Balance between macrophage migration inhibitory factor and sCD74 predicts outcome in patients with acute decompensation of cirrhosis.

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and an important regulator of innate immune responses. We hypothesised that serum concentrations of MIF are associated with disease severity and outcome in patients with decompensated cirrhosis and acute-on-chronic liver failure (ACLF). METHODS: Circulating concentrations of MIF and its soluble receptor CD74 (sCD74) were determined in sera from 292 patients with acute decompensation of cirrhosis defined as new onset or worsening of ascites requiring hospitalisation. Of those, 78 (27%) had ACLF. Short-term mortality was assessed 90 days after inclusion. RESULTS: Although serum concentrations of MIF and sCD74 did not correlate with liver function parameters or ACLF, higher MIF (optimum cut-off >2.3 ng/ml) and lower concentrations of sCD74 (optimum cut-off <66.5 ng/ml) both indicated poorer 90-day transplant-free survival in univariate analyses (unadjusted hazard ratio [HR] 2.01 [1.26-3.22]; p = 0.004 for MIF; HR 0.59 [0.38-0.92]; p = 0.02 for sCD74) and after adjustment in multivariable models. Higher MIF concentrations correlated with surrogates of systemic inflammation (white blood cells, p = 0.005; C-reactive protein, p = 0.05) and were independent of genetic MIF promoter polymorphisms. Assessment of MIF plasma concentrations in portal venous blood and matched blood samples from the right atrium in a second cohort of patients undergoing transjugular intrahepatic portosystemic shunt insertion revealed a transhepatic MIF gradient with higher concentrations in the right atrial blood. CONCLUSIONS: Serum concentrations of MIF and its soluble receptor CD74 predict 90-day transplant-free survival in patients with acute decompensation of cirrhosis. This effect was independent of liver function and genetic predispositions, but rather reflected systemic inflammation. Therefore, MIF and sCD74 represent promising prognostic markers beyond classical scoring systems in patients at risk of ACLF. LAY SUMMARY: Inflammatory processes contribute to the increased risk of death in patients with cirrhosis and ascites. We show that patients with high serum levels of the inflammatory cytokine macrophage migration inhibitory factor (MIF) alongside low levels of its binding receptor sCD74 in blood indicate an increased mortality risk in patients with ascites. The cirrhotic liver is a relevant source of elevated circulating MIF levels.

Immunoglobulin A deficiency in children, an undervalued clinical issue.

Immunoglobulin A (IgA) is the principal antibody in secretions that bathe the gastrointestinal and respiratory mucosal surfaces and acts as an important first line of defense against invasion of pathogenic micro-organisms. The reported prevalence rate of complete IgA deficiency in healthy children ranges from 1:170 to 1:400, and as a solitary condition, it is often considered of limited clinical importance. However, patients with IgA deficiency can develop recurrent respiratory and gastrointestinal infections, as well as allergic and autoimmune diseases. In children referred for recurrent respiratory tract infections, the observed prevalence rate increases more than tenfold. This review discusses several aspects of IgA deficiency in children, including immunologic and microbiome changes in early childhood and the potential consequences of this condition in later life. It illustrates the importance of early identification of children with impaired IgA production who deserve appropriate clinical care and follow-up.

Methanonatronarchaeum thermophilum gen. nov., sp. nov. and 'Candidatus Methanohalarchaeum thermophilum', extremely halo(natrono)philic methyl-reducing methanogens from hypersaline lakes comprising a new euryarchaeal class Methanonatronarchaeia classis nov.

Methanogenic enrichments from hypersaline lakes at moderate thermophilic conditions have resulted in the cultivation of an unknown deep lineage of euryarchaeota related to the class Halobacteria. Eleven soda lake isolates and three salt lake enrichment cultures were methyl-reducing methanogens that utilize C1 methylated compounds as electron acceptors and H2 or formate as electron donors, but they were unable to grow on either substrates alone or to form methane from acetate. They are extreme halophiles, growing optimally at 4 M total Na+ and the first representatives of methanogens employing the 'salt-in' osmoprotective mechanism. The salt lake subgroup is neutrophilic, whereas the soda lake isolates are obligate alkaliphiles, with an optimum around pH 9.5. Both grow optimally at 50 °C. The genetic diversity inside the two subgroups is very low, indicating that the soda and salt lake clusters consist of a single genetic species each. The phylogenetic distance between the two subgroups is in the range of distant genera, whereas the distance to other euryarchaea is below 83 % identity of the 16S rRNA gene. These isolates and enriched methanogens, together with closely related environmental clones from hypersaline habitats (the SA1 group), form a novel class-level clade in the phylum Euryarchaeota. On the basis of distinct phenotypic and genetic properties, the soda lake isolates are classified into a new genus and species, Methanonatronarchaeum thermophilum, with the type strain AMET1T (DSM 28684T=NBRC 110805T=UNIQEM U982T), and the salt lake methanogens into a candidate genus and species 'Candidatus Methanohalarchaeum thermophilum'. These organisms are proposed to form novel family, order and class Methanonatronarchaeaceae fam. nov., Methanonatronarchaeales ord. nov. and Methanonatronarchaeia classis nov., within the phylum Euryarchaeota.

