ABSTRACT
Epigenetics is one of the key areas of future research that can elucidate how genomes work. It combines genetics and the environment to address complex biological systems such as the plasticity of our genome. While all nucleated human cells carry the same genome, they express different genes at different times. Much of this is governed by epigenetic changes resulting in differential methylation of our genome--or different epigenomes. Individual studies over the past decades have already established the involvement of DNA methylation in imprinting, gene regulation, chromatin structure, genome stability and disease, especially cancer. Now, in the wake of the Human Genome Project (HGP), epigenetic phenomena can be studied genome-wide and are giving rise to a new field, epigenomics. Here, we review the current and future potential of this field and introduce the pilot study towards the Human Epigenome Project (HEP).
Subject(s)
DNA Methylation , Gene Expression Regulation , Genome, Human , Autoimmune Diseases/genetics , Chromosome Mapping , CpG Islands , Cytosine/metabolism , DNA Modification Methylases/metabolism , Human Genome Project , Humans , Major Histocompatibility Complex , Neoplasms/genetics , Neoplasms/metabolism , Oligonucleotide Array Sequence AnalysisABSTRACT
A 3.5-kb segment of the alcohol dehydrogenase (Adh) region that includes the Adh and Adh-related genes was sequenced in 139 Drosophila pseudoobscura strains collected from 13 populations. The Adh gene encodes four protein alleles and rejects a neutral model of protein evolution with the McDonald-Kreitman test, although the number of segregating synonymous sites is too high to conclude that adaptive selection has operated. The Adh-related gene encodes 18 protein haplotypes and fails to reject an equilibrium neutral model. The populations fail to show significant geographic differentiation of the Adh-related haplotypes. Eight of 404 single nucleotide polymorphisms (SNPs) in the Adh region were in significant linkage disequilibrium with three ADHR protein alleles. Coalescent simulations with and without recombination were used to derive the expected levels of significant linkage disequilibrium between SNPs and 18 protein haplotypes. Maximum levels of linkage disequilibrium are expected for protein alleles at moderate frequencies. In coalescent models without recombination, linkage disequilibrium decays between SNPs and high frequency haplotypes because common alleles mutate to haplotypes that are rare or that reach moderate frequency. The implication of this study is that linkage disequilibrium mapping has the highest probability of success with disease-causing alleles at frequencies of 10%.
Subject(s)
Alcohol Dehydrogenase/genetics , Alleles , Drosophila/genetics , Linkage Disequilibrium , Polymorphism, Single Nucleotide , Animals , Drosophila/enzymology , Evolution, Molecular , Haplotypes , PhylogenyABSTRACT
Molecular portraits, such as mRNA expression or DNA methylation patterns, have been shown to be strongly correlated with phenotypical parameters. These molecular patterns can be revealed routinely on a genomic scale. However, class prediction based on these patterns is an under-determined problem, due to the extreme high dimensionality of the data compared to the usually small number of available samples. This makes a reduction of the data dimensionality necessary. Here we demonstrate how phenotypic classes can be predicted by combining feature selection and discriminant analysis. By comparing several feature selection methods we show that the right dimension reduction strategy is of crucial importance for the classification performance. The techniques are demonstrated by methylation pattern based discrimination between acute lymphoblastic leukemia and acute myeloid leukemia.
