Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are the most prevalent metabolic liver disorders and a serious global health burden. NAFLD/NASH pathogenesis and progression are highly multi-factorial and likely demand a combination of multiple mechanisms to provide a more effective treatment. We have developed a dual farnesoid X receptor agonist (FXRA)/soluble epoxide hydrolase inhibitor (sEHi) to simultaneously address two validated and complementary modes of action in NASH treatment. Here we report the in vivo profiling for this FXRA/sEHi in toxin- and diet-induced rodent NASH models. In streptozotocin-induced NASH as a proof-of-concept study, the experimental FXRA/sEHi drug robustly prevented hepatic steatosis and fibrosis, and improved lipid homeostasis as well as biochemical markers of liver health. In methionine-choline-deficient high-fat diet-induced NASH, FXRA/sEHi treatment reduced hepatic steatosis and fibrosis to levels similar to healthy animals and demonstrated anti-inflammatory activity confirming that dual FXRA/sEHi modulation produces a triad of complementary anti-NASH effects. Our results validate dual FXRA/sEHi modulation as an effective therapeutic strategy to treat NASH and advocates for a combinational drug therapeutic approach for multifactorial liver diseases.
Nonalcoholic steatohepatitis arising from Western diet and lifestyle is characterized by accumulation of fat in liver causing inflammation and fibrosis. It evolves as serious health burden with alarming incidence, but there is no satisfying pharmacological therapy to date. Considering the disease's multifactorial nature, modulation of multiple targets might provide superior therapeutic efficacy. In particular, farnesoid X receptor (FXR) activation that revealed antisteatotic and antifibrotic effects in clinical trials combined with inhibition of soluble epoxide hydrolase (sEH) as anti-inflammatory strategy promises synergies. To exploit this dual concept, we developed agents exerting partial FXR agonism and sEH inhibitory activity. Merging known pharmacophores and systematic exploration of the structure-activity relationship on both targets produced dual modulators with low nanomolar potency. Extensive in vitro characterization confirmed high dual efficacy in cellular context combined with low toxicity, and pilot in vivo data revealed favorable pharmacokinetics as well as engagement on both targets in vivo.
Polyurethanes (PU) are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC), TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU) Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 degreesC, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.
TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60 degrees C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.
The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.
Title: A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate Then J, Wei R, Oeser T, Gerdts A, Schmidt J, Barth M, Zimmermann W Ref: FEBS Open Bio, 6:425, 2016 : PubMed
Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7 degreesC (wild-type TfCut2: 69.8 degreesC) and its half-inactivation temperature to 84.6 degreesC (TfCut2: 67.3 degreesC). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 degreesC compared to 65 and 70 degreesC of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/- 0.1%) at 70 degreesC after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.
Title: Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition Wei R, Oeser T, Schmidt J, Meier R, Barth M, Then J, Zimmermann W Ref: Biotechnol Bioeng, 113:1658, 2016 : PubMed
Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65 degrees C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5-fold lower substrate binding constant than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658-1665. (c) 2016 Wiley Periodicals, Inc.
Several bacterial polyester hydrolases are able to hydrolyze the synthetic polyester polyethylene terephthalate (PET). For an efficient enzymatic degradation of PET, reaction temperatures close to the glass transition temperature of the polymer need to be applied. The esterases TfH, BTA2, Tfu_0882, TfCut1, and TfCut2 produced by the thermophilic actinomycete Thermobifida fusca exhibit PET-hydrolyzing activity. However, these enzymes are not sufficiently stable in this temperature range for an efficient degradation of post-consumer PET materials. The addition of Ca2+ or Mg2+ cations to the enzymes resulted in an increase of their melting points between 10.8 and 14.1 degreesC determined by circular dichroism spectroscopy. The thermostability of the polyester hydrolases was sufficient to degrade semi-crystalline PET films at 65 degreesC in the presence of 10 mM Ca2+ and 10 mM Mg2+ resulting in weight losses of up to 12.9% after a reaction time of 48 h. The residues Asp174, Asp204, and Glu253 were identified by molecular dynamics simulations as potential binding residues for the two cations in TfCut2. This was confirmed by their substitution with arginine, resulting in a higher thermal stability of the corresponding enzyme variants. The generated variants of TfCut2 represent stabilized catalysts suitable for PET hydrolysis reactions performed in the absence of Ca2+ or Mg2+.
