RESUMO
OBJECTIVES: In patients with celiac disease, enteropathy is caused by the entry of gluten peptides into the lamina propria of the intestine, in which their immunogenicity is potentiated by tissue transglutaminase (tTG) and T-helper type 1-mediated immune responses are triggered. Tight junction disassembly and paracellular permeability are believed to have an important role in the transport of gluten peptides to the lamina propria. Larazotide acetate is a tight-junction regulator peptide that, in vitro, prevents the opening of intestinal epithelial tight junctions. The aim of this study was to evaluate the efficacy and tolerability of larazotide acetate in protecting against gluten-induced intestinal permeability and gastrointestinal symptom severity in patients with celiac disease. METHODS: In this dose-ranging, placebo-controlled study, 86 patients with celiac disease controlled through diet were randomly assigned to larazotide acetate (0.25, 1, 4, or 8 mg) or placebo three times per day with or without gluten challenge (2.4 g/day) for 14 days. The primary efficacy outcome was the urinary lactulose/mannitol (LAMA) fractional excretion ratio. Secondary endpoints included gastrointestinal symptom severity, quality-of-life measures, and antibodies to tTG. RESULTS: LAMA measurements were highly variable in the outpatient setting. The increase in LAMA ratio associated with the gluten challenge was not statistically significantly greater than the increase in the gluten-free control. Among patients receiving the gluten challenge, the difference in the LAMA ratios for the larazotide acetate and placebo groups was not statistically significant. However, larazotide acetate appeared to limit gluten-induced worsening of gastrointestinal symptom severity as measured by the Gastrointestinal Symptom Rating Scale at some lower doses but not at the higher dose. Symptoms worsened significantly in the gluten challenge-placebo arm compared with the placebo-placebo arm, suggesting that 2.4 g of gluten per day is sufficient to induce reproducible gluten toxicity. Larazotide acetate was generally well tolerated. No serious adverse events were observed. The most common adverse events were headache and urinary tract infection. CONCLUSIONS: LAMA variability in the outpatient setting precluded accurate assessment of the effect of larazotide acetate on intestinal permeability. However, some lower doses of larazotide acetate appeared to prevent the increase in gastrointestinal symptom severity induced by gluten challenge.
Assuntos
Doença Celíaca/prevenção & controle , Fármacos Gastrointestinais/uso terapêutico , Glutens/administração & dosagem , Oligopeptídeos/uso terapêutico , Doença Aguda , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevenção Secundária/métodos , Índice de Gravidade de Doença , Junções Íntimas/efeitos dos fármacosRESUMO
We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.
Assuntos
Amoeba/genética , Genes , Miosinas/genética , Fragmentos de Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon , Enzimas de Restrição do DNA , Microscopia Eletrônica , Subfragmentos de Miosina , SoftwareRESUMO
Nerve growth factor (NGF) is essential for the development and differentiation of sympathetic or sensory neurons. A complementary DNA was cloned that corresponds to a gene sequence induced more than 50-fold in a cultured target cell line of pheochromocytoma cells (PC12 cells) 5 hours after the addition of NGF. The induced messenger RNA encodes a 90,000-dalton polypeptide that may represent one of the primary events in NGF-induced differentiation of neurons.
Assuntos
Clonagem Molecular , Genes , Fatores de Crescimento Neural/fisiologia , Actinas/genética , Neoplasias das Glândulas Suprarrenais/genética , Animais , Sequência de Bases , Linhagem Celular , Galinhas , DNA/genética , Regulação da Expressão Gênica , Hibridização de Ácido Nucleico , Feocromocitoma/genética , RNA Mensageiro/genética , Coelhos , RatosRESUMO
A primer extension assay that is diagnostic for the messenger RNA's (mRNA's) transcribed from the beta-cytoplasmic, alpha-cardiac, and alpha-skeletal actin genes of the chicken was used to measure the mRNA levels for these actin isoforms. Measurements were made in chicken breast muscle during myogenesis in vivo and in vitro. alpha-Cardiac actin mRNA accounts for more than 90 percent of the sarcomeric actin transcripts expressed in avian embryonic breast muscle. Five weeks after hatching, alpha-skeletal actin mRNA is the only detectable sarcomeric actin transcript.
