoskeleton by multiple mechanisms. Truncation of the central rod domain leads to normal DAP complex formation and almost fully prevents development of dystrophy. Deletion analysis of the COOH-terminal regions indicates that a broad cysteine-rich domain is indispensable for dystrophin function. This region coincides with the in vitro identified beta-dystroglycan binding domain. Mice lacking this latter domain express very low levels of the sarcoglycans, indicating that the sarcoglycan complex binds dystrophin via dystroglycan. All deletion constructs tested lead to normal expression of the syntrophins, indicating that syntrophin associates with the DAP complex via multiple binding partners.

Actins↗
PubMed1996

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Animals↗
PubMed1996

Forced expression of dystrophin deletion constructs reveals structure-function correlations.

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.

Animals↗
PubMed1996

Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a "mild Becker" phenotype.

The functional significance of the actin-binding domain of dystrophin, the protein lacking in patients with Duchenne muscular dystrophy, has remained elusive. Patients with deletions of this domain (domain I) typically express low levels of the truncated protein. Whether the moderate to severe phenotypes associated with such deletions result from loss of an essential function, or from reduced levels of a functional protein, is unclear. To address this question, we have generated transgenic mice that express wild-type levels of a dystrophin deleted for the majority of the actin-binding domain. The transgene derived protein lacks amino acids 45-273, removing 2 of 3 in vitro identified actin interacting sites and part of hinge 1. Examination of the effect of this deletion in mice lacking wild-type dystrophin (mdx) suggests that a functional domain I is not essential for prevention of a dystrophic phenotype. However, in contrast to deletions in the central rod domain and to full-length dystrophin, both of which are functional at only 20% of wild-type levels, proteins with a deletion in domain I must be expressed at high levels to prevent a severe dystrophy. These results are also in contrast to the severe dystrophy resulting from truncation of the COOH-terminal domain that links dystrophin to the extracellular matrix. The mild phenotype observed in mice with domain I-deletions indicates that an intact actin-binding domain is not essential, although it does contribute to an important function of dystrophin. These studies also suggest the link between dystrophin and the subsarcolemmal cytoskeleton involves more than a simple attachment of domain I to actin filaments.

Actinin↗
PubMed1995

Identification of alpha-syntrophin binding to syntrophin triplet, dystrophin, and utrophin.

Syntrophin represents three cytoplasmic components of the dystrophin-glycoprotein complex that links the cytoskeleton to the extracellular matrix in skeletal muscle. alpha-Syntrophin has now been translated in vitro and shown to associate directly with all three components of the syntrophin triplet and with dystrophin. The in vitro translated 71-kDa non-muscle dystrophin isoform, containing the cystein-rich/C-terminal domain, can also interact with the syntrophin triplet. The syntrophin binding motif in dystrophin was localized to exons 73 and 74 including amino acids 3447-3481 by comparing the interactions of alpha-syntrophin and seven overlapping human dystrophin fusion proteins. More than one syntrophin interaction site in this binding motif was suggested. alpha-Syntrophin also interacts directly with a C-terminal utrophin fusion protein. alpha-Syntrophin is localized to the muscle sarcolemma as well as to the neuromuscular junction in control mouse muscle. However, similar to utrophin, alpha-syntrophin is only present at the neuromuscular junction in mdx mouse muscle in which dystrophin is absent. Our data suggest that alpha-syntrophin binds all syntrophin isoforms, and syntrophin directly interacts with dystrophin through more than one binding site in dystrophin exons 73 and 74 including amino acids 3447-3481.

ATP-Binding Cassette Transporters↗
PubMed1995

Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.

Duchenne and Becker muscular dystrophy are caused by defects in the dystrophin gene, and are candidates for treatment by gene therapy. We have shown previously that overexpression of a full-length dystrophin cDNA prevents the development of dystrophic symptoms in mdx mice. We show here that this functional correction can be achieved by expressing the full-length muscle isoform at a lower level than is present in control animals. Gene therapy for DMD may necessitate the use of truncated dystrophin mini-genes to accommodate the limited cloning capacity of current-generation viral delivery vectors. We have constructed both murine and human mini-genes deleted for exons 17-48, and have demonstrated that expression of either mini-gene can almost completely prevent the development of dystrophic symptoms in transgenic mdx mice. These results suggest that viral-mediated expression of moderate levels of a truncated dystrophin could be an effective treatment for DMD.