Motor neurons, which relay neural commands to drive skeletal muscle movements, encompass types ranging from "slow" to "fast," whose biophysical properties govern the timing, gradation, and amplitude of muscle force. Here we identify the noncanonical Notch ligand Delta-like homolog 1 (Dlk1) as a determinant of motor neuron functional diversification. Dlk1, expressed by ~30% of motor neurons, is necessary and sufficient to promote a fast biophysical signature in the mouse and chick. Dlk1 suppresses Notch signaling and activates expression of the K(+) channel subunit Kcng4 to modulate delayed-rectifier currents. Dlk1 inactivation comprehensively shifts motor neurons toward slow biophysical and transcriptome signatures, while abolishing peak force outputs. Our findings provide insights into the development of motor neuron functional diversity and its contribution to the execution of movements.
Title: Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata Wei R, Oeser T, Then J, Kuhn N, Barth M, Schmidt J, Zimmermann W Ref: AMB Express, 4:44, 2014 : PubMed
Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60 degrees C and 55 degrees C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55 degrees C. Tcur0390 showed a higher hydrolytic activity against poly(epsilon-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50 degrees C. At 55 degrees C and 60 degrees C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of alpha/beta serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.
The chemical composition of the essential oil obtained from the leaves of Pulicaria undulata Gamal Ed Din (syn P. orientalis sensu Schwartz and P. jaubertii Gamal Ed Din) was analyzed by GC-MS. Major compounds of P. undulata oil were the oxygenated monoterpenenes, carvotanacetone (91.4%) and 2,5-dimethoxy-p-cymene (2.6.%). The antimicrobial activity of the essential oil was evaluated against six microorganisms, Escherichia coli Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans, using disc diffusion and broth microdilution methods. The oil showed the strongest bactericidal activity against Staphylococcus aureus and methicillin-resistant S. aureus, as well as Candida albicans. The essential oil showed moderate cytotoxic activity against MCF-7 breast tumor cells, with an IC50 of 64.6 +/- 13.7 microg/mL. Bioautographic assays were used to evaluate the acetylcholinesterase inhibitory effect as well as antifungal activity of the oil against Cladosporium cucumerinum.
Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein-coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca(2+) or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1-2 d.
Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.
Anastomoses between the jejunum and the bile duct are an important component of many surgical procedures; however, risk factors for clinically relevant bile leaks have not yet been adequately defined. The objective of this study was to describe the incidence of bile leaks after hepaticojejunostomy and to define predictive factors associated with this risk and with surgical morbidity. Between October 2001 and April 2004, hepaticojejunostomies were performed in 519 patients in a standardized way. Patient- and treatment-related data were documented prospectively. A bile leak was defined as bilirubin concentration in the drains exceeding serum bilirubin with a consecutive change of clinical management or occurrence of a bilioma necessitating drainage. Surgical morbidity occurred in 15% of patients, the incidence of a bile leak was 5.6%. Multivariate analysis confirmed preoperative radiochemotherapy, preoperative low cholinesterase levels, biliary complications after liver transplantation necessitating a hepaticojejunostomy, and simultaneous liver resection as risk factors for bile leakages, whereas biliary complications after liver transplantation necessitating hepaticojejunostomy, simultaneous liver resection, and diabetes mellitus were significantly associated with postoperative surgical morbidity. Our results demonstrate that hepaticojejunostomy is a safe procedure if performed in a standardized fashion. The above found factors may help to better predict the risk for complications after hepaticojejunostomy.
Four beta-carboline alkaloids, brunneins A-C (1-3) and 3-(7-hydroxy-9H-beta-carboline-1-yl)propanoic acid (4), were isolated from fruiting bodies of the agaricoid fungus Cortinarius brunneus. The structures of 1-3 were determined by analysis of NMR and MS data, and the structure of compound 4 was determined by comparison with published data. Brunnein A (1) exhibited very low cholinesterase inhibitory effects and no cytotoxicity.
The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.
Title: Cloning and analysis of the simocyclinone biosynthetic gene cluster of Streptomyces antibioticus Tu 6040 Galm U, Schimana J, Fiedler HP, Schmidt J, Li SM, Heide L Ref: Arch Microbiol, 178:102, 2002 : PubMed
The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTu 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.
