A specific decrease of the fluorescence depolarization of perylene in muscle membranes from mice with muscular dystrophy

The microviscosity of erythrocyte membranes and muscle microsomes from age matched 6-week old control mice REJ 129 Dy/Dy, and mice with muscular dystrophy REJ 129 dy/dy has been estimated by measuring the fluorescence depolarization of perylene. There was no difference between the erythrocyte membranes. The muscle microsomes from dystrophic animals had about 20% lower values than the controls. The temperature dependence indicated that a transition occurs in both sets of muscle microsomes, but the transition temperature was lower in the dystrophic microsomes. Cholesterol, phospholipid and triglyceride analyses of the membranes showed no difference between the erythrocyte membranes. The largest difference in the muscle microsomes was a two-fold increase in cholesterol level found in the dystrophic microsomes. No simple correlation could be made between the lipid analysis and the microviscosity measurements. Since the change in microviscosity is found in membranes isolated from the tissue primarily affected by the dy gene, we suggest that the change in microviscosity may be important in the development of the disease.     

Isolation and characterization of transverse tubule from normal and dystrophic mice

I have recently reported the isolation and characterization of sarcoplasmic reticulum from normal and dystrophic mice. These sarcoplasmic reticulum fractions were similar in calcium pump function, calcium release properties, and lipid composition. In this report, I describe the isolation of mouse muscle transverse tubule membranes using a calcium phosphate-loading technique. When the relative purity of normal and dystrophic preparations was considered, transverse tubule from normal and dystrophic mice were similar in calcium-insensitive ATPase activity, cholesterol content, and membrane microviscosity (as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene); transverse tubule yield from dystrophic muscle, however, was twice that from normal muscle, while sarcoplasmic reticulum yield from these same dystrophic muscles was only 60% that from normal muscle. This result may reflect a difference in the relative quantities of these membranes in situ.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia.

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the non-dystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of proteins in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the nondystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of protein in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Erythrocyte membrane fluidity in chicken muscular dystrophy.

An increased rigidity of erythrocyte membranes in four-week old dystrophic chickens compared to closely related normal controls has been suggested using electron spin resonance. These findings suggest that similar to the case of human Duchenne muscular dystrophy, chicken muscular dystrophy may be associated with a generalized membrane defect.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae I. Isolation of a unique lipid in dystrophic erythrocyte membranes

Several structural and functional properties are characterized in nucleated erythrocyte plasmalemmae of age and sex-matched dystrophic (line 413) and normal (line 412) chickens obtained from the University of California at Davis. Plasmalemma purity is assessed through marker enzymes. Significant differences are observed in the phospholipid content between dystrophic and normal chickens. The dystrophic chicken erythrocyte plasmalemma has an increased concentration of phosphatidylserine and a decreased concentration of phosphatidylethanolamine compared with control birds. Also, a measurable and distinct polar lipid, observed only on thin-layer chromatography (TLC) plates spotted with dystrophic preparations, is visualized adjacent to phosphatidylethanolamine. These abnormalities in the dystrophic chicken erythrocyte may signal a general defect in membrane structure for chicken dystrophy.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae. I. Isolation of a unique lipid in dystrophic erythrocyte membranes

Several structural and functional properties are characterized in nucleated erythrocyte plasmalemmae of age- and sex-matched dystrophic (line 413) and normal (line 412) chickens obtained from the University of California at Davis. Plasmalemma purity is assessed through marker enzymes. Significant differences are observed in the phospholipid content between dystrophic and normal chickens. The dystrophic chicken erythrocyte plasmalemma has an increased concentration of phosphatidylserine and a decreased concentration of phosphatidylethanolamine compared with control birds. Also, a measurable and distinct polar lipid, observed only on thin-layer chromatography (TLC) plates spotted with dystrophic preparations, is visualized adjacent to phosphatidylethanolamine. These abnormalities in the dystrophic chicken erythrocyte may signal a general defect in membrane structure for chicken dystrophy.     