Assuntos
Actinas/metabolismo , Músculos/embriologia , Miocárdio/metabolismo , Actinas/genética , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Coração/embriologia , RNA Mensageiro/metabolismo , Sarcômeros/embriologiaRESUMO
Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Músculos/embriologia , Fatores de Transcrição/genética , Animais , Sequência de Bases , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/fisiologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila , Expressão Gênica , Genes Homeobox , Genes de Insetos , Genes Reguladores , Teste de Complementação Genética , Fatores de Transcrição MEF2 , Mesoderma/metabolismo , Dados de Sequência Molecular , Músculos/citologia , Músculos/metabolismo , Mutagênese , Fatores de Regulação Miogênica , Miosinas/biossíntese , Miosinas/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/análise , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: Lifelong adherence to a strict gluten-free diet is the cornerstone of coeliac disease treatment. Elucidation of disease pathogenesis has created opportunities for novel therapeutic approaches to coeliac disease. AT-1001 is an inhibitor of paracellular permeability whose structure is derived from a protein secreted by Vibrio cholerae. AIM: To determine the safety and tolerability of 12 mg doses of AT-1001 in coeliac disease subjects challenged with gluten. METHODS: An in-patient, double-blind, randomized placebo-controlled safety study utilizing intestinal permeability, measured via fractional excretions of lactulose and mannitol, as an exploratory measure of drug efficacy. RESULTS: Compared to placebo, no increase in adverse events occurred in patients exposed to AT-1001. Following acute gluten exposure, a 70% increase in intestinal permeability was detected in the placebo group, while none was seen in the AT-1001 group. Interferon-gamma levels increased in four of seven patients (57%) of the placebo group, but only in four of 14 patients (29%) of the AT-1001 group. Gastrointestinal symptoms were more frequently detected in the placebo group when compared to the AT-1001 group (P = 0.018). CONCLUSIONS: AT-1001 is well tolerated and appears to reduce intestinal barrier dysfunction, proinflammatory cytokine production, and gastrointestinal symptoms in coeliacs after gluten exposure.
Assuntos
Doença Celíaca/dietoterapia , Glutens/efeitos adversos , Receptores de Superfície Celular/uso terapêutico , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Oligopeptídeos/uso terapêutico , Placebos , Qualidade de Vida , Receptores de Superfície Celular/antagonistas & inibidoresRESUMO
Approximately 1 kilobase of genomic DNA from the chicken fast myosin light-chain 1f/3f gene 5' to the transcriptional start sites for each light-chain mRNA was sufficient for differentiation-dependent, tissue-restricted expression. This was determined in primary chick myoblast cultures transfected with the chloramphenicol acetyltransferase (CAT) expression vector p8CAT containing these 5'-flanking sequences. The expression of CAT activity from both light-chain promoters was 10- to 20-fold higher in differentiated myotubes than in fibroblasts or myoblasts grown in bromodeoxyuridine. In contrast, the beta-actin and Rous sarcoma virus promoters joined to the CAT gene were expressed equally in all cell backgrounds tested. Even though the relative timing of light-chain 1f and 3f expression was altered, tissue-restricted, differentiation-dependent expression of the light-chain mRNAs was maintained with these 5' cis-acting sequence elements.