Title: Interaction of the myogenic determination factor myogenin with E12 and a DNA target: mechanism and kinetics Spinner DS, Liu S, Wang SW, Schmidt J Ref: Journal of Molecular Biology, 317:431, 2002 : PubMed
The myogenic determination factors MyoD, myogenin, myf5, and MRF4 are members of the basic helix-loop-helix (bHLH) family of transcription factors and crucial agents of myogenesis. The bHLH regions of these proteins enable them to dimerize with E proteins, another class of the bHLH family, and to bind a specific DNA element known as an E box (CANNTG consensus sequence), which results in the activation of muscle-specific gene expression. As a model for such assembly of the myogenic determination factor/E protein-DNA ternary complex, we have studied the physiologically relevant association of myogenin, E12, and the 3' E box of the acetylcholine receptor (AChR) alpha-subunit gene enhancer. Using the technique of electrophoretic mobility shift assay combined with order-of-addition and time-course experiments, we find that heterodimerization of myogenin with E12 occurs prior to DNA-binding. In addition, we deduce the dissociation (Kd) and rate (k) constants for each step in the formation of the myogenin/E12-DNA ternary complex. Kinetic simulations indicate that at 37 degrees C myogenin and E12 heterodimerize with a Kd of 36 microM (k(on) of 573 M(-1) x s(-1) and k(off )of 0.0205 x s(-1)), and that subsequently the heterodimer binds the AChR alpha-subunit gene enhancer 3' E box with a Kd of 8.8 nM (with possible k(on) and k(off) values ranging from 1.0x10(8) to 14.1x10(8) M(-1) x s(-1), and 0.875 to 12.3 s(-1), respectively).
The myogenic determination factors (MDFs) are transcriptional activators that target E boxes in many muscle-specific promoters, including those of the genes coding for the subunits of the acetylcholine receptor. It is not known, however, if in vivo a given E box in a transcriptionally active gene is occupied, either uniquely by one MDF or randomly by all MDFs. We have analyzed expression of MDF and acetylcholine receptor subunits in cultured mouse muscle cells and, using chromatin immunoprecipitation, have determined which individual MDFs reside at promoters of several receptor subunit genes. We find that before fusion, C2C12 cells express myf-5, MyoD, and myogenin, all of which take up residence at promoters of all subunits except epsilon. At this stage, herculin is present in limited amounts and is detected mainly at the gamma and delta subunit genes. On myotube formation, herculin reaches high levels; concomitantly, the epsilon subunit gene becomes a common MDF target and begins to be expressed. In general, any MDF protein that is expressed also is present on transcriptionally active receptor genes; transcriptional activity of target genes correlates with occupancy by MDF, in particular, herculin.
Title: The role of the CANNTG promoter element (E box) and the myocyte-enhancer-binding-factor-2 (MEF-2) site in the transcriptional regulation of the chick myogenin gene Malik S, Huang CF, Schmidt J Ref: European Journal of Biochemistry, 230:88, 1995 : PubMed
We have carried out an analysis of 833 bp of the chick myogenin gene 5' flanking sequence. A 131-bp segment of this upstream region, which contains a CANNTG promoter element (E box) and a myocyte-enhancer-binding-factor-2 (MEF-2) site, acts as a full promoter. It resembles the human and the mouse myogenin promoters in the structure and disposition of regulatory elements, including a TATA box and the transcription start site. Examination of eight myogenic factor/E protein combinations cotransfected with several myogenin promoter constructs into HeLa cells, reveals that the chick myogenin/E2-5 combination maximally activates the myogenin promoter. This activation is mediated through the E box motif; the MEF-2 site limits the factor combinations that can activate the myogenin promoter and enhances activation by myogenin/E2-5. We have found previously that activation of protein kinase C inactivates the transcription of the chick myogenin gene [Huang, C.-F., Neville, C. M. & Schmidt, J. (1993) Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle, FEBS Lett. 319, 21-25]. In this study, we show that the activation of protein kinase C inhibits the factor bound to the E box, which thereupon negatively regulates the activity of the MEF-2 binding protein.
Title: Depolarization-transcription signals in skeletal muscle use calcium flux through L channels, but bypass the sarcoplasmic reticulum Huang CF, Flucher BE, Schmidt MM, Stroud SK, Schmidt J Ref: Neuron, 13:167, 1994 : PubMed
Membrane depolarization inactivates acetylcholine receptor (AChR) genes in skeletal muscle. We have studied this process in C2C12 cells, focusing on the role of calcium. Cytoplasmic calcium was monitored with fluo-3, and the activity of receptor genes was measured with a sensitive transcript elongation assay. Removal of extracellular calcium or blockage of L-type calcium channels disrupts signaling, even when release of calcium from the sarcoplasmic reticulum (SR) is not impeded, whereas L channel agonists induce signaling without membrane depolarization or release of calcium from intracellular stores. Activators of calcium release from the SR do not inhibit AChR genes, either in C2C12 or in chicken skeletal muscle in vivo. It appears that calcium ions do not act as messengers between sarcolemma and nucleus but target a sensor near their port of entry where they initiate a signal that bypasses the SR.