Defect in regulation of membrane transport of monosaccharides in dystrophic muscle.

The penetration of a nonmetabolized glucose analogue, 3--O-methyl-D-glucose, across the plasma membranes of tissues from dystrophic mice and cardiomyopathic (dystrophic) hamsters has been compared with that of normal controls. Under basal conditions the penetration of test sugar was similar in lens and diaphragm of normal and dystrophic 129/ReJ mice. Stimulation of sugar transport by 2,4-dinitrophenol did occur in normal but not in dystrophic diaphragm. A submaximal concentration of insulin had a more variable effect in dystrophic than in normal muscle while a supramaximal concentration of the hormone increased the uptake of the glucose analogue to an equal extent in the two tissues. In the BIO 14.6 strain of cardiomyopathic hamsters, uncoupling of oxidative phosphorylation did not increase sugar transport in extensor digitorum longus muscles, while the normal effect was observed in dystrophic soleus and in both these muscles of the random bred controls. The absence of an effect by a condition simulating anoxia suggests that in dystrophy, certain muscles are unable to accelerate the entry of glucose when this is required.     

Analysis of erythrocyte membrane proteins from dystrophic hamsters

To search for potentially mutant proteins, we have investigated erythrocyte ghost proteins from normal and dystrophic hamster by two-dimensional gel electrophoresis. No significant differences are observed between dystrophic and normal erythrocytes in their peptide patterns on SDS-polyacrylamide gel electrophoresis while on two-dimensional gels a protein spot of approximate Mr 20 000 with an approximate isoelectric point of 4.5 is found in erythrocytes from dystrophic animals and is consistently absent in normal erythrocytes. A large population of erythrocyte (60%) from dystrophic hamsters shows distorted shape as visualized by scanning electron microscopy. The nature of this protein and its relevance in hamster muscular dystrophy are at present not known.     

The levels of both lipid rafts and raft-located acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency

Wild type and dystrophic (merosin-deficient) Lama2dy mice muscles were compared for their density of lipid rafts. The 5-fold higher level of caveolin-3 and the 2-3 times higher level of ecto-5’-nucleotidase activity in raft preparations (Triton X-100-resistant membranes) of dystrophic muscle supported expansion of caveolar and non-caveolar lipid rafts. The presence in rafts of glycosylphosphatidylinositol (GPI)-linked acetylcholinesterase (AChE) dimers, which did not arise from erythrocyte or nerve, not only revealed for the first time the capacity of the myofibre for translating the AChE-H mRNA but also an unrecognized pathway for targeting AChE-H to specialized membrane domains of the sarcolemma. Rafts of dystrophic muscle contained a 5-fold higher AChE activity/mg protein. RT-PCR for 3’-alternative mRNAs of AChE revealed AChE-T mRNA prevailing over AChE-R and AChE-H mRNAs in wild type mouse muscle. It also displayed principal 5’-alternative AChE mRNAs with exons E1c and E1e (the latter coding for N-terminally extended subunits) and fewer with E1d, E1a and E1b. The levels of AChE and butyrylcholinesterase mRNAs were unaffected by dystrophy. Finally, the decreased level of proline-rich membrane anchor (PRiMA) mRNA in Lama2dy muscle provided for a rational explanation to the loss of PRiMA-bearing AChE tetramers in dystrophic muscle.     

Fatty acid metabolism and mitochondrial proteins in the C57BL/6J-dy2Jdy2J dystrophic mice

Measurements of [1-14C]oleoyl CoA oxidation in homogenates and isolated mitochondria of skeletal muscles, hearts and livers from dystrophic C57BL/6J mice and unaffected litter-mates indicate that rates are lower in dystrophic mitochondria, but are comparable in whole homogenates. These findings are not due to differences in the endogenous concentrations of the fatty CoA esters. Qualitative and quantitative differences in the molecular weight distribution of the proteins in the mitochondria and post-mitochondrial supernatants are found in the skeletal muscles with increases in the post-mitochondrial supernatant and decreases in the mitochondria in dystrophic samples. We suggest that these results are due to increased leakiness of the mitochondrial membranes in dystrophic muscles. Such differences were not observed in preparations from normal animals.     

