Increased expression of dystrophin, β-dystroglycan and adhalin in denervated rat muscles

Summary To evaluate a potential regulatory role of the nerve, the distribution and expression of dystrophin, of -dystroglycan (43DAG) and adhalin (50DAG), two of the dystrophin-associated proteins and utrophin (dystrophin related protein or DRP) were studied in rat muscles after 2 weeks of denervation. We found that dystrophin, -dystroglycan and adhalin were overexpressed in denervated muscle, whereas utrophin did not increase and was found only in the post-synaptic membrane. The study of the distribution of dystrophin in the sarcolemma of single muscle fibres indicates that the molecular organization of dystrophin was maintained after denervation. Dystrophin in addition of forming a scaffold around the fibre was found around the clusters of AChR that reappeared in the extra-synaptic membrane after denervation. Also -dystroglycan colocalises at these clusters. These results suggest that the increase in dystrophin, -dystroglycan and adhalin is correlated with the reappearance of AChRs in the extra synaptic membrane.     

Modulation of rat skeletal muscle chloride channels by activators and inhibitors of protein kinase C

The membrane electrical parameters and component conductances of rat extensor digitorum longus muscle fibres were studied in vitro at 30 °C with standard two microelectrode square pulse cable analysis in the presence of protein kinase C (PKC) activators and inhibitors. The PKC activator, 4--phorbol-12,13 dibutyrate (4--PDB), (2–90nM) blocked up to 67% chloride conductance (G _Cl) in rat skeletal muscle fibres and induced myotonic hyperexcitability. The concentration necessary to produce a 50% block of the membrane G _Cl was 23 nM. The inactive 4--phorbol-12,13 dibutyrate had no effect at 2 M. The blocking effect of 4--PDB on G _Cl was prevented by preincubation of the preparations with the PKC inhibitors, staurosporine (1–5 M) and tetrahydropapaverolone (50–100 M). The blocking effects on membrane G _Cl of 4--PDB and its antagonism by the inhibitors used support the concept of the involvement of PKC in regulating Cl channels of mammalian skeletal muscle fibres.     

Histochemical staining of clonal mammalian cell lines expressingE. coli β galactosidase indicates heterogeneous expression of the bacterial gene

An evaluation has been made of the E. coli -galactosidase (-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter--gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of -gal under control of this promoter element was observed.     

Interaction of transforming growth factor β1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator

The effect of transforming growth factor- (TGF-) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF- treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF- favors the development of sclerosis.Abbreviations FN Fibronectin - GEC Glomerular epithelial cells - TGF- Transforming growth factor - uPA Urokinase-type plasminogen activator     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Glucocorticoids and TGF-β1 Synergize in Augmenting Fibroblast Mediated Contraction of Collagen Gels

TGF- plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF- and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF- in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF- for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E₂ (PGE₂) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE₂ release by HFL-1 fibroblasts. TGF- significantly augmented gel contraction but also induced a 30% increase in PGE₂ release and increased the expression of COX-1. Glucocorticoids inhibited TGF-1 induced-PGE₂ release, and enhanced TGF- augmented gel contraction without significantly affecting TGF- augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF- augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF- in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE₂ production. Such interactions between glucocorticoids and TGF- may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.     

Course of denervation atrophy in type I and type II fibres of rat extensor digitorum longus muscle

Summary In the denervated extensor digitorum longus muscle of the rat type I and type II muscle fibres were differentiated histochemically and their course of atrophy was studied. Until 42 days after denervation type I and type II fibres could be identified by means of the myofibrillar ATPase reaction. Up to that time an exclusive atrophy of type II fibres was found. Type I fibres, the smallest of the normal muscle, did not change their diameters and therefore represented the largest fibres 42 days after denervation. Type II fibres of the white muscle portion, in which the larger IIB fibres are predominant, showed a higher rate of atrophy than those of the red muscle portion, in which the smaller IIA fibres are predominant: by 42 days the diameters of all type II fibres had gone down to equal values. Combined with a further progress of atrophy at later stages, there was a dedifferentiation of the histochemical properties, and the type I fibres exhibited atrophy as well. 120 days after denervation all muscle fibres were found to be highly atrophied.Dedicated to Prof. Dr. A. Faller on the occasion of his 65th birthday.Supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich.Miss F. Schramm provided skilful technical assistance.     