Subject(s)
DNA Methylation , Neoplasms/chemistry , Neoplasms/classification , Computational Biology , CpG Islands , DNA, Neoplasm/chemistry , Humans , Leukemia, Myeloid, Acute/metabolism , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Principal Component AnalysisABSTRACT
A beta-D-glucan exohydrolase was purified from the cell walls of developing maize (Zea mays L.) shoots. The cell wall enzyme preferentially hydrolyzes the non-reducing terminal glucosyl residue from (1-->3)-beta-D-glucans, but also hydrolyzes (1-->2)-, (1-->6)-, and (1-->4)-beta-D-glucosyl units in decreasing order of activity. Polyclonal antisera raised against the purified exo-beta-D-glucanase (ExGase) were used to select partial-length cDNA clones, and the complete sequence of 622 amino acid residues was deduced from the nucleotide sequences of the cDNA and a full-length genomic clone. Northern gel-blot analysis revealed what appeared to be a single transcript, but three distinct polypeptides were detected in immunogel-blot analyses of the ExGases extracted from growing coleoptiles. Two polypeptides appear in the cell wall, where one polypeptide is constitutive, and the second appears at the time of the maximum rate of elongation and reaches peak activity after elongation has ceased. The appearance of the second polypeptide coincides with the disappearance of the mixed-linkage (1-->3), (1-->4)-beta-D-glucan, whose accumulation is associated with cell elongation in grasses. The third polypeptide of the ExGase is an extrinsic protein associated with the exterior surface of the plasma membrane. Although the activity of the membrane-associated ExGase is highest against (1-->3)-beta-D-glucans, the activity against (1-->4)-beta-D-glucan linkages is severely attenuated and, therefore, the enzyme is unlikely to be involved with turnover of the (1-->3), (1-->4)-beta-D-glucan. We propose three potential functions for this novel ExGase at the membrane-wall interface.
Subject(s)
Cell Wall/enzymology , Glycoside Hydrolases/metabolism , Zea mays/enzymology , Amino Acid Sequence , Base Sequence , Cell Membrane/enzymology , Cloning, Molecular , DNA Primers , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/genetics , Glycoside Hydrolases/isolation & purification , Molecular Sequence Data , Sequence Homology, Amino Acid , Zea mays/growth & developmentABSTRACT
DNA methylation is essential for the control of a number of biological mechanisms in mammals [1]. Mammalian development is accompanied by two major waves of genome-wide demethylation and remethylation: one during germ-cell development and the other after fertilisation [2] [3] [4] [5] [6] [7]. Most previous studies have suggested that the genome-wide demethylation observed after fertilisation occurs passively, that is, by the lack of maintenance methylation following DNA replication and cell division [6] [7], although one other study has reported that replication-independent demethylation may also occur during early embryogenesis [8]. Here, we report that genes that are highly methylated in sperm are rapidly demethylated in the zygote only hours after fertilisation, before the first round of DNA replication commences. By contrast, the oocyte-derived maternal alleles are unaffected by this reprogramming. They either remain methylated after fertilisation or become further methylated de novo. These results provide the first direct evidence for active demethylation of single-copy genes in the mammalian zygote and, moreover, reveal a striking asymmetry in epigenetic methylation reprogramming. Whereas paternally (sperm)-derived sequences are exposed to putative active demethylases in the oocyte cytoplasm, maternally (oocyte)-derived sequences are protected from this reaction. These results, whose generality is supported by findings of Mayer et al. [9], have important implications for the establishment of biparental genetic totipotency after fertilisation, the establishment and maintenance of genomic imprinting, and the reprogramming of somatic cells during cloning.
Subject(s)
DNA Methylation , Spermatozoa/chemistry , Zygote/metabolism , Actins/genetics , Animals , CpG Islands/genetics , DNA/genetics , DNA/metabolism , Female , Insulin-Like Growth Factor II/genetics , Male , Mice , Oocytes/chemistry , Transgenes , Zygote/chemistryABSTRACT
Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.