Acoustic radiation force impulse elastography in distinguishing hepatic haemangiomata from metastases: preliminary observations.

OBJECTIVE: The increasing quality of diagnostic ultrasound has resulted in the detection of greater numbers of potentially benign hepatic lesions. Current radiological practice requires contrast enhanced ultrasound, CT or MRI to confirm the diagnosis. Acoustic radiation force impulse (ARFI) elastography is an imaging technique measuring the elasticity of biological tissues. Recent technical advances in ultrasound have made it possible to generate shear waves, whose velocity in the liver is proportional to the degree of hepatic elasticity. METHODS: This shear wave velocity (SWV) may be used as a marker for both focal and diffuse liver pathology.We used this technique to examine patients with normal livers and those with haemangiomata and metastases. RESULTS: Patients with normal ultrasound examinations and normal liver enzymes, n = 99, had SWVs of 1.24 ± 0.23 m s(-1) (mean ± standard deviation) independent of site of measurement, age or gender. Results of SWV measurements in haemangiomata, n = 35, produced values of the same order, 1.35 ± 0.48 m s(-1). In contrast, patients with metastases, n = 10, had SWVs of 4.23 ± 0.59 m s(-1). With a cut-off value of 2.5 m s(-1), the sensitivity and specificity for haemangiomata were 97.1% and 100%, respectively, with an area under the curve of 0.999. CONCLUSION: ARFI elastography with SWV measurements is a promising new technique which could replace invasive investigations for benign hepatic lesions.

Cannabinoid type 1 receptors in human skeletal muscle cells participate in the negative crosstalk between fat and muscle.

AIMS/HYPOTHESIS: Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. METHODS: The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyte-conditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. RESULTS: We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulin-stimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt(Ser473) phosphorylation, but had no effect on Akt(Thr308) and glycogen synthase kinase 3alpha/beta phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. CONCLUSIONS/INTERPRETATION: Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance.

IGF-1 receptor signalling determines the mitogenic potency of insulin analogues in human smooth muscle cells and fibroblasts.

AIMS/HYPOTHESIS: Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues. METHODS: DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively. RESULTS: Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin. CONCLUSIONS/INTERPRETATION: These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome.

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.

Expression of the IKr components KCNH2 (rERG) and KCNE2 (rMiRP1) during late rat heart development.

To understand molecular mechanisms that regulate formation and maintenance of cardiac IKr (rapidly activating component of the delayed rectifier K+ current), we have investigated the spatiotemporal expression pattern of two rat potassium voltage-gated channels, namely subfamily H (eag-related), member2 (KCNH2) (alias name: rERG) and Isk-related family, member2 (KCNE2) (alias name: rMiRP1) during late embryonic development by means of the in situ hybridization technique. KCNE2 is transcribed predominantly in atrial und ventricular myocardium at stages E14.5-E18.5dpc and only a minor signal emerged in the tongue at E16.5dpc. In contrast, KCNH2 transcripts appeared in a less confined pattern with intense signals in atrial and ventricular myocardium, somites, spinal cord, bowel system, central nervous system and thymus at stages E14.5-E18.5dpc. Non-cardiac expression even exceeds the intensity of the cardiac signal, indicating that KCNH2 contributes to K+ currents in non-cardiac tissue as well. Transcription of the rat b-subunit KCNE2 is present in all regions of the fetal myocardium and co-distributes perfectly with transcription of the pore forming a-subunit KCNH2. It seems likely that KCNH2 and KCNE2 are linked to form cardiac IKr channels, associated to cardiogenesis and cardiomyocyte excitability.

Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens.

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

Molecular cloning and expression of cERG, the ether à go-go-related gene from canine myocardium.

Given the anatomical and physiological similarities to the human heart, canine in vivo heart models may facilitate the analysis of molecular mechanisms underlying cardiac repolarization abnormalities. The development of such models depends, however, on information about canine K+ channels responsible for the establishment of IK currents. In this context, we isolated and sequenced the reverse transcript of the canine ether à go-go-related gene (cERG). The complementary deoxyribonucleic acid (cDNA-derived cERG polypeptide consists of 1,158 amino acids, the sequence of which shows striking homology to human, rat and mouse ERG subunits (97%, 94% and 95% identity respectively). In highly conserved peptide domains like the PAS domain, the membrane-spanning segments S1, S3-S6 and the pore-forming region, there was 100% identity. Analysis of cERG transcription revealed abundant expression of cERG messenger ribonucleic acid (mRNA) in heart and brain and low expression in liver, spleen and kidney. Membrane currents recorded in Xenopus oocytes expressing cERG channels showed functional properties very similar to the human K+ channel hERG, which encodes the alpha-subunit of the cardiac rapidly activating, delayed rectifier (IKr) channel.

Different functions of fetal and adult AChR subtypes for the formation and maintenance of neuromuscular synapses revealed in epsilon-subunit-deficient mice.

