scholarly journals Acetylcholine receptor-associated 43K protein contains covalently bound myristate.

1988 ◽  
Vol 107 (3) ◽  
pp. 1113-1121 ◽  
Author(s):  
L S Musil ◽  
C Carr ◽  
J B Cohen ◽  
J P Merlie
Keyword(s):  
Fatty Acid ◽  
Acetylcholine Receptor ◽  
Lipid Bilayers ◽  
Neuromuscular Junctions ◽  
Consensus Sequence ◽  
Internal Standard ◽  
Covalent Attachment ◽  
Mouse Muscle ◽  
Prolonged Incubation ◽  
Amino Terminal

Torpedo electroplaque and vertebrate neuromuscular junctions contain high levels of a nonactin, 43,000-Mr peripheral membrane protein referred to as the 43K protein. 43K protein is associated with the cytoplasmic face of postsynaptic membranes at areas of high acetylcholine receptor density and has been implicated in the establishment and/or maintenance of these receptor clusters. Cloning of cDNAs encoding Torpedo 43K protein revealed that its amino terminus contains a consensus sequence sufficient for the covalent attachment of the rare fatty acid myristate. To examine whether 43K protein is, in fact, myristoylated, mouse muscle BC3H1 cells were metabolically labeled with either [35S]cysteine or [3H]myristate and immunoprecipitated with a monospecific antiserum raised against isolated Torpedo 43K protein. In cells incubated with either precursor, a single labeled species was specifically recovered that comigrated on SDS-PAGE with 43K protein purified from Torpedo electric organ. Approximately 95% of the 3H labeled material released from [3H]myristate-43K protein by acid methanolysis was extractable in organic solvents and eluted from a C18 reverse-phase HPLC column exclusively at the position of the methyl myristate internal standard. Thus, 43K protein contains authentic myristic acid rather than an amino or fatty acid metabolite of [3H]myristate. Myristate appears to be added to 43K protein cotranslationally and cannot be released from it by prolonged incubation in SDS, 2-mercaptoethanol, or hydroxylamine (pH 7.0 or 10.0), characteristics consistent with amino terminal myristoylation. Covalently linked myristate may be responsible for the high affinity of purified 43K protein for lipid bilayers despite the absence of a notably hydrophobic amino acid sequence.

scholarly journals Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

The polyunsaturated fatty acid status of foetal and neonatal ruminants

1978 ◽  
Vol 39 (1) ◽  
pp. 53-59 ◽  
Author(s):  
E. Payne
Keyword(s):  
Fatty Acid ◽  
Soft Tissues ◽  
Placental Transfer ◽  
Total Concentration ◽  
Young Animal ◽  
Internal Standard ◽  
Mature Animal ◽  
Neonatal Lambs ◽  
Total Body ◽  
Total Pufa

1. Information on the fatty acid composition of tissues of foetal calves, neonatal lambs, deer and piglets reported by Payne (1978) has been quantified by the use of an internal standard during analysis, to give concentrations of total polyunsaturated fatty acids (PUFA) derived from linoleic acid (ω6) and linolenic acid (ω3) expressed on a per kg tissue basis. The total concentration of both acids (ω6+ω3) was similar in all tissues examined except brain. Because muscle, the main constituent of the soft tissues of young animals, contains about 40–50 % of the total body content of these acids, it is considered that muscle concentrations are a reflection of total body status of these acids.2. Concentrations in muscle of both ω6 derivatives and total PUFA were significantly lower in the neonatal lamb and foetal calf than in the mature animal whereas in pigs and deer the concentrations in the young animal were similar to those in the mature animal. Concentrations of ω6 derivatives and total PUFA in lambs were significantly lower than those in calves; the presence of ω3 derivatives reduced the level of significance for total PUFA. Again, total PUFA content did not differ significantly between the piglet and the young ruminants.3. There was a substantial placental transfer, with apparently a preferential transfer of ω3 derivatives.4. In brain the levels of ω3 acids were as high in the foetal and neonatal animals as in mature animals. The levels of ω6 acids were lower in young animals.5. Calculations of ω6 intake from milk showed that the total deficit of ω6 could be made up within a few days.6. It was concluded that the extent of deficiency of ω6 in young ruminants raised in a grazing situation, as in New Zealand, is marginal and any feeding to overcome this is unlikely to be of any benefit.