Title: Rapid inhibition of myogenin-driven acetylcholine receptor subunit gene transcription Huang CF, Lee YS, Schmidt MM, Schmidt J Ref: EMBO Journal, 13:634, 1994 : PubMed
In investigating the coupling of depolarization and transcription in skeletal muscle we have focused on how protein kinase C suppresses acetylcholine receptor subunit genes. The activity of acetylcholine receptor subunit promoters in non-muscle cells co-transfected with myogenic factors and E proteins was measured, and their response to protein kinase C activation analyzed. To simplify interpretation of results, gene activities rather than levels of reporter enzymes were assayed, transcriptional effects of phorbol esters were determined, with drug exposures brief enough to preclude kinase depletion, and analysis was carried out with HeLa cells, which are not liable to myogenic conversion. Myogenin, which had been postulated previously to play a role in denervation supersensitivity (Neville et al., Mol. Cell. Neurobiol., 12, 511-527, 1992), was found to be the only myogenic factor whose inactivation kinetics can account for the plasma membrane-protein kinase C-receptor gene cascade observed in intact muscle (Huang et al., Neuron, 9, 671-678, 1992).
Title: Calcium influx blocks the skeletal muscle acetylcholine receptor alpha-subunit gene in vivo Huang CF, Schmidt J Ref: FEBS Letters, 338:277, 1994 : PubMed
The transcriptional activity of the acetylcholine receptor alpha-subunit gene was measured in denervated chick skeletal muscle in response to calcium-active drugs, using a ribonuclease protection version of the conventional run-off assay. The L-channel agonist (-)Bay-K6844 and the calcium ionophore A23187 mimicked, and the intracellular chelator BAPTA and the calcium channel blockers D600 and nifedipine blocked, the effect of electrostimulation. These results suggest that influx of extracellular calcium is an integral component of the membrane depolarization-receptor gene inactivation cascade.
Title: Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle Huang CF, Neville CM, Schmidt J Ref: FEBS Letters, 319:21, 1993 : PubMed
Myogenic factor genes were found to respond differentially to electrical stimulation of denervated chick skeletal muscle. Myogenin gene activity declined rapidly (t1/2: approximately 2 min), comparable to the rate of acetylcholine receptor (AChR) gene inactivation, while other myogenic bHLH genes either lost activity more slowly (MyoD) or not at all (myf5, herculin). Protein kinase C (PKC) is known to couple membrane activity to AChR gene inactivation; myogenin gene transcription was also rapidly blocked by the PKC activator PMA, whereas electrostimulation remained without effect on myogenin gene activity in muscle that was either exposed to the kinase inhibitor staurosporine or chronically treated with PMA to deplete PKC. These results attest to a special role for myogenin in the activation of AChR genes in denervation supersensitivity.
Title: Protein kinase C couples membrane excitation to acetylcholine receptor gene inactivation in chick skeletal muscle Huang CF, Tong J, Schmidt J Ref: Neuron, 9:671, 1992 : PubMed
The signaling pathway connecting membrane depolarization and gene activity in skeletal muscle remains largely unknown. Using transcription elongation (run-on) analysis we have found that electrical stimulation of denervated chick skeletal muscle in vivo rapidly and selectively results in inactivation of acetylcholine receptor (AChR) subunit genes. We have studied the possible involvement of protein kinase C (PKC) in this response and have observed that electrical stimulation increases the activity of PKC in the nucleus by over two orders of magnitude within 10 min; phorbol esters, within minutes after intramuscular application, block AChR subunit genes in the absence of electrical activity; and the activity-triggered gene inactivation is blocked by the protein kinase inhibitor staurosporine or by enzyme depletion resulting from chronic pretreatment of muscle with phorbol esters. We conclude that PKC is an integral component of the pathway coupling membrane excitation and AChR gene control.
Title: Analysis of binding and activating functions of the chick muscle acetylcholine receptor gamma-subunit upstream sequence Jia HT, Tsay HJ, Schmidt J Ref: Cellular Molecular Neurobiology, 12:241, 1992 : PubMed
1. The skeletal muscle acetylcholine receptor comprises several subunits whose coordinated expression during myogenesis is probably controlled by cis elements in the individual subunit genes. We have previously analyzed promoter regions of the alpha and delta genes (Wang et al., 1988, 1990); to gain further insight into receptor regulation, we have now studied the promoter of the chick muscle gamma-subunit gene. 2. This analysis was faciliated by the close upstream proximity of the coding region of the delta-subunit gene and the consequent brevity (740 bp) of the untranslated linker connecting the two genes (Nef et al., 1984). Nuclease protection and primer extension analysis revealed that transcription of the gamma-subunit gene starts at position 56 upstream of the translational initiation site. 3. Nested deletions of the promoter region were employed to identify functionally important elements. A 360-bp sequence (-324 to +36) was found to activate transcription, in a position- and orientation-independent manner, during myotube formation. This sequence comprises 5 M-CAT (Nikovits et al., 1986) similarities and contains, at positions -52/-47 and -33/-28, two CANNTG (Lassar et al., 1989) motifs. 4. Binding experiments were performed by means of gel retardation, gel shift competition, and footprint analysis. The CANNTG motifs were found to bind MyoD and myogenin fusion proteins and to interact with proteins in nuclear extracts from cultured myotubes. 5. Point mutations in the CANNTG motifs revealed that these elements are crucial for full promoter activity in myotubes and essential in fibroblasts cotransfected with a myogenin expression vector. 6. We conclude that the activity of the gamma-subunit gene is determined largely by E boxes, which in vivo are likely to be activated by MyoD family proteins; in addition, other transactivators such as the M-CAT binding protein presumably play a role. Both CANNTG elements and M-CAT motifs are also present in the alpha- and delta-subunit enhancer and may therefore account for the coordinate expression of the three subunits during muscle differentiation.
