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1.
Dev Biol ; 414(2): 207-18, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27108394

RESUMO

In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity.


Assuntos
Proteínas Aviárias/fisiologia , Moela das Aves/embriologia , Músculo Liso/embriologia , Proteínas de Ligação a RNA/fisiologia , Alelos , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Moela das Aves/citologia , Humanos , Mesoderma/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Cultura Primária de Células , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Splicing de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
2.
Gene Expr Patterns ; 13(8): 287-92, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23727297

RESUMO

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.


Assuntos
Proteínas Aviárias/genética , Colo/metabolismo , Genes Homeobox , Moela das Aves/metabolismo , Fatores de Transcrição/genética , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha , Colo/citologia , Colo/embriologia , Mucosa Gástrica/embriologia , Mucosa Gástrica/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Moela das Aves/citologia , Moela das Aves/embriologia , Mucosa Intestinal/embriologia , Mucosa Intestinal/metabolismo , Miócitos de Músculo Liso/metabolismo , Especificidade de Órgãos , Piloro/citologia , Piloro/embriologia , Piloro/metabolismo , Reto/citologia , Reto/embriologia , Reto/metabolismo , Fatores de Transcrição/metabolismo
3.
Gen Comp Endocrinol ; 166(1): 12-8, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19914253

RESUMO

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus-gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus-gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm(2)) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid-late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.


Assuntos
Patos , Duodeno/metabolismo , Epitélio/metabolismo , Grelina/genética , Moela das Aves/metabolismo , Proventrículo/metabolismo , Animais , Duodeno/citologia , Regulação da Expressão Gênica no Desenvolvimento , Grelina/metabolismo , Moela das Aves/citologia , Moela das Aves/embriologia , Moela das Aves/crescimento & desenvolvimento , Imuno-Histoquímica , Proventrículo/citologia , Proventrículo/embriologia , Proventrículo/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Acta Histochem ; 111(1): 83-92, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18799201

RESUMO

The main purpose of this study was to evaluate the regional distribution pattern and relative frequency of some endocrine cells in the three portions of the gastrointestinal tract (GIT)--the proventriculus, gizzard and duodenum- of the rufous-collared sparrow (Zonotrichia capensis subtorquata), by immunohistochemical methods using six types of polyclonal antisera, specific for serotonin (5-HT), somatostatin (D cells), glucagon, motilin, polypeptide YY (PYY) and insulin. In the proventriculus, endocrine cells immunoreactive for all of these markers were observed. The somatostatin-immunoreactive cells were found with greater frequency, with the presence of cytoplasmic processes. In the gizzard, endocrine cells secreting somatostatin, 5-HT and PYY were detected, while those secreting glucagon and insulin were not. In the final part of the gizzard, endocrine cells secreting 5-HT were more frequent, and cells secreting somatostatin and insulin were not detected. All of the cell types studied were observed in the duodenum in different frequencies, except for cells immunoreactive for glucagon and insulin. The somatostatin-positive (D cells) were the most numerous, being more prevalent in the intestinal glands. The other endocrine cells were identified in smaller numbers, some of them located in the intestinal villi and Lieberkuhn glands. The finding of these cell types in the duodenum confirms their preferential location in the final portions of the principal segments of the digestive system and suggests control by feedback of its functions. In conclusion, some interesting distributional patterns of gastrointestinal endocrine cells were found in this species of sparrow.


Assuntos
Duodeno/citologia , Células Endócrinas/citologia , Passeriformes , Estômago/citologia , Animais , Biomarcadores/análise , Duodeno/química , Células Endócrinas/química , Moela das Aves/química , Moela das Aves/citologia , Glucagon/análise , Imuno-Histoquímica , Insulina/análise , Motilina/análise , Peptídeo YY/análise , Serotonina/análise , Somatostatina/análise , Estômago/química
5.
J Vet Med Sci ; 69(11): 1203-5, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18057841

RESUMO

This work was done to identify a fowl adenovirus (FAV) binding protein in the gizzard, a known target organ for certain strains of FAV serotype 1. By using a virus overlay protein binding assay (VOPBA), a putative FAV binding protein of approximately 200 kDa expressed in the gizzard was detected.