Subject(s)
Biological Evolution , CpG Islands/genetics , DNA Methylation , Factor VIII/genetics , Point Mutation , Protein-Tyrosine Kinases , Receptors, Fibroblast Growth Factor/genetics , Achondroplasia/genetics , Cloning, Molecular , Female , Hemophilia A/genetics , Humans , Male , Ovum , Polymerase Chain Reaction , Receptor, Fibroblast Growth Factor, Type 3 , Sequence Analysis, DNA , Sex Factors , SpermatozoaABSTRACT
5-Azacytidine inhibits DNA synthesis and to a lesser proportion RNA synthesis in S. antibioticus. The biosynthesis of proteins is not affected. The main inhibitory effect of 5-azacytidine on DNA and RNA synthesis is probably caused by its incorporation into newly synthesized DNA or RNA and the formation of covalent complexes between cytosine-specific methyltransferases and the modified DNA or RNA templates. To analyze whether such effects could occur at the oriC region of S. antibioticus we analyzed the methylation status of this region using the bisulphite assisted genomic sequencing method. One of the cytosine residues found to be partially methylated was contained within an unique NaeI sequence (GCCGGC) in oriC. Subsequent analysis shows chromosomal DNA from S. antibioticus to be resistant to R.NaeI restriction indicating that this strain contains a NaeI-specific cytosine C5-methyltransferase activity. Following 5-azacytidine treatment the NaeI site within the oriC region becomes partially demethylated. Our results suggest that some of the 5-azacytidine effects on DNA and RNA synthesis might indeed be related to the complex formation and inhibition of a cytosine-specific DNA methyltransferase.
Subject(s)
Azacitidine/pharmacology , DNA Methylation , DNA, Bacterial/biosynthesis , Nucleic Acid Synthesis Inhibitors/pharmacology , Replication Origin , Streptomyces antibioticus/genetics , DNA-Cytosine Methylases/metabolism , Streptomyces antibioticus/drug effectsABSTRACT
Although most patients are discharged following traumatic brain injury (TBI) with "good recovery', recent reports indicate that many have persistent neuropsychological deficits. The purposes of this study were to: (1) determine if functional, neuropsychological and social outcome at 3 and 6 months in patients hospitalized following TBI could be ascertained via telephone follow-up, and (2) assess use of rehabilitation services in this population. Patients were identified through acute hospital admissions. A trained nurse practitioner abstracted data from acute charts. Using the Functional Independence Measure (FIM), Neurobehavioral Rating Scale (NRS), Telephone Interview for Cognitive Status (TICS), and a social questionnaire she obtained follow-up information at 3 and 6 months post-injury. Of 74 patients initially identified, 51 and 48 were available at 3 and 6 months, respectively. Most experienced mild to moderate TBI. Physical disability was minimal at follow-up as indicated by the FIM. Approximately half of the patients were rated cognitively impaired on the TICS at 3 months and over one-third remained impaired at 6 months. At 6 months 60.5% remained unemployed. Only eight of the 67 discharged survivors received any rehabilitation services. A brief telephone follow-up appears to be a cost-effective way to ascertain functional and neuropsychological outcome in TBI survivors. Since few of these patients received rehabilitation, a telephone follow-up may identify those who would potentially benefit from additional rehabilitation services.
Subject(s)
Brain Damage, Chronic/rehabilitation , Brain Injuries/rehabilitation , Neuropsychological Tests , Social Adjustment , Activities of Daily Living/classification , Activities of Daily Living/psychology , Adult , Brain Damage, Chronic/diagnosis , Brain Damage, Chronic/psychology , Brain Injuries/diagnosis , Brain Injuries/psychology , Cohort Studies , Disability Evaluation , Female , Follow-Up Studies , Glasgow Coma Scale , Humans , Length of Stay , Male , Multiple Trauma/diagnosis , Multiple Trauma/psychology , Multiple Trauma/rehabilitation , Rehabilitation, Vocational/psychology , Telephone , Treatment OutcomeABSTRACT
Sequencing of bisulphite modified genomic DNA is the most powerful method to determine methylation patterns in chromosomal DNA. In many experimental systems, the amount of material available for analysis is very small which makes it necessary to perform experiments at extreme levels of sensitivity and reproducibility. In this communication, we present an improved modification of the bisulphite based sequencing method. Our strategy is to perform the bisulphite treatment and subsequent PCR steps on material embedded into agarose beads. This prevents loss of DNA during the experimental procedure and ensures an optimal bisulphite reactivity by maintaining the DNA in the single stranded form. The modification improves previously published protocols in that it facilitates the handling of probes and reproducibly reaches a very high level of sensitivity.