Title: Expression of myogenic factors in skeletal muscle and electric organ of Torpedo californica Neville CM, Schmidt J Ref: FEBS Letters, 305:23, 1992 : PubMed
Fish electric organ is a skeletal muscle homolog in which many muscle-specific genes are inhibited while acetylcholine receptor is expressed at high levels. The molecular mechanisms underlying this discoordinate regulation have not yet been explored. We have obtained partial sequences for MyoD, myogenin, and myf5 from Torpedo californica and have measured their mRNAs in several organs, using ribonuclease protection. We have found that MyoD and myf5 are expressed at comparable levels in muscle and electric organ, whereas myogenin transcripts could not be detected in either tissue. Acetylcholine receptor alpha subunit mRNA, on the other hand, is two orders of magnitude more abundant in electric tissue. We conclude that neither the loss of contractile proteins from, nor the enhanced expression of acetylcholine receptor genes in, the differentiating electrocyte is a simple consequence of the abundance of myogenic factor messages.
Title: Response of myogenic determination factors to cessation and resumption of electrical activity in skeletal muscle: a possible role for myogenin in denervation supersensitivity Neville CM, Schmidt M, Schmidt J Ref: Cellular Molecular Neurobiology, 12:511, 1992 : PubMed
1. We have prepared probes specific for the chicken myogenic determination genes MyoD, myogenin, myf5, and herculin and have investigated the expression of these genes in response to denervation and acute electrical stimulation in neonate chick muscle, using ribonuclease protection. 2. Upon denervation, herculin mRNA remains essentially unchanged, myf5 transcript levels approximately double, and MyoD message is up-regulated by two- to fivefold. In contrast, the message coding for myogenin, barely detectable in innervated muscle, rises dramatically (approximately 200-fold) on the second day after nerve section; in this respect it resembles acetylcholine receptor (AChR) alpha-, gamma- and delta-subunit mRNAs. Cohybridization experiments reveal that the increase in myogenin mRNA slightly precedes the rise in AChR alpha-subunit message. 3. Electrical stimulation of denervated muscle leads to an immediate decline in myogenin and AChR alpha-subunit mRNAs, with half-lives of less than an hour and approximately 4 hr, respectively; message stability measurements suggest that this is effected through a rapid shutdown of transcription. Messages coding for MyoD, myf5, and herculin decay much more slowly, as a result of slower turnover. 4. Previous experiments have indicated the involvement of a de novo induced (Tsay, H.-J., Neville, C. M., and Schmidt, J., FEBS Lett. 274:69-72, 1990) autocatalytic (Neville, C. M., Schmidt, M., and Schmidt, J., NeuroReport 2:655-657, 1991) transcription factor in the denervation-triggered up-regulation of AChR alpha-subunit expression; the denervation and electrical stimulation experiments reported here are compatible with the notion that myogenin is that factor.
Title: Kinetics of expression of ACh receptor alpha-subunit mRNA in denervated and stimulated muscle Neville C, Schmidt M, Schmidt J Ref: Neuroreport, 2:655, 1991 : PubMed
Levels of the acetylcholine receptor (AChR) alpha-subunit mRNA were quantified in chick leg muscle, both after section of the sciatic nerve and following electrical stimulation of the denervated leg musculature. Whereas a lag period of approximately 17 h intervenes between the nerve section and the increase in message level, electrical stimulation leads to an immediate decline, which proceeds with a half-life of 3-4 h, similar to the decay induced by treatment with actinomycin D. The asymmetry in the kinetics of activation and repression can be accommodated by several regulatory schemes of which the simplest contains an autocatalytic loop as recently proposed by Changeux (The New Biologist 3: 413-429, 1991).