Assuntos
Aviadenovirus/fisiologia , Galinhas/virologia , Moela das Aves/citologia , Moela das Aves/virologia , Receptores Virais/fisiologia , Animais , Membrana Celular , Ligação Proteica
6.
Development ; 131(15): 3795-804, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15240557

RESUMO

Gastrointestinal (GI) development is highly conserved across vertebrates. Although several transcription factors and morphogenic proteins are involved in the molecular controls of GI development, the interplay between these factors is not fully understood. We report herein the expression pattern of Sox9 during GI development, and provide evidence that it functions, in part, to define the pyloric sphincter epithelium. SOX9 is expressed in the endoderm of the GI tract (with the exclusion of the gizzard) and its derivate organs, the lung and pancreas. Moreover, SOX9 is also expressed at the mesoderm of the pyloric sphincter, a structure that demarcates the gizzard from the duodenum. Using retroviral misexpression technique, we show that Sox9 expression in the pyloric sphincter is under the control of the BMP signaling pathway, known to play a key role in the development of this structure. By misexpressing SOX9 in the mesoderm of the gizzard, we show that SOX9 is able to transdifferentiate the adjacent gizzard epithelium into pyloric sphincter-like epithelium through the control of mesodermal-epithelial signals mediated in part by Gremlin (a modulator of the BMP pathway). Our results suggest that SOX9 is necessary and sufficient to specify the pyloric sphincter epithelial properties.


Assuntos
Epitélio/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mesoderma/fisiologia , Músculo Liso/embriologia , Transdução de Sinais/fisiologia , Estômago/embriologia , Fatores de Transcrição/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Embrião de Galinha , Epitélio/anatomia & histologia , Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Moela das Aves/citologia , Moela das Aves/embriologia , Moela das Aves/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/citologia , Morfogênese , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Piloro/anatomia & histologia , Piloro/embriologia , Piloro/metabolismo , Fatores de Transcrição SOX9 , Estômago/anatomia & histologia , Fatores de Transcrição/genética
7.
J Biol Chem ; 279(14): 13668-76, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-14736875

RESUMO

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.


Assuntos
Músculo Liso/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/genética , Proteínas/metabolismo , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Galinhas , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Moela das Aves/citologia , Moela das Aves/fisiologia , Dados de Sequência Molecular , Músculo Liso/citologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteínas/genética , Sítios de Splice de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Retículo Sarcoplasmático/metabolismo
8.
Cell Struct Funct ; 27(5): 383-91, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12502893

RESUMO

Arp2/3 protein complex consists of seven subunits (Arp2, Arp3, p41-Arc, p34-Arc, p21-Arc, p20-Arc and p16-Arc) in apparent 1:1 stoichiometry. This complex has been shown to promote the formation of Y-branch structures of F-actin in cultured cells. We generated specific antibodies against chicken Arp2, Arp3, and p34-Arc to analyze the distribution of these subunits in chicken tissues. In whole samples of brain and gizzard, antibodies against each recombinant protein reacted with single bands of predicted molecular mass based on their cDNA sequences of the antigens. Anti-p34-Arc antibody detected at least two neighboring spots in 2D-PAGE, which might suggest the existence of isoforms or modified forms. Arp2/3 complex bound to an F-actin affinity column from gizzard extract. However, Arp2/3 complex did not tightly bind major actin cytoskeleton because the complex was extracted easily when gizzard smooth muscle was homogenized in PBS. Immunoblot analysis of various tissues revealed that the amounts of Arp2/3 subunits were lower in striated muscle than in non-muscle and smooth muscle tissues. Amounts and ratio of the three subunits varied in tissues, as estimated by quantitative immunoblotting. With immunofluorescence microscopy, we also observed localization of Arp3 and p34-Arc in frozen sections of gizzard with different staining patterns around blood vessels. These results suggest that the Arp2/3 complex exists also in places where rapid actin polymerization does not occur, and that a part of the subunits may exist in different forms from the complex containing the seven subunits in some tissues.