Subject(s)
Cytosine/metabolism , DNA Methylation , Sulfites/metabolism , Cell Line , Sensitivity and SpecificityABSTRACT
It has been suggested that at the time of innervation, developing neurites release one or more soluble factors that locally induce acetylcholine receptor (AChR) aggregate formation at the synaptic site. To test this hypothesis, we developed a model system that mimics the local, neural induction of AChR aggregation at developing synapses. Partially purified protein derived from fetal pig brain was applied locally to the surface of cultured myotubes via a micropipet. We found that local application of this factor for as little as 30 min induced the formation of AChR aggregates that were restricted to a region within 30 microns around the release site. In addition, the locally applied factor induced a local AChR aggregation response, but did not cause a detectable change in the myotube resting membrane potential at the release site. Our data support a soluble factor hypothesis and suggest that neither cell-cell contact nor local electric fields are necessary for the initial induction of AChR aggregation.
Subject(s)
Muscles/ultrastructure , Nerve Tissue Proteins/pharmacology , Receptors, Nicotinic/metabolism , Animals , Brain Chemistry , Cells, Cultured , In Vitro Techniques , Muscles/metabolism , Rats , Rats, Sprague-Dawley , Receptor Aggregation , Swine , Video RecordingABSTRACT
A soluble fetal brain extract (EBX) induces acetylcholine receptor (AChR) aggregation in cultured rat myotubes within 4 hr at 36 degrees C in a defined medium containing 1.8 mM (normal) extracellular Ca2+ (Olek et al., 1983). The activity of EBX was Ca2+ dependent; reducing extracellular Ca2+ significantly inhibited EBX-induced AChR aggregation and a 15-50% increase in extracellular Ca2+ synergistically enhanced the activity of EBX. Synergism was specific for Ca2+ as increases in other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) had no effect. A large increase (300-500%) in extracellular Ca2+ alone also induced AChR aggregation within 4 hr at 36 degrees C. An equivalent increase in other cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) did not promote AChR aggregation. An initial 15-min pulse of increased extracellular Ca2+ alone or with EBX was adequate to induce AChR aggregation. Aggregates induced by EBX, Ca2+ alone, or EBX/Ca2+ were found predominantly on the top surface of the myotube. These treatments did not detectably alter preexisting aggregates present at substrate contact sites on the bottom surface of myotubes. AChR aggregation induced by any treatment was not inhibited by cycloheximide, Ca2+ channel blockers, or protease inhibitors but was blocked by Co2+ and sodium azide.
Subject(s)
Calcium/pharmacology , Nerve Tissue Proteins/pharmacology , Receptors, Cholinergic/metabolism , Animals , Azides/pharmacology , Brain Chemistry , Calcium Channel Blockers/pharmacology , Cells, Cultured , Fetus/chemistry , Muscles/drug effects , Muscles/metabolism , Nerve Tissue Proteins/isolation & purification , Rats , Rats, Inbred Strains , Receptors, Cholinergic/drug effects , Sodium AzideABSTRACT
Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.
Subject(s)
Brain/cytology , Cell Extracts/pharmacology , Cytoskeletal Proteins/metabolism , Receptors, Cholinergic/metabolism , Tissue Extracts/pharmacology , Actinin/metabolism , Actins/metabolism , Animals , Brain/ultrastructure , Cells, Cultured , Contractile Proteins/metabolism , Culture Media/analysis , Culture Media/pharmacology , Cytoskeletal Proteins/analysis , Filamins , Fluorescent Antibody Technique , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Molecular Weight , Muscles/embryology , Muscles/metabolism , Muscles/ultrastructure , Rats , VinculinABSTRACT
A soluble toxic extract derived from spine tissue of the lionfish (Pterois volitans) decreased heart rate and force of contraction in isolated clam and frog hearts. These actions were due to the presence of micromolar concentrations of acetylcholine in the extract. Toxicity was retained after hydrolysis of acetylcholine by exogenous acetylcholinesterase, but heart function was no longer affected. Toxin treated in this way induced muscle fibrillation in an isolated nerve-muscle preparation, followed by blockade of neuromuscular transmission. Bursts of transient depolarizations were recorded at the muscle endplate shortly after toxin addition that correlated in time with the duration of toxin-induced muscle fibrillation. These effects are thought to be due to the increased release and then depletion of acetylcholine from the nerve terminal.