Title: Protein synthesis is required for the denervation-triggered activation of acetylcholine receptor genes Tsay HJ, Neville CM, Schmidt J Ref: FEBS Letters, 274:69, 1990 : PubMed
The effect of cycloheximide (CHX) on denervation-induced acetylcholine receptor (AChR) expression was investigated in chickens one day after nerve section, using probe excess solution hybridization to quantitate AChR alpha-subunit gene transcript levels and run-on analysis to measure subunit gene activity. The increase in alpha-subunit transcripts that normally follows denervation was prevented when drug treatment was commenced 2 h before or after denervation but was not blocked when CHX administration was begun 6 h after the operation. Drug-induced reduction of transcript levels results from decreased activity of genes coding for the alpha-, delta-, and gamma-subunits; in contrast, the transcription rates of several non-receptor genes are not affected by CHX. The results suggest that the de novo synthesis of a transcriptional activator is required as a mediating event in the signalling pathway linking the plasma membrane and AChR gene expression.
Title: Expression of the acetylcholine receptor delta-subunit gene in differentiating chick muscle cells is activated by an element that contains two 16 bp copies of a segment of the alpha-subunit enhancer Wang XM, Tsay HJ, Schmidt J Ref: EMBO Journal, 9:783, 1990 : PubMed
The acetylcholine receptor is a multimeric membrane protein whose expression is activated during muscle differentiation and upon denervation of adult muscle. To gain insight into the coordinate expression of receptor subunits during myogenesis we have analyzed the chick muscle receptor delta-subunit gene upstream region. The delta-subunit gene lacks canonical promoter elements (CCAAT and TATA boxes). Nuclease protection and primer extension analysis revealed that transcription starts at six major and several minor sites between -110 and -30 upstream of the translational initiation site; two sites, at positions -77 and -66, give rise to approximately 50% of all transcripts. Using nested deletions of the proximal 960 bp of the 5' flanking region of this gene we have identified a 62 bp sequence (-207 to -146) that activates transcription in a position independent manner. This enhancer-like element is activated during myotube formation; it contains two distinct functional moieties, each resembling the same 16 bp portion of the stage and tissue specific alpha-subunit gene enhancer which we have characterized previously [Wang et al. (1988) Neuron, 1, 527-534]. This common element, which also comprises several previously proposed skeletal muscle specific motifs [Buskin, J. N. and Hauschka, S. D. (1989) Mol. Cell Biol., 9, 2627-2640; Mar, J. H. and Ordahl, C. P. (1988) Proc. Natl. Acad. Sci. USA, 85, 6404-6408], may account for the coordinate expression of the two subunits. The cell specificity of the delta-subunit gene 5' flanking region is partly due to the enhancer, partly to an inhibitory element upstream of -207.
Transcriptional activity of acetylcholine receptor subunit genes was investigated in innervated and denervated chick skeletal muscle. The sciatic nerve of 3-d-old White Leghorn chicks was sectioned unilaterally; after various intervals, nuclei were isolated from operated and sham-operated animals, and run-on assays performed. Nuclei were incubated with 32P-UTP, and total RNA was extracted and hybridized onto filters containing an excess of subunit-specific DNA. Specific transcripts were detected by autoradiography and quantitated densitometrically. A sharp increase in transcriptional activity was observed to begin approximately 1/2 d after the operation and peak 1 d later when transcriptional rates reached approximately seven-, six-, and fivefold control levels for the alpha-, delta-, and gamma-subunit genes, respectively. The specificity of the effect was ascertained by normalization to total RNA synthesis and by the demonstration that several nonreceptor genes respond differently to denervation. These results suggest that a denervation signal reaches the genome to induce receptor expression. In addition, since the increase in mRNA levels significantly exceeds what can be accounted for by increased gene activity, posttranscriptional effects are suggested.
Title: Binding sites for [3H]-acetylcholine and 125I-alpha-bungarotoxin in the optic ganglion of Loligo pealii Chen SJ, Spathis R, Schmidt J Ref: Comparative Biochemistry & Physiology C Pharmacol Toxicol, 90:317, 1988 : PubMed
1. In the optic ganglion of Loligo pealii, binding sites for [3H]-acetylcholine (KD: 5.2 x 10(-7) M; Bmax: 1.7 x 10(-11) mol/g tissue) and 125I-alpha-bungarotoxin (KD: 3.3 x 10(-9) M; Bmax: 9.7 x 10(-11) mol/g tissue) were observed. 2. Both sites are blocked by nicotinic compounds, but differ significantly in their affinity for individual ligands, with the acetylcholine site preferentially binding agonists, and the toxin site, antagonists. 3. The acetylcholine site is substantially more thermolabile than the toxin site. 4. A partial separation of the two binding activities is accomplished by sucrose density centrifugation. 5. These observations and a comparison with other tissues (Torpedo californica electroplaque; chick optic lobe; rat brain) suggest the presence, in the squid, of more than one kind of neuronal nicotinic receptor.