Assuntos
Actinas/metabolismo , Encéfalo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Moela das Aves/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Animais , Galinhas , Moela das Aves/citologia , Imuno-Histoquímica , Polímeros/metabolismo , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo
9.
J Biochem ; 130(3): 335-40, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11530008

RESUMO

An arginine-specific ADP-ribosyltransferase activity was detected in chicken gizzard smooth muscle, and the specific activity is highest in the membrane fraction. This transferase is released from the membrane fraction by phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it is a glycosylphosphatidylinositol (GPI)-anchored protein. When primary cultured gizzard smooth muscle cells (SMCs) were incubated with [adenylate-(32)P]NAD, several proteins were labeled. The labeling was inhibited by preincubation of the cells with PI-PLC, or by the addition of L-arginine to the reaction, and was sensitive to hydroxylamine treatment. The activity of the transferase was maintained in differentiated SMCs cultured with insulin, but was dramatically decreased concomitantly with cell dedifferentiation induced by serum or a specific PI3-kinase inhibitor, LY294002. These results indicate that the GPI-anchored arginine-specific ADP-ribosyltransferase is expressed on the surface of differentiated SMCs and can modify several cell surface proteins. Our results also suggest that PI3-kinase is involved in the regulation of transferase activity during differentiation.


Assuntos
Arginina/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Músculo Liso/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/metabolismo , Animais , Sítios de Ligação/fisiologia , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Galinhas , Cromonas/farmacologia , Moela das Aves/citologia , Moela das Aves/enzimologia , Insulina/metabolismo , Morfolinas/farmacologia , Músculo Liso/citologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Poli(ADP-Ribose) Polimerases , Fosfolipases Tipo C/metabolismo
10.
Am J Physiol Cell Physiol ; 279(6): C1722-32, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11078686

RESUMO

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.


Assuntos
Aorta/fisiologia , Moela das Aves/fisiologia , Contração Muscular/fisiologia , Cadeias Leves de Miosina/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Biomarcadores , Embrião de Galinha , Moela das Aves/citologia , Moela das Aves/embriologia , Técnicas In Vitro , Isomerismo , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/enzimologia , Cadeias Leves de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Vasoconstrição/fisiologia
11.
J Biol Chem ; 274(49): 35095-8, 1999 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-10574990

RESUMO

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.


Assuntos
Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Aorta/citologia , Embrião de Galinha , Moela das Aves/citologia , Cinética , Contração Muscular/genética , Músculo Liso/embriologia , Cadeias Leves de Miosina/genética , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Transdutores , Transfecção
12.
J Biochem ; 122(2): 344-51, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9378712

RESUMO

Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent K(d) value of calponin to tubulin was calculated to be 5.2 microM with 2 mol of calponin maximally bound per 1 mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270 mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4 mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization.


Assuntos
Encéfalo/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Moela das Aves/metabolismo , Proteínas dos Microfilamentos , Microtúbulos/metabolismo , Músculo Liso/metabolismo , Actinas/metabolismo , Ácidos Alcanossulfônicos , Animais , Química Encefálica , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Galinhas , Reagentes de Ligações Cruzadas , Etildimetilaminopropil Carbodi-Imida , Moela das Aves/citologia , Cinética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfolinas , Proteínas Musculares/metabolismo , Ligação Proteica , Ratos , Proteínas S100/metabolismo , Cloreto de Sódio/farmacologia , Tropomiosina/metabolismo , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/metabolismo , Calponinas
13.
Cell Tissue Res ; 285(3): 395-401, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8772153