Mice deficient in epsilon-subunits of the acetylcholine receptor (AChR) channel die prematurely due to severe AChR deficiency that leads to the progressive reduction in AChR density at the neuromuscular endplate [Witzemann, V., Schwarz, H., Koenen, M., Berberich, C., Villarroel, A., Wernig, A., Brenner, H.R. & Sakmann, B. (1996) Proc. Natl Acad. Sci. USA, 93, 13286-13291]. The mice may serve as a model for studying AChR-related myasthenic diseases. The postnatal development of the subsynaptic apparatus takes place in the absence of the adult type, epsilon-subunit-containing receptors which normally replace the fetal gamma-subunit-containing receptors. During later development the secondary folds of the postsynaptic membrane disappear concomitant with the decrease in AChR density, so that the flattened-out membrane with its remaining nicotinic receptors is in close proximity to the subsynaptic cytoplasmatic compartment and the subsynaptic myonuclei. The decrease in AChR concentration is correlated with a decrease of postsynaptic rapsyn, but has less effect on agrin, a neuronally released aggregating factor for AChRs. Thus, despite the presence of agrin at the synapse, AChR expression is not maintained at the level required to stabilize normal synaptic structure comprising secondary postsynaptic membrane folds. Collectively the results suggest that the postnatal switch from the global, activity-sensitive gamma-subunit gene transcription to the synapse-specific, activity-independent epsilon-subunit gene transcription is not required for the formation and differentiation of synapses but is essential for the maintenance of the highly organized structure of the neuromuscular endplate.

N-Terminal deletions of rKv1.4 channels affect the voltage dependence of channel availability.

Rat Kv1.4 potassium channels undergo rapid inactivation, which is mediated by the N-terminal structure of the polypeptide. This inactivation can be removed by N-terminal deletion of about 20 residues. However, more substantial deletion (e.g. 37 residues) restores inactivation suggesting a second inactivating domain [Kondoh et al. J Biol Chem 272:19333-19338, 1997]. Here we provide evidence that this inactivation shares all properties with N-type inactivation. Pore mutations, which are supposed to affect C-type inactivation, have no effect. In addition, the redox regulation of inactivation, which is typical for Kv1.4 channels, can be conferred to the inactivation of the deleted constructs by incorporation of an N-terminal cysteine residue. The most remarkable feature of this secondary inactivation is the existence of two components in the steady-state voltage dependence of inactivation. For mutant rKv1. 4Delta2-37 about 90% of the channels only activate when the holding membrane potential is more negative than about -120 mV; the remaining 10% show the typical threshold at -60 mV. Mutagenesis of the truncated channel affected the relative amplitudes of these two components, but not the voltage dependence. The results suggest that the secondary ball structures of rKv1.4 channels interact with the protein structures responsible for activation.

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor.

We have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272-1292; Ser-1309, amino acids 1305-1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor beta-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.

Acetylcholine receptor epsilon-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice.

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the gamma-subunit of the fetal acetylcholine receptor (AChR) by the epsilon-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the epsilon-subunit gene caused in homozygous mutant mice the persistence of gamma-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of epsilon-subunit containing AChRs is essential for normal development of skeletal muscle.

Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma-subunit of the fetal muscle acetylcholine receptor (gamma-AChR) is replaced by the epsilon-subunit in the adult muscle receptor (epsilon-AChR), was investigated by analysing the structure of gamma- and epsilon-subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single-channel conductance. 2. The gamma- and epsilon-subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)- or gamma(epsilon)-subunits were constructed from the parental epsilon- and gamma-subunits, respectively. Exchanging the four hydrophobic segments (M1-M4) of the gamma-subunit for those of the epsilon-subunit and vice versa completely reversed the difference in conductance between gamma-AChR and epsilon-AChR channels. 4. Effects of single- and multiple-point mutations in M1-M4 segments of gamma- and epsilon-subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma-subunit) for serine/isoleucine (epsilon-subunit) in M2, and the lysine (gamma-subunit) for glutamine (epsilon-subunit) exchanges in the regions flanking the M2 segment.

Organisation of the murine 5-HT3 receptor gene and assignment to human chromosome 11.

We have isolated the murine gene encoding the 5-HT3 receptor (5-HT3R), a member of the ligand-gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5-HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use of exon 9 splice acceptor sites generated two 5-HT3R variants. The 5-HT3R gene, whose structure is closely related to neuronal and muscle AChR alpha genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.

Two adjacent E box elements and a M-CAT box are involved in the muscle-specific regulation of the rat acetylcholine receptor beta subunit gene.

We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle-specific promoter element carrying a M-CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.

Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression.

Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma- and epsilon-subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5' flanking sequences of the gamma- and epsilon-subunit genes that confer muscle-specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma-subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle-specific transcriptional activity suggesting transactivation by helix-loop-helix proteins. The epsilon-subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non-muscle cells and thus may control the muscle-specific expression of the epsilon-subunit gene. The results suggest that CANNTG motifs together with their 3' and 5' flanking nucleotides provide binding sites for both activating as well as repressing trans-acting factors. These elements could thus contribute to the muscle-specific expression of AChR subunit genes.