Title: Biochemistry of nicotinic acetylcholine receptors in the vertebrate brain Schmidt J Ref: International Review of Neurobiology, 30:1, 1988 : PubMed
Levels of mRNAs specific for the alpha-, gamma- and delta-subunit of the nicotinic acetylcholine receptor were measured in chick skeletal muscle by solution hybridization, using a genomic DNA probe containing the intramembrane segments M2 and M3 of the alpha-subunit and probes comprising exons 2-6 and exons 4-8, respectively, of the gamma- and delta-subunit. In the innervated calf musculature of adult chickens, receptor-specific messages were detected in approx. 100-fold excess over the amount required to account for the observed synthesis rate. Within 1 week after section of the sciatic nerve, alpha-, gamma- and delta-subunit message levels rose 112-, 42- and 24-fold, respectively, while receptor expression rate increased about 150-fold. The rise in message levels preceded the denervation-induced increase in receptor concentration. In differentiating myogenic cells all three messages were found in excess over the amounts required for the observed rate of receptor synthesis. Treatment of differentiated myotubes with drugs that change receptor synthesis rate selectively affects alpha-subunit mRNA. In all situations in vitro and in vivo the alpha-subunit mRNA was found to reach final levels faster, and to be from 3 to over 30 times more abundant, than the other messages. These observations corroborate earlier evidence for a regulatory mechanism in which the supply of mRNA determines acetylcholine receptor synthesis rate. They also suggest that receptor expression is not simply proportional to acetylcholine receptor subunit mRNA concentrations, but rather is controlled, to a considerable extent, by the efficiency with which the receptor-specific mRNAs and/or the subunits they code for are subsequently utilized.
Title: A cell type-specific enhancer drives expression of the chick muscle acetylcholine receptor alpha-subunit gene Wang Y, Xu HP, Wang XM, Ballivet M, Schmidt J Ref: Neuron, 1:527, 1988 : PubMed
The regulation of acetylcholine receptor alpha-subunit gene expression was analyzed by transient expression assays. Using rabbit beta-globin cDNA as a reporter gene, we have confirmed that the 5'-flanking sequence of the chicken acetylcholine receptor alpha-subunit gene directs specific expression in differentiated C2C12 cells, a mouse muscle cell line, but not in undifferentiated C2C12 cells and mouse 3T3 fibroblasts. Testing chimeric plasmids containing Bal31 deletion mutants of the alpha-subunit gene upstream sequence, we found the -116 to -81 region of the alpha-subunit to be responsible for tissue- and stage-specific expression. This 36 bp fragment stimulates the activity of both alpha-subunit and SV40 promoters in a distance- and orientation-independent manner, thus fulfilling the criteria of an enhancer.
Title: Quantitation of an alpha subunit splicing intermediate: evidence for transcriptional activation in the control of acetylcholine receptor expression in denervated chick skeletal muscle Shieh BH, Ballivet M, Schmidt J Ref: Journal of Cell Biology, 104:1337, 1987 : PubMed
We have investigated the mechanisms responsible for the increase in acetylcholine receptor subunit mRNAs during the induction of denervation supersensitivity in skeletal muscle. Using a cRNA probe specific for exon 7 (224 nucleotides; with flanking intron sequences of 105 nucleotides on the 3' end, and of 70 nucleotides on the 5' end) of the alpha subunit of the chicken muscle acetylcholine receptor gene, we were able to quantitate the concentration of mature alpha subunit mRNA and its precursor. In 3-wk-old chicks, the concentration of alpha subunit message in leg muscle was found to be 4.0 attomoles per microgram total RNA, and to increase 40-fold within 1 wk after section of the sciatic nerve. The molar ratio of precursor/mature mRNA, which was approximately 0.023 in innervated as well as denervated muscle, transiently rose to 0.047 at the beginning of the second postoperative day when mature message content increased 20-fold; the rise in precursor level preceded the increase in mature message content. These findings suggest that an accelerated rate of transcription of the message coding for the alpha subunit causes increased message content and the stimulation of receptor synthesis characteristic of denervated muscle.
Title: Biochemical characterization of two nicotinic receptors from the optic lobe of the chick Schneider M, Adee C, Betz H, Schmidt J Ref: Journal of Biological Chemistry, 260:14505, 1985 : PubMed
We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.