Assuntos
Genes , Músculos/metabolismo , Miosinas/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , Acetiltransferases/genética , Animais , Diferenciação Celular , Células Cultivadas , Galinhas , Cloranfenicol O-Acetiltransferase , Regulação da Expressão Gênica , Músculos/citologia , Miosinas/biossíntese , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The mouse histone H4 gene, when stably transformed into L cells on the PSV2gpt shuttle vector, is cell cycle regulated in parallel with the endogenous H4 genes. This was determined in exponentially growing pools of transformants fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cell cycle without the use of treatments that arrest growth. Linker additions in the 5' noncoding region of the H4 RNA or in the coding region of the gene did not affect the cell cycle-regulated expression of the modified H4 gene even though the overall level of expression was altered. However, replacing the H4 promoter with the human alpha-2 globin promoter, so that the histone transcript produced by the chimeric gene remains essentially unchanged, resulted in the constitutive expression of H4 mRNA during all phases of the cell cycle with no net increase in H4 mRNA levels during the G1-to-S transition. From these results we conclude that all the information necessary for the cell cycle-regulated expression of the H4 gene is contained in the 5.2-kilobase subclone used in these studies with 228 nucleotides of 5'-flanking DNA and that the increase in H4 mRNA during the G1-to-S transition in the cell cycle is mediated by the H4 promoter and not by the increased stability of the H4 RNA.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Regiões Promotoras Genéticas , Animais , Ciclo Celular , Células L/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transformação GenéticaRESUMO
The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis.
Assuntos
Actinas/genética , Proteínas de Ligação a DNA/análise , Miocárdio/análise , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Núcleo Celular/análise , Embrião de Galinha , Análise Mutacional de DNA , Enzimas de Restrição do DNA , Desoxirribonuclease I , Regulação da Expressão Gênica , Vetores Genéticos , Dados de Sequência Molecular , Fatores de Transcrição/análise , Transcrição GênicaRESUMO
Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.
Assuntos
Regulação da Expressão Gênica , Músculos/fisiologia , Proteína MyoD/genética , Regiões Promotoras Genéticas , Transativadores , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Galinhas , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Genes , Sequências Hélice-Alça-Hélice , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Proteínas Musculares/fisiologia , Fator Regulador Miogênico 5 , Fatores de Regulação Miogênica/fisiologia , Miogenina/fisiologia , Fatores de Transcrição/metabolismoRESUMO
The control and eventual eradication of bovine tuberculosis (TB) poses major challenges in New Zealand, given the variety of wildlife species susceptible to TB, many of which are capable of onwards transmission of Mycobacterium bovis infection. Here we discuss the role of feral ferrets (Mustela furo), focussing on potential transmission or risk pathways that have implications for management of TB. Firstly inter-specific transmission to ferrets. Ferrets scavenge potentially infected wildlife, including other ferrets, thus prevalence of TB can be amplified through ferrets feeding on tuberculous carcasses, particularly brushtail possums (Trichosurus vulpecula). Secondly intra-specific transmission between ferrets. The rate of ferret-ferret transmission depends on population density, and in some places ferret densities exceed the estimated threshold for disease persistence. TB can therefore potentially be maintained independently of other sources of infection. Thirdly transmission from ferrets to other wildlife. These include the main wildlife maintenance host, brushtail possums, that will occasionally scavenge potentially tuberculous ferret carcasses. Fourthly transmission from ferrets to livestock. This is considered to occur occasionally, but the actual rate of transmission has never been measured. Fifthly geographical spread. M. bovis-infected ferrets can travel large distances and cause new outbreaks of TB at locations previously free of TB, which may have caused an expansion of TB-endemic areas. Ferrets play a complex role in the TB cycle in New Zealand; they are capable of contracting, amplifying and transmitting M. bovis infection, sometimes resulting in ferret populations with a high prevalence of TB. However, ferret population densities are usually too low to sustain infection independently, and transmission to other wildlife or livestock appears a rarer event than with possums. Nevertheless, management of ferrets remains a key part of the National Pest Management Strategy for TB. Control is prudent where M. bovis-infected ferret populations exist in high numbers, to reduce the onward transmission risk of any self-sustained infection to livestock. When ferret numbers are well below the theoretical disease maintenance threshold, ferret control is still sometimes warranted because of the animals' ability to acquire infection when young and, through dispersal, transport it outside TB-endemic areas. Ferrets can also be used as disease sentinels for TB, especially in areas where alternative sentinel species are rare or expensive to survey, and when sampling of possums is not cost-effective.
