Dystrophin in a membrane skeletal network: localization and comparison to other proteins

We studied the location, relative abundance, and stability of dystrophin in clusters of ACh receptors (AChRs) isolated from primary cultures of neonatal rat myotubes. Although variable amounts of dystrophin were found at receptor clusters, dystrophin was always associated with organized, receptor-rich domains (AChR domains). Dystrophin was occasionally seen in focal contact domains, but never in clathrin-coated domains. Dystrophin was also present in a diffuse, punctate distribution in regions of myotube membrane that did not contain AChR clusters. Immunogold labeling at the ultrastructural level localized dystrophin in a spectrin-rich filamentous network closely applied to the cytoplasmic surface of the cell membrane at AChR domains. Dystrophin was not associated with overlying actin filaments. Semiquantitative immunofluorescence studies indicated that dystrophin was present in relatively small amounts in these preparations, with only one molecule of dystrophin for every approximately 5 AChR, 43 kDa and 58 kDa molecules, and for every approximately 20-35 beta-spectrin molecules. Clusters were disrupted, but the total amount of dystrophin was not significantly reduced, when myotubes were incubated with sodium azide or in Ca(2+)-free medium, and when isolated AChR clusters were extracted at low ionic strength, at high pH, or in 6 M urea. These treatments extract other peripheral membrane proteins from AChR clusters. Labeling for dystrophin was completely eliminated when clusters were incubated with chymotrypsin, however. Thus, dystrophin forms part of a membrane skeleton at AChR clusters, but it is more difficult to remove than other proteins in the network. This suggests that dystrophin attaches to cluster membrane in a unique way.     

Alkaline phosphatase (ALP) activity in rheumatoid arthritis (RA): its clinical significance and synthesis of ALP in RA synovium

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.     

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

Dystrophin, utrophin and beta-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

The precise localization and semiquantitative correlation of dystrophin, utrophin and beta-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of beta-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of beta-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of beta-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to beta-dystroglycan.     

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the gamma subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the gamma subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the gamma subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the gamma subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.     

Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors

We analyzed the kinetics and pharmacology of EPSCs in two kinds of neurons in the embryonic avian ciliary ganglion. Whole-cell voltage-clamp recordings revealed that the singly innervated ciliary neurons had large-amplitude (1.5-8.0 nA) EPSCs that could be classified according to the kinetics of their falling phases. Most of the neurons responded with an EPSC the falling phase of which followed a double exponential time course with time constants of approximately 1 and 10 msec. The EPSCs of the remaining ciliary neurons followed a single time constant ( approximately 8 msec). Multiple innervated choroid neurons had smaller-amplitude responses (0.2-1.5 nA when all inputs were activated) that appeared to contain only a slowly decaying component (tau = 12 msec). The fast and slow components of EPSC decay seen in most ciliary neurons could be pharmacologically isolated with two toxins against nicotinic acetylcholine receptors (AChRs). The fast component was blocked by 50 nM alpha-bungarotoxin (alpha-BuTx), which binds alpha7-subunit-containing AChRs. The slow component was selectively blocked by 50 nM alpha-conotoxin MII (alpha-CTx-MII), which blocks mammalian AChRs containing an alpha3/beta2 subunit interface. A combination of both alpha-BuTx and alpha-CTx-MII abolished nearly all evoked current. Similar pharmacological results were found for ciliary neurons with monoexponentially decaying EPSCs and for choroid neurons. These results suggest that nerve-evoked transmitter acts on at least two different populations of AChRs on autonomic motor neurons in the ciliary ganglion.     

Genomic organization of the human dystrophin gene across the major deletion hot spot and the 3'' region

The genomic organization of most of the human dystrophin gene has not been defined at single-exon level, owing to its enormous size (2300 kb). By taking advantage of a YAC-based restriction map of the gene previously constructed, we have localized individual dystrophin exons from 42 to 79 along the central and 3' regions of the gene. These data elucidate the general organization of this large portion of the gene (1250 kb) and, in particular, characterize the genomic region most frequently involved in deletion mutations responsible for Duchenne and Becker muscular dystrophies.     

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p < 0.0001), and by 50% in skeletal muscle (p < 0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labelling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.     

Acetylcholine receptor and calcium leakage activity in nondystrophic and dystrophic myotubes (MDX)

To determine whether the lack of dystrophin alters the occurrence of calcium leakage activity (CLA) and acetylcholine receptor (AChR) activity, the frequency of each event class was determined from several cell attached patches on nondystrophic and dystrophic (mdx) myotubes. The frequency of CLA observed in the presence of ACh was significantly (P < 0.05) elevated in mdx myotubes, an effect which was partly due to a significant (P < 0.05) increase in the proportion of cell attached patches that exhibited 100% CLA with no AChR activity. Areas of mdx and nondystrophic membrane that exhibited reduced or absent AChR activity had significantly (P < 0.01) and substantially elevated calcium leakage event frequencies. This inverse and discontinuous relationship between CLA and AChR activity provides further evidence that some CLA in dystrophic muscle is produced by clusters of AChRs that form unusual physical associations with the dystrophic cytoskeleton during the processes associated with receptor localization and stabilization.     