Animals↗
PubMed1994

Prevention of dystrophic pathology in mdx mice by a truncated dystrophin isoform.

The C-terminal domain of dystrophin is alternatively spliced to produce a variety of tissue and developmental stage-specific isoforms. Recent studies suggest that the C-terminal domain binds to the dystrophin-associated glycoprotein complex (DGC) in muscle, but little is known about the functional significance of the alternative splicing or what role individual isoforms may play in specific tissues. The major dystrophin transcript in brain lacks exons 71-74, and encodes an isoform not observed in skeletal muscle. To explore the capacity of this truncated isoform to function in muscle, we have generated transgenic mice expressing a murine dystrophin mini-gene missing exons 71-74. Uniform expression of this construct on a mutant mdx mouse background results in normal muscle morphology and physiology, and prevents the development of muscular dystrophy. These mice also display normal expression and localization of the DGC, suggesting that the alternatively spliced exons are not required for dystrophin function in skeletal muscle. An additional line of mice was analyzed that had a mosaic pattern of expression. These mice display a markedly milder phenotype than mdx mice, despite the expression of dystrophin in only half the muscle fibers. These results indicate that viral delivery of dystrophin to a simple majority of fibers in a muscle group would greatly reduce the dystrophic pathology associated with Duchenne muscular dystrophy.

Alternative Splicing↗
PubMed1993

Evaluation of pulmonary systemic blood flow using ECG gated acquisition.

We propose a functional parametric analysis method using ECG-gated 99mTc-labeled red blood cell (RBC) imaging for detection and characterization of periodic variations in local blood activity in the lungs during cardiac cycle. We validated in animal experiments that such count variations correlate with cyclical pulmonary blood flow and may be used for evaluation of systemic-to-pulmonary shunts. Clinical studies were performed in 48 patients. After labeling the RBC pool with 99mTc, ECG-gated gamma camera images of both lung fields were acquired and processed to obtain Fourier transforms of time/activity functions in selected regions. The first harmonic parametric images of amplitude and phase were derived. There was an excellent correlation (r = 0.92) between activity variations and pulsatile flow measured by our method with that obtained by the thermodilution method in dog experiments (n = 10) after implantation of a systemic-to-pulmonary shunt. Patient studies showed the technique to be sensitive in detecting and quantifying abnormal systemic-to-pulmonary blood flow. Lung pulsatile flow can thus be noninvasively measured from functional parametric phase and amplitude images; the technique may be useful for detecting and quantifying abnormal systemic-to-pulmonary blood flow in man.

Animals↗
PubMed1992

[Image processing in medicine].

In this paper some basic concepts of Digital Image processing are presented, such as image enhancement and segmentation, and some of the activities in Medical Imaging of the Image Processing group of the University of Aveiro/INESC are described.

Diagnostic Imaging↗
PubMed1991

Ventilation and perfusion display in a single image.

A new method of ventilation and perfusion display onto a single image is presented. From the data on regions of interest of the lungs, three-dimensional histograms are created, containing as parameters X and Y for the position of the pixels, Z for the perfusion and colour for local ventilation. The perfusion value is supplied by sets of curves having Z proportional to the local perfusion count rate. Ventilation modulates colour. Four perspective views of the histogram are simultaneously displayed to allow visualization of the entire organ. Information about the normal ranges for both ventilation and perfusion is also provided in the histograms.

Adult↗
PubMed1986

Dynamic imaging of the compliant perfusion of the lungs.

A method for the study of the dynamics of the perfusion of the compliant vascular tree of the lungs using in vivo labelled 99mTc red cells and ECG gating is presented. Following blood pool labelling, posterior, ECG gated gamma camera images were acquired in time intervals of about 40 ms for 1,000-2,000 cardiac cycles. The images show the lungs plus the heart and great vessels. The image sequence containing the periodic variation of the blood activity in the lungs during the cardiac cycle is analyzed to obtain volume curves and functional images of the amplitude and phase of the lung perfusion. The interference of the heart and great vessels with the left lung can be avoided by excluding the superimposed areas in the ROIs with the sacrifice of part of the left lung. This technique allows dynamic visualization of the perfusion process in the compliant arterial and venous vascular trees of the lungs. The results obtained with this method in normals and some pathologies are discussed.

Electrocardiography↗