Assuntos
Reservatórios de Doenças/veterinária , Furões/microbiologia , Vigilância de Evento Sentinela , Tuberculose Bovina/epidemiologia , Animais , Bovinos , Espécies Introduzidas , Nova Zelândia/epidemiologiaRESUMO
The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.
Assuntos
Actinas/genética , Genes , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , Enzimas de Restrição do DNA , Miocárdio/metabolismo , Biossíntese de Proteínas , Capuzes de RNA/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Saccharomyces cerevisiae/genética , Animais , Sequência de Bases , Galinhas/genética , Mapeamento Cromossômico , DNA Fúngico/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Hibridização de Ácido Nucleico , RNA Fúngico/biossíntese , Especificidade da Espécie , Transcrição GênicaRESUMO
Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing. Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation. Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation. Co-expression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter. Similarly, transfection of myoblasts with the cyclin-dependent kinase (cdk) inhibitors p16 and p21 supports some muscle-specific gene expression even in growth medium. Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks. We review evidence coupling growth status, the cell cycle and myogenesis. We describe a novel mitogen-sensitive mechanism that involves the cyclin D1-dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen-sensitive manner.
Assuntos
Divisão Celular , Músculos/citologia , Músculos/metabolismo , Proteína MyoD/metabolismo , Animais , Diferenciação Celular , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Fase G1 , Regulação da Expressão Gênica , Proteína MyoD/químicaRESUMO
Drugs specifically designed to block cellular signalling proteins are currently evaluated as a new way to treat gastrointestinal tumours. One such "new targeted agent" is aprinocarsen, an antisense oligonucleotide that specifically blocks the mRNA of protein kinase C-alpha (PKC-alpha). Blocking PKC-alpha, an important cellular signalling molecule associated with tumour growth, is anticipated to result in tumour cell arrest and achieve clinical benefits. However, it is not known which patients may benefit most from a specific inhibition of PKC-alpha. Past experience with other novel targeted agents suggests that expression of the target molecule is an important factor for the success of such a specific therapy. Therefore, reviewing the specific role of PKC-alpha in various gastrointestinal tumours may contribute to focus the clinical development of selective or specific PKC-alpha inhibitors, such as aprinocarsen, on those patients with a distinctive PKC-alpha expression pattern.
Assuntos
Neoplasias Gastrointestinais/enzimologia , Proteínas de Neoplasias/fisiologia , Proteína Quinase C/fisiologia , Inibidores Enzimáticos/uso terapêutico , Neoplasias Gastrointestinais/tratamento farmacológico , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-alfaRESUMO
Herein we outline three methods that, when coupled with cell-free protein synthesis, permit the identification of recombinant DNA molecules encoding specific polypeptides. RNAs enriched by fractionation on methylmercury hydroxide agarose gels are used to prepare sequence-specific probes. Recombinant DNA clones thus identified are further characterized as to their encoded polypeptides by either hybrid selection or hybrid arrest of translation.
Assuntos
DNA Recombinante/análise , Genes , Biossíntese de Proteínas , RNA Mensageiro/genética , Actinas/genética , Animais , Sistema Livre de Células , Cromatografia em Gel/métodos , DNA/análise , Engenharia Genética/métodos , Indicadores e Reagentes , Cinética , Hibridização de Ácido Nucleico , RNA Mensageiro/isolamento & purificaçãoRESUMO
Isolated chicken myoblasts had previously been utilized in many studies aiming at understanding the emergence and regulation of the adult myogenic precursors (satellite cells). However, in recent years only a small number of chicken satellite cell studies have been published compared to the increasing number of studies with rodent satellite cells. In large part this is due to the lack of markers for tracing avian myogenic cells before they become terminally differentiated and express muscle-specific structural proteins. We previously demonstrated that myoblasts isolated from fetal and adult chicken muscle display distinct schedules of myosin heavy-chain isoform expression in culture. We further showed that myoblasts isolated from newly hatched and young chickens already possess the adult myoblast phenotype. In this article, we report on the use of polyclonal antibodies against the chicken myogenic regulatory factor proteins MyoD and myogenin for monitoring fetal and adult chicken myoblasts as they progress from proliferation to differentiation in culture. Fetal-type myoblasts were isolated from 11-day-old embryos and adult-type myoblasts were isolated from 3-week-old chickens. We conclude that fetal myoblasts express both MyoD and myogenin within the first day in culture and rapidly transit into the differentiated myosin-expressing state. In contrast, adult myoblasts are essentially negative for MyoD and myogenin by culture Day 1 and subsequently express first MyoD and then myogenin before expressing sarcomeric myosin. The delayed MyoD-to-myogenin transition in adult myoblasts is accompanied by a lag in the fusion into myotubes, compared to fetal myoblasts. We also report on the use of a commercial antibody against the myocyte enhancer factor 2A (MEF2A) to detect terminally differentiated chicken myoblasts by their MEF2+ nuclei. Collectively, the results support the hypothesis that fetal and adult myoblasts represent different phenotypic populations. The fetal myoblasts may already be destined for terminal differentiation at the time of their isolation, and the adult myoblasts may represent progenitors that reside in an earlier compartment of the myogenic lineage.