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

Progressive muscular dystrophy type Duchenne. II. Viscosity and resistence to erythrolytic compounds of erythrocyte membranes

The transition temperature of erythrocyte ghosts of normal subjects is about 18-20 degrees C. We have studied the viscosity of erythrocyte ghosts of dystrophic children, showing that the transition shifts to lower temperatures (17-18 degrees C). After treatment with erythrocytic compounds like L-Lyso phosphatidyl-Choline dystrophic erythrocytes hemolize at lower Lysophosphatidyl-Choline concentration and at a greater extents than these of normal and carriers subjects.     

RAPID AXOPLASMIC TRANSPORT IN DYSTROPHIC MICE

Abstract— Rapid axoplasmic transport was studied in dystrophic mice of the 129/ReJ-dy strain. Proteins transported in vivo through α-motoneurons of the sciatic nerve were labeled by injections of [³H] or [³⁵S] amino acids into the ventral horn of the lumbar spinal cord. Following an 18 h incubation, axoplasmic transport was quantitated by summing the radioactivity in the 10 mm length of sciatic nerve proximal to a ligation. Although the amount of transported radioactivity was small, transport appeared depressed when adult dystrophic mice were compared to controls. Transport was also studied in the sensory fibers of the sciatic nerve under in vitro conditions, resulting in high levels of transported radioactivity. In this system transport was strongly depressed. The severity of the deficiency varied with age, being small in animals with early clinical signs and becoming maximal (80–90%) in animals over 60 days of age. Proteins transported by adult dy/dy and +/+ animals were compared by gel electrophoresis using double-label techniques. Transport of nearly all proteins was depressed in dy/dy mice, although the possibility exists that small differences occur. The data suggest that the dystrophic state produces a significant deficiency in rapid axoplasmic transport in both motor and sensory fibers, and may interfere with transport processes in all neurons. Since rapid axoplasmic transport has been associated with membranes, the data are consistent with a general alteration of cellular membranes in dystrophic animals.     

Antisera to synthetic peptides of bovine rhodopsin: use as site-specific probes of disc membrane changes in retinal dystrophic dogs

Based on the amino acid sequence of bovine rhodopsin, five peptides corresponding to the carboxyl terminus and one loop region have been synthesized. Rabbit antisera to these peptides recognize rhodopsin in whole bovine and dog retinas. Antisera were used to detect differences in specific regions of rhodopsin in dystrophic vs normal dog retinas. As detected on both "dot blots" and Western blots, rhodopsin from retinas of dystrophic dogs has a reduced reaction with antisera to peptides, Rhod-4 and Rhod-10 (# 341-348 and 232-239, respectively). Since these sites on rhodopsin are possible binding sites for transducin and rhodopsin kinase, an alteration in these regions would have profound effects in the dystrophic state.     

Systematic characterization of sperm-specific membrane proteins in swine

To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with M(r) 7500-66,000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2-4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae II. Characterization of a unique lipid from dystrophic erythrocyte membranes as ethanolamine plasmalogen

The phospholipid content of normal (line 412) and dystrophic (line 413) chicken erythrocyte plasmalemmae has been quantified on a developmental basis using sex matched controls. A specific minor phospholipid component, ethanolamine plasmalogen, is identified from dystrophic erythrocyte membrane preparations. To arrive at this identification, data from studies utilizing gas-liquid chromatography, thin-layer chromatography, [¹⁴C]ethanolamine incorporation, and biochemical assay for specific organic moieties were correlated. This phospholipid has the potential to alter and regulate membrane fluidity and thus membrane function. The possible presence of significant concentrations of plasmalogen in human dystrophic tissues may serve as a marker for dystrophy and thus be of clinical importance.