Voltage-sensitive Na+ channels in mammalian peripheral nerves detected using scorpion toxins

Summary The localization of voltage-sensitive sodium channels was investigated in mouse, rat and rabbit sciatic nerves using iodinated - and -Scorpion toxins (ScTx) as specific probes. Saturable specific binding for a -ScTx was detected in mouse sciatic nerve homogenates (K_d=90 pM, binding site capacity=90 fmol mg^–1 protein). LM autoradiographic studies demonstrated that the two types of ScTx stained the Ranvier nodes of the myelinated fibres, and also showed a clear but weaker labelling of the unmyelinated Remak bundles. In the sciatic nerve, which is widely considered as a model myelinated nerve, the nodal membrane represented only a small fraction of the total axonal membranes (0.2% and 0.05% for mouse and rabbit sciatic nerves respectively). Therefore, despite their high channel density, nodal membranes contribute only a small proportion of the total labelling by -ScTx (15% and 2.3% for mouse and rabbit sciatic nerves respectively), with the major contribution to labelling arising from unmyelinated axons.The distribution of specific binding sites for a -Scorpion toxin was then analysed in cross-sections of rabbit sciatic nerve at the EM level. The quantitative analysis of autoradiograms involved three methods, the 50% probability circle method, and two cross-fire analyses using either systematically distributed hypothetical sources or hypothetical sources only located on the plasma membranes of axons and of Schwann cells associated with unmyelinated Remak bundles. No specific -Scorpion toxin binding sites were detected at the plasma membrane of Schwann cells from either myelinated fibres or unmyelinated bundles, or at the internodal surface of myelinated axons. Sites were only detected at the surface of unmyelinated axons and at nodal axolemma. Their density in unmyelinated axons was found to be in the range of 1–6 per m² of plasma membrane surface area by combining quantitative EM autoradiography and stereological measurements.     

Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression

Transforming growth factor-beta (TGF-), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF- appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF- and are intimately involved in lung inflammation. We therefore chose to study TGF- regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF- mRNA expression. We conclude that IFN- downregulates TGF- mRNA expression in murine macrophages.This work was supported by the Tobacco-related Disease Research Program (#1KT110), the University of California Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation and Veterans Administration Merit Review Research Funds. Min Huang was supported by a Jaye Haddad/Concern Foundation Tumor Immunology fellowship.     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Cytokine mRNA in the Joints and Draining Lymph Nodes of Rats with Adjuvant Arthritis and Effects of Cyclosporin A

TNF- and IL-1 promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-, IL-1, IL-6, IFN-, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF- and IFN- mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1 and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1 and TNF- throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-, IL-1, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF- levels were reached on d20, while IL-1 peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN- mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1 or TNF- was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN- mRNA in synovium being analogous to human rheumatoid arthritis.     

Involvement of β-adrenoreceptors in the shift of ocular dominance after monocular deprivation

Summary In the visual cortex of young kittens, the extent of ocular dominance shift following brief monocular lid suture was less than expected in the hemisphere which had been locally perfused with -adrenergic receptor blockers at a moderate concentration. The present result was not due to the effect similar to binocular deprivation, since local anesthetic effect of -adrenergic blockers was unlikely to be involved in the present paradigm. Thus, the present results are consistent with the idea that -adrenergic receptors within the visual cortex are necessary, at least in part, for the regulation of visuocortical plasticity.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Co-localization of the β-subtype of protein kinase C and phosphorylation-dependent immunoreactivity of neurofilaments in intact, decentralized and axotomized rat peripheral neurons