scholarly journals Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatjana Straka ◽  
Charlotte Schröder ◽  
Andreas Roos ◽  
Laxmikanth Kollipara ◽  
Albert Sickmann ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Ribosomal Proteins ◽  
Neuromuscular Junctions ◽  
Sympathetic Neurons ◽  
Sympathetic Innervation ◽  
Regulatory Function ◽  
Hindlimb Muscles ◽  
Nerve Muscle

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.

trans activation of gene expression by v-myb

1990 ◽  
Vol 10 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
C E Ibanez ◽  
J S Lipsick
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Vp16 Fusion ◽  
Acute Myelomonocytic Leukemia ◽  
Simplex Virus

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

scholarly journals Improved Method for Gas Chromatographic–Mass Spectrometric Analysis of 1-13C-labeled Long-Chain Fatty Acids in Plasma Samples

2002 ◽  
Vol 48 (6) ◽  
pp. 906-912 ◽  
Author(s):  
José M Hernández-Pérez ◽  
Eduard Cabré ◽  
Lourdes Fluvià ◽  
Ágata Motos ◽  
Cruz Pastor ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Isotopic Ratio ◽  
Internal Standard ◽  
Mass Spectrometric ◽  
Spectrometric Analysis ◽  
Calibration Curves ◽  
Long Chain ◽  
Chromatographic Mass

Abstract Background: Gas chromatographic–mass spectrometric (GC/MS) tracking of stable-isotope-labeled substrates is useful in metabolic studies. However, GC/MS analysis of long-chain fatty acid methyl esters yields results that mostly depend on their concentration in the system. We describe a protocol aimed to obviate this and other drawbacks in plasma [1-13C]palmitic and [1-13C]oleic acid measurements. Methods: Lipoproteins were separated by sequential ultracentrifugation. Free or esterified heptadecanoic acid was used as internal standard. Fatty acids were derivatized to trimethylsilyl (TMS) esters. GC separation was in isothermal mode at 210 °C for 27 min. For both TMS-palmitate and TMS-oleate, M and [M + 1] signals were simultaneously acquired with a dual acquisition program in single-ion monitoring mode. Calibration mixtures containing increasing amounts of labeled fatty acids were prepared gravimetrically to construct calibration curves for isotopic enrichment. Likewise, five calibration curves (for increasing concentrations) were constructed for each fatty acid; this allowed selection of the most appropriate curve for the concentration in a plasma sample. Results: Oleic acid-TMS ester was clearly separated from that of its stereoisomer, elaidic acid. Within a 10-fold concentration range, the isotopic ratio was independent on the amount of the analyte in the sample, with a maximum uncertainty of 0.34% in terms of molar percent excess. In addition, the within- and between-day imprecision (CV) of the method was <1%. Conclusion: Results obtained with this method are independent of concentration and sufficiently precise for tracking 1-13C-labeled palmitic and oleic acids in biological samples

scholarly journals The proto-oncogene HLF and the related basic leucine zipper protein TEF display highly similar DNA-binding and transcriptional regulatory properties

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4607-4617 ◽  
Author(s):  
SP Hunger ◽  
S Li ◽  
MZ Fall ◽  
L Naumovski ◽  
ML Cleary
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Basic Leucine Zipper ◽  
Amino Terminal ◽  
Transcriptional Regulatory ◽  
Regulatory Properties

Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To address these questions, we cloned the human homologue of TEF/VBP, a bZIP protein closely related to HLF. Using a binding site selection assay, we found that TEF bound preferentially to the consensus sequence 5′-GTTACGTAAT-3′, which is identical to the previously determined HLF recognition site. TEF and HLF activated transcription of consensus site-containing reporter genes in several different cell types with similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to a discrete approximate 40 amino acid region of TEF and HLF within which they share 72% amino acid identity and 85% similarity. The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs. The THAD contained most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogated transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF and HLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important.

Sign in / Sign up

Export Citation Format

Share Document