Subject(s)
Acetylcholine/toxicity , Fish Venoms/toxicity , Neurotoxins/toxicity , Tissue Extracts/toxicity , Acetylcholine/isolation & purification , Animals , Biological Assay , Bivalvia , Fish Venoms/isolation & purification , In Vitro Techniques , Membrane Potentials/drug effects , Mice , Mice, Inbred BALB C , Myocardial Contraction/drug effects , Neuromuscular Junction/drug effects , Neurotoxins/isolation & purification , Rana pipiens , Synaptic Transmission/drug effects , Tissue Extracts/isolation & purificationABSTRACT
We have studied the effects of temperature and sodium azide on the formation and stability of embryonic brain extract (EBX)2-induced acetylcholine receptor (AChR) aggregates on myotubes. Sequential changes in AChR distribution were studied on living myotubes in culture by video-intensified fluorescence microscopy. Aggregate formation was temperature dependent, increasing sharply from 24-36 degrees, maximal at 36-37 degrees, and virtually blocked at 38-40 degrees. Whereas aggregate size increased rapidly with time (up to 4 hr) at 36 degrees, at 18-24 degrees small (less than or equal to 1 micron) "microaggregates" formed and accumulated for up to 10 hr. Aggregates formed within 1.5 hr at the sites of microaggregates (formed after 4 hr at 23 degrees) if the temperature was raised to 36 degrees. However, if EBX was removed, the microaggregates on 50% of myotubes disassembled within 1.5 hr. The formation of microaggregates at 23 degrees and aggregates at 36 degrees was reversibly inhibited by sodium azide. These results show that clusters of microaggregates are the precursors of aggregates, and suggest that microaggregate clouds represent a discrete, labile, ATP-dependent stage in aggregate formation. Aggregates that had formed after 4 hr in the presence of EBX disassembled slowly (within 12-14 hr) following removal of EBX at 36 degrees, and even more slowly at 23-30 degrees. However, a temperature shift to 38 degrees, or the addition of azide, resulted in a rapid but reversible disassembly of aggregates (within 4 hr). Thus, newly formed aggregates appear to be relatively stable structures, while microaggregate clouds are labile, tending to disassemble or evolve into aggregates.
Subject(s)
Muscles/physiology , Receptors, Nicotinic/metabolism , Animals , Azides/pharmacology , Brain/physiology , Bungarotoxins , Cell Compartmentation , Cell Differentiation , Cells, Cultured , Macromolecular Substances , Motion Pictures , Muscles/cytology , Rats , Sarcolemma/physiology , Sarcolemma/ultrastructure , TemperatureABSTRACT
[125I]alpha-Bungarotoxin specifically binds to a subpopulation of rodent spinal cord neurons in vitro. Binding first becomes apparent between 1 and 2 weeks in culture and then increases dramatically after 3 weeks. Similarly, cell suspensions from freshly dissociated embryonic spinal cords do not bind toxin whereas cell suspensions from 1 week old neonates demonstrate specific binding of [125I]alpha-bungarotoxin. In vitro, binding is inhibited more effectively in the presence of nicotinic rather than muscarinic agents. Autoradiography of [125I]alpha-bungarotoxin binding to 4-week-old cultures revealed a uniform labeling pattern over cell somas and processes. Although the relation of toxin binding to functional acetylcholine receptors is not known, the appearance of toxin binding sites may have some developmental significance for the maturation of cholinergic transmission or the maintenance of synaptic connections.