Summary The localizations of protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments were compared in rat dorsal root, hypogastric, and superior cervical ganglia. In all the ganglia studied, protein kinase C- and phosphorylation-dependent immunoreactivity of neurofilaments were co-localized in nerve fibres, and no fibres with only protein kinase C--immunoreactivity or phosphorylation-dependent immunoreactivity of neurofilaments were observed. Most intense perikaryal protein kinase c- and phosphorylation-dependent neurofilament-staining were seen in large dorsal root ganglion neurons, whereas in the superior cervical ganglion only very faint protein-kinase C- and no phosphorylation-dependent staining was seen in the neuronal cell bodies. Both decentralization and axotomy of the superior cervical ganglion induced an accumulation of protein-kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments in the majority of neuronal perikarya. The accumulation was first observed at 1–2 days postoperation and it persisted up to 6–10 days postoperation. In strongly labelled decentralized neuronal perikarya, precipitation of immunoreactivity was seen near the cell and nuclear membranes, whereas in axotomized neurons, immunoreactivity was often concentrated as a unipolar clump in the cytoplasm. The results show that protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments are colocalized in intact rat peripheral ganglia and that both accumulate transiently in cell bodies of the superior cervical ganglion after decentralization and axotomy.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

The role of interferon β in human cytomegalovirus-mediated inhibition of HLA DR induction on endothelial cells

Summary Human cytomegalovirus (HCMV), a member of the virus familyHerpesviridae that is associated with extensive worldwide morbidity and mortality in immunocompromised hosts, inhibits interferon- (IFN)-mediated induction of human leukocyte antigen (HLA) class II antigens on endothelial cells. In this study, the ability of HCMV-infected endothelial cells to synthesize interferon- (IFN), and the role of IFN in HCMV-mediated inhibition of HLA class II induction, was investigated. As detemined by an encephalomyocarditis virus protection assay, HCMV-infected endothelial cell culture supernatants contained 240 IU/ml of IFN type I activity, of which 99.9% was IFN, as compared to the absence of IFN in mock-infected culture supernatants. UV-irradiated supernatants from HCMV-infected cultures inhibited induction of HLA class II in noninfected cultures by 24%. This inhibition could be abolished with 500 NU/ml of anti-IFN antibody. Addition of anti-IFN antibody directly to HCMV-infected cultures mitigated but did not abolish HLA class II antigen inhibition. Dual immunohistochemistry for HCMV and HLA DR demonstrated that infected cells, in contrast to noninfected cells, were rarely induced to express HLA class II even in the presence of anti-IFN antibody. These findings suggest that HCMV inhibits induction of HLA class II antigens by IFN dependent and independent mechanisms.     

Differential distribution of dystrophin and β-spectrin at the sarcolemma of fast twitch skeletal muscle fibers

We used double label immunofluorescence and confocal microscopy to examine the organization of -spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both -spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable -spectrin. The dystrophin-associated proteins, syntrophin and -dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without -spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on -spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.     

α-cardiac-like myosin heavy chain MHCIα is not upregulated in transforming rat muscle

The expression of MHCI, an -cardiac-like myosin heavy chain isoform, was studied in extensor digitorum longus (EDL) and tibialis anterior (TA) rat muscles undergoing fast-to-slow transition by chronic low-frequency stimulation (CLFS), a condition inducing a transient upregulation of MHCI in rabbit muscle. In order to enhance the transformation process, CLFS was applied to hypothyroid rats. mRNA analyses were performed by RT-PCR, and studies at the protein level by immunoblotting and immunohistochemistry, using the F88 antibody (F88 12F8,1) demonstrated in the accompanying paper to be specific for MHCI. In total RNA preparations from slow- and fast-twitch muscles, MHCI mRNA was present at minute levels, at least three orders of magnitude lower than in cardiac atrium. As verified immunohistochemically, MHCI is present only in intrafusal fibres of rat muscle. Moreover, MHCI is not expressed in extrafusal fibres and, contrary to the rabbit, was not upregulated at both the mRNA and protein levels by CLFS. These results support our notion of species-specific responses to CLFS. Another antibody reported to be specific to MHCI, BA-G5, was also investigated by immunoblot and immunohistochemical analyses. Its specificity could not be validated for skeletal muscles of the rat. BG-A5 was shown to cross-react with MHCIIb and MHCI. These results question an upregulation of MHCI in transforming rat muscles as reported in studies based on the use of this antibody.