Assuntos
Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos , Células Cultivadas , Galinhas , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Fatores de Transcrição MEF2 , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/imunologia , Fatores de Regulação Miogênica , Miogenina/imunologia , Miosinas/imunologia , Miosinas/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
The intermediate filament gene vimentin exists in a single copy in the chick haploid genome. However, it exhibits the curious property of producing at least three functional vimentin mRNA transcripts in vivo through the differential utilization of multiple polyadenylylation sites. According to one group in erythroid cells there may be a tissue-specific utilization of one of these poly A addition sites. The chicken and hamster vimentin genes exhibit remarkable nucleotide sequence homology both within coding and 3'-noncoding regions (82%). This nucleotide homology extends both to the size and juxtaposition of exons. With the noted exception of valine, even the frequency of codons utilized is strongly conserved across the widely different species. Of course, this strong homology at the DNA level extends to an amino acid homology of 92% between vimentins and 65% between related proteins in the same species.
Assuntos
Músculos/embriologia , Transcrição Gênica , Vimentina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Códon , Cricetinae , DNA/análise , Desmina/análise , Músculos/metabolismo , Especificidade da Espécie , Vimentina/análiseRESUMO
BACKGROUND: Coeliac disease, an autoimmune disorder triggered by gluten ingestion, is managed by a gluten-free diet (GFD), which is difficult for many patients. Larazotide acetate is a first-in-class oral peptide that prevents tight junction opening, and may reduce gluten uptake and associated sequelae. AIM: To evaluate the efficacy and tolerability of larazotide acetate during gluten challenge. METHODS: This exploratory, double-blind, randomised, placebo-controlled study included 184 patients maintaining a GFD before and during the study. After a GFD run-in, patients were randomised to larazotide acetate (1, 4, or 8 mg three times daily) or placebo and received 2.7 grams of gluten daily for 6 weeks. Outcomes included an experimental biomarker of intestinal permeability, the lactulose-to-mannitol (LAMA) ratio and clinical symptoms assessed by Gastrointestinal Symptom Rating Scale (GSRS) and anti-transglutaminase antibody levels. RESULTS: No significant differences in LAMA ratios were observed between larazotide acetate and placebo groups. Larazotide acetate 1-mg limited gluten-induced symptoms measured by GSRS (P = 0.002 vs. placebo). Mean ratio of anti-tissue transglutaminase IgA levels over baseline was 19.0 in the placebo group compared with 5.78 (P = 0.010), 3.88 (P = 0.005) and 7.72 (P = 0.025) in the larazotide acetate 1-, 4-, and 8-mg groups, respectively. Adverse event rates were similar between larazotide acetate and placebo groups. CONCLUSIONS: Larazotide acetate reduced gluten-induced immune reactivity and symptoms in patients with coeliac disease undergoing gluten challenge and was generally well tolerated; however, no significant difference in LAMA ratios between larazotide acetate and placebo was observed. Results and design of this exploratory study can inform the design of future studies of pharmacological interventions in patients with coeliac disease.