Evidence for in situ and in vitro association between beta-dystroglycan and the subsynaptic 43K rapsyn protein. Consequence for acetylcholine receptor clustering at the synapse

The accumulation of dystrophin and associated proteins at the postsynaptic membrane of the neuromuscular junction and their co-distribution with nicotinic acetylcholine receptor (AChR) clusters in vitro suggested a role for the dystrophin complex in synaptogenesis. Co-transfection experiments in which alpha- and beta-dystroglycan form a complex with AChR and rapsyn, a peripheral protein required for AChR clustering (Apel, D. A., Roberds, S. L., Campbell, K. P., and Merlie, J. P. (1995) Neuron 15, 115-126), suggested that rapsyn functions as a link between AChR and the dystrophin complex. We have investigated the interaction between rapsyn and beta-dystroglycan in Torpedo AChR-rich membranes using in situ and in vitro approaches. Cross-linking experiments were carried out to study the topography of postsynaptic membrane polypeptides. A cross-linked product of 90 kDa was labeled by antibodies to rapsyn and beta-dystroglycan; this demonstrates that these polypeptides are in close proximity to one another. Affinity chromatography experiments and ligand blot assays using rapsyn solubilized from Torpedo AChR-rich membranes and constructs containing beta-dystroglycan C-terminal fragments show that a rapsyn-binding site is present in the juxtamembranous region of the cytoplasmic tail of beta-dystroglycan. These data point out that rapsyn and dystroglycan interact in the postsynaptic membrane and thus reinforce the notion that dystroglycan could be involved in synaptogenesis.     

Specific retinal diacylglycerol and protein kinase C beta isoform modulation mimics abnormal retinal hemodynamics in diabetic rats

PURPOSE: Elevation of diacylglycerol (DAG) and protein kinase C (PKC) levels in diabetic vascular tissue is associated with abnormalities of retinal and renal hemodynamics. The object of this study was to determine whether direct elevation of retinal DAG levels, in the absence of diabetes or hyperglycemia, can mimic the hemodynamic abnormalities normally observed in diabetic rats. Retinal DAG levels were elevated using an inhibitor of DAG kinase that converts DAG to phosphatidic acid. The effectiveness of a specific PKC-beta isoform inhibitor introduced directly into the retinas of diabetic rats in reversing diabetes-related abnormal retinal hemodynamics was also investigated. METHODS: For retinal blood flow studies, diacylglycerol kinase (DGK) inhibitor R59949, at various concentrations, was injected into the vitreous of nondiabetic Sprague-Dawley rats (n = 33), and a PKC-beta isoform-selective inhibitor LY333531 was injected into the vitreous of rats with streptozotocin (STZ)-induced diabetes of 2 weeks' duration (n = 21). Retinal hemodynamic changes were quantitated using video-based fluorescein angiography. Total DAG levels were assayed from five nondiabetic rat retinas after DGK inhibition and retinal PKC activities were assayed from six diabetic rat retinas after PKC-beta inhibition. RESULTS: DGK inhibitor R59949 injected into the vitreous dose dependently increased the mean circulation time (MCT) and decreased retinal blood flow (EC50 = 10(-8) M). After 30 minutes, 10(-5) M R59949 induced a 1.7-fold increase in total retinal DAG levels, compared with the levels in vehicle-injected eyes, an increase in MCT from 0.87 +/- 0.05 seconds to 1.44 +/- 0.12 seconds (P < 0.01) and a decrease in retinal blood flow from 105.3 +/- 6.5 pixel2/second to 64.1 +/- 5 pixel2/second (P < 0.01). The effect of R59949 was sustained for 60 minutes after injection. These retinal hemodynamic parameters after DGK inhibition were comparable to those measured at baseline in rats with STZ-induced diabetes of 2 weeks' duration (MCT = 1.38 +/- 0.20 seconds; retinal blood flow = 68 +/- 11.2 pixel2/second). Intravitreal injection of the PKC-beta inhibitor (LY333531) at 10(-5) M in diabetic rats decreased by a factor of 1.6 the diabetes-related increased PKC activation, decreased the prolonged MCT (0.98 +/- 0.13 seconds; P < 0.01) and increased retinal blood flow (93.4 +/- 14.2 pixel2/second; P < 0.01). The measured retinal circulatory parameters after PKC inhibition in the retina were comparable to those measured at baseline in the nondiabetic rats. CONCLUSIONS: These results provide direct evidence that DAG elevation and subsequent PKC-beta isoform activation are the primary biochemical sequelae responsible for the development of the abnormal retinal hemodynamics observed in diabetic rats.     

Memory performance among women with parental abuse histories: enhanced directed forgetting or directed remembering?

Progesterone interaction with human spermatozoa promotes a rise in intracellular Ca2+ and can trigger acrosomal exocytosis in capacitated cells. We have used nifedipine, a 1,4-dihydropyridine Ca2+ channel antagonist, to investigate the possibility that Ca2+ channels play a role in the progesterone-stimulated exocytotic response. Cells were assessed biochemically for the generation of diacylglycerol (DAG) and microscopically for acrosome loss using chlortetracycline fluorescence. When motile cells were preincubated for 5 hr using culture conditions similar to those used for successful human in vitro fertilization, a short exposure to progesterone significantly stimulated DAG formation and acrosomal exocytosis. The addition of nifedipine (10 and 100 nM), either at time 0 or just prior to progesterone introduction, significantly inhibited both DAG formation and exocytosis, suggesting that Ca2+ channels are involved in the responses observed. Treatment of capacitated cells with a synthetic permeant DAG stimulated exocytosis irrespective of whether nifedipine was present, indicating that Ca2+ channels function prior to DAG generation. The possibility that an influx of Na+, as well as Ca2+, might be involved in the exocytotic pathway was investigated using the monovalent cation ionophores monensin and nigericin. Both significantly stimulated DAG generation and acrosome loss, but the prior inclusion of nifedipine significantly inhibited all responses. These results strongly suggest that the entry of Ca2+ through Ca2+ channels, with characteristics similar to those of L-type, voltage-sensitive Ca2+ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to exocytosis in progesterone-stimulated human spermatozoa. An influx of Na+ also may play a role, but at a point prior to the opening of Ca2+ channels.     

Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.     

Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation

Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.