Title: An immunochemical approach to the identification of the MBTA binding site of the nicotinic acetylcholine receptor of Torpedo californica Cahill S, Schmidt J Ref: Biochemical & Biophysical Research Communications, 122:602, 1984 : PubMed
Monospecific anti-[4-(N-maleimidobenzyl) trimethylammonium] (MBTA) antibodies were prepared from sera of rabbits immunized with an albumin-MBTA conjugate and used to synthesize an MBTA-specific immunosorbent. Torpedo californica acetylcholine receptor was affinity labeled with [3H]-MBTA and proteolyzed extensively with pronase, and the peptide fraction of the digest chromatographed on the anti-MBTA resin. The amino acid composition of the purified MBTA-peptide fraction was compared with the sequences flanking the seven cysteinyl residues of the alpha-subunit. The best fit was observed with the segment containing cysteine 142.
Title: Destruction of acetylcholine receptor by decaying 125I-alpha-bungarotoxin Schmidt J Ref: Journal of Biological Chemistry, 259:14033, 1984 : PubMed
Decay of 125I in 125I-alpha-bungarotoxin bound to detergent-solubilized acetylcholine receptor from Torpedo californica electric tissue results in the inactivation of virtually all of the directly occupied and of the second toxin-binding sites. This finding establishes the usefulness of 125I-labeled ligands for binding-site multiplicity analysis of proteins.
Title: Trifluoperazine stimulates acetylcholine receptor synthesis in cultured chick myotubes Schneider M, Shieh BH, Pezzementi L, Schmidt J Ref: Journal of Neurochemistry, 42:1395, 1984 : PubMed
Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I-alpha-bungarotoxin. At drug concentrations of 5 to 10 X 10(-6) M, receptor appearance rate was significantly enhanced while receptor half-life, cellular protein, net protein synthesis rate, and acetylcholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 X 10(-5) M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KCl. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D-600, phenothiazines may stimulate receptor synthesis by blocking a voltage-gated calcium channel.
Title: Search for ligands of neuronal alpha-bungarotoxin receptors Polz-Tejera G, Schmidt J Ref: Biochemical & Biophysical Research Communications, 111:82, 1983 : PubMed
Extracts of calf brain were analyzed for substances capable of blocking the binding of [125I]-alpha-bungarotoxin to chick brain membrane preparations, and shown to contain blocking activity that was insensitive to heating and trypsin. Fractionation on Sephadex G-25 yielded two components, one representing nonspecific inhibition by inorganic cations, the other identified as choline by co-chromatography experiments and analysis of the purified inhibitor using thin layer chromatography and mass spectrometry. These results support the notion that the alpha-bungarotoxin binding macromolecule in the central nervous system is an acetylcholine receptor.
Title: Extracellular potassium and the regulation of acetylcholine receptor synthesis in embryonic chick muscle cells Shieh BH, Pezzementi L, Schmidt J Ref: Brain Research, 263:259, 1983 : PubMed
The effect of elevated extracellular potassium on acetylcholine receptor synthesis was studied in chick embryonic muscle cultures. At physiological ionic strength, potassium chloride, in the 3.3 to 50 mM range, gave rise to a complex dose-response curve whose prominent features are a considerable reduction of receptor appearance rate at 20 mM and a more than 2-fold increase at higher concentrations. The effect of potassium chloride on receptor synthesis appears to be fairly specific: neither was there a duplication of its effect by other electrolytes or solutes, nor did it alter total protein synthesis or receptor stability by more than 30% at any concentration tested; cellular acetylcholinesterase levels actually declined with increasing KCl concentrations. In order to explore the mechanism of the potassium effect, tetrodotoxin (10(-6) M), veratridine (3 X 10(-6) M), D-600 (1.6 X 10(-5) M), and ryanodine (3 X 10(-7) M) were tested in the presence of various concentrations of potassium. Sodium channel toxins as well as calcium effectors modified the potassium response. Based on these findings we propose that the effects of potassium are due to: (a) cessation of spontaneous muscle activity upon raising KCl from 3 to 10 mM; (b) depolarization of the muscle membrane and persistent activation of a calcium channel as concentration is raised from 10 to 20 mM; (c) finally, inactivation or desensitization of the calcium channel, or some other signaling element proximal to the sarcoplasmic reticulum, upon further depolarization.
Title: Distribution of alpha-bungarotoxin binding sites in the central nervous system and peripheral organs of the rat Schechter N, Handy IC, Pezzementi L, Schmidt J Ref: Toxicon, 16:245, 1978 : PubMed
Title: The cation sensitivity of the acetylcholine receptor from Torpedo californica Schmidt J, Raftery MA Ref: Journal of Neurochemistry, 23:617, 1974 : PubMed
Title: Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax Moody T, Schmidt J, Raftery MA Ref: Biochemical & Biophysical Research Communications, 53:761, 1973 : PubMed
Title: Use of affinity chromatography for acetylcholine receptor purification Schmidt J, Raftery MA Ref: Biochemical & Biophysical Research Communications, 49:572, 1972 : PubMed