Subject(s)
Bungarotoxins/metabolism , Neurons/metabolism , Receptors, Cholinergic/analysis , Receptors, Nicotinic , Spinal Cord/metabolism , Animals , Autoradiography , Cell Survival , Cells, Cultured , Embryo, Mammalian , Mice , Neurons/physiology , Rats , Receptors, Cholinergic/physiology , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
The ultrastructure of cultured rat myotubes was examined at stages in the initial assembly of acetylcholine receptor (AChR) aggregates in order to elucidate the role of cell-surface specializations in aggregate formation. Within 4-6 hr, embryonic brain extract (EBX) induces the formation of sites of AChR density elevated 5-9 X above that of surrounding regions, and the appearance of these aggregates is preceded by the formation of clouds of punctate microaggregates (Olek et al., 1983). A video image-intensification system was used to monitor this redistribution of fluorescently labeled AChR, and sites of aggregation were mapped on identified myotubes. After processing the cultures for electron microscopy, thin sections were taken through identified aggregate sites at various stages in assembly. Specializations, including a basal lamina, mound-shaped plasma membrane contours with occasional deep infoldings, and a subjacent dense cytoskeletal specialization, which tended to exclude other cytoplasmic organelles, were associated with newly formed aggregates found 4-6 hr after adding EBX to the cultures. Analysis of random thin sections through EBX-treated and untreated myotubes showed that the extent of specializations of the basal lamina and cytoplasm was approximately threefold greater in cells exposed to EBX for 4 hr, suggesting a concurrent, and possibly interdependent, organization of such specializations with AChR aggregate assembly. Examination of sections through clouds of microaggregates, which formed within 90 min, revealed mound-shaped plasma membrane contours and underlying cytoplasm depleted of organelles but relatively little basal lamina and submembrane cytoskeletal density. These results suggest that the initial stage of AChR aggregate assembly involves relatively subtle changes in the structure of the cell cortex and that the evolution of microaggregates to aggregates may require the formation of additional cytoskeletal and extracellular matrix structures.
Subject(s)
Muscles/ultrastructure , Receptors, Cholinergic/analysis , Animals , Female , Horseradish Peroxidase/metabolism , Microscopy, Electron , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Muscles/embryology , Pregnancy , Rats , Rats, Inbred Strains , Time FactorsABSTRACT
We have directly observed the redistribution of acetylcholine receptors (AChR) on the surface of cultured myotubes, induced by a soluble brain extract. The AChR were fluorescently labeled with rhodamine-conjugated alpha-bungarotoxin and viewed under low incident illumination with a video image intensification system. The results of our sequential observations indicate that AChR aggregates can be assembled rapidly (30-120 min) from mobile, diffuse AChR. This assembly was characterized by the initial formation of microaggregates (less than 1 micron diameter) that increased in number and coalesced or grew to form larger aggregates. The redistribution of fluorescently labeled AChR was completely inhibited by illumination of cells at levels used for conventional fluorescence micrography and could be observed only by using low light levels.
Subject(s)
Brain Chemistry , Muscles/embryology , Receptors, Cholinergic/metabolism , Tissue Extracts/pharmacology , Animals , Cells, Cultured , Light , Microscopy, Fluorescence , Muscles/metabolism , Rats , Rats, Inbred StrainsABSTRACT
It was previously found that the number of junctional acetylcholine receptors of rat diaphragm, as measured with [125I]alphabungarotoxin binding, suddenly increased 2 days after denervation in vivo or in vitro. Organ culture was used here to characterize further this unusual class of junctional receptors. The 'new' acetylcholine receptors were physiologically functional and were functionally located only in the junctional region. The rate of degradation of new receptors was slower than that of extrajunctional receptors and similar (in the first 24 h) to that of typical junctional receptors. In addition, the appearance of new junctional receptors was inhibited by cycloheximide and actinomycin D given at critical periods, implicating a protein synthetic step. Finally, nerve stimulation in the presence of a post-synaptic blocker (pancuronium) advanced the time of appearance of new junctional receptors. This last finding coupled with our previous report of nerve stump length effects on junctional acetylcholine receptors reinforces the suggestion that under certain conditions the level of junctional receptors can be regulated by the motor neuron.