RESUMO

We examined the in vitro differentiation of smooth muscle cells from undifferentiated cells of embryonic chicken gizzard. We used the gizzard from 7-day-old embryos (Hamburger and Hamilton's stage 26-28) as a source of culture, since the expression of myosin is extremely low, and the gizzard consists of round cells, which are not stained by anti-smooth muscle myosin antiserum. When the dissections of the gizzard were cultured, they attached to the dish, and fibroblast-like cells migrated within 4 days after the culture. Then round cells migrated from the transplants over the layer of fibroblast-like cells. At 12-14 days after the culture, smooth muscle cells, which were ribbon-shaped and stained by anti-smooth muscle myosin antiserum, appeared in the layer of round cells. The dissected transplant itself was not stained by anti-smooth muscle myosin antiserum even after being cultured for 15 days. We concluded then that the smooth muscle cells were differentiated from the round cells, which spread on the layer of the fibroblast-like cells. We also observed the differentiation of smooth muscle cells when the cells separated from 7-day-old embryo cultured on the layer of cloned fibroblast cells. We suggest that the fibroblast-like cell may play an important role in the differentiation of smooth muscle cells.


Assuntos
Fibroblastos/citologia , Moela das Aves/citologia , Músculo Liso/citologia , Células-Tronco/citologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Células Cultivadas/citologia , Embrião de Galinha
14.
Int J Dev Neurosci ; 14(3): 297-314, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8842806

RESUMO

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.


Assuntos
Laminina/fisiologia , Crista Neural/citologia , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Galinhas , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Moela das Aves/citologia , Moela das Aves/inervação , Moela das Aves/metabolismo , Immunoblotting , Imuno-Histoquímica , Isomerismo , Laminina/isolamento & purificação , Laminina/metabolismo , Microscopia Eletrônica , Crista Neural/fisiologia
15.
Anat Embryol (Berl) ; 192(6): 547-55, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8751112

RESUMO

The ontogeny and the distribution of chromogranin A (CgA)- and chromogranin B (CgB)-immunoreactive endocrine cells was studied in the chicken gizzard and gizzard-duodenal junction (also called pylorus or antrum) during embryonic and postnatal life. The same tissue sections were then double-immunostained to identify the CgA-and CgB-immunoreactive cells, with a panel of polyclonal antibodies raised against main gut amine/peptides. In the gizzard, positive cells were observed only in its two diverticula (proximal and distal caeca), where the first CgA- and CgB-immunoreactive cells were found on day 12 of incubation. They always remained moderate in number and co-stored mainly serotonin, gastrin/CCK and neurotensin. A few also co-stored somatostatin, but only during the embryonic period. Others co-stored PYY, but only after hatching. Co-localization with motilin was rare and never occurred with bombesin. In the chicken antrum, the first CgA- and CgB-immunoreactive cells were observed on day 12 of incubation and soon reached very high numbers. Antral positive cells showed almost the same co-localization pattern as the gizzard diverticula. Despite their high chromogranin content, the antral cells had weak argyrophilia, whereas in the gizzard diverticula the two staining patterns corresponded.


Assuntos
Galinhas/anatomia & histologia , Cromograninas/análise , Grânulos Citoplasmáticos/química , Moela das Aves/química , Antro Pilórico/química , Aminas/imunologia , Animais , Especificidade de Anticorpos , Embrião de Galinha , Cromogranina A , Cromograninas/imunologia , Moela das Aves/citologia , Imuno-Histoquímica , Peptídeos/imunologia , Antro Pilórico/citologia , Coloração pela Prata
16.
Dev Dyn ; 201(3): 236-44, 1994 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7881127

RESUMO

The enteric nervous system is largely formed from the vagal neural crest which arises from the neuroaxis between somites 1-7. In order to evaluate the contribution of different regions of the vagal crest to the enteric nervous system, we marked crest cells by injecting somites 1-10 with a replication-defective spleen necrosis virus vector which contains the marker gene lacZ. After incubation in X-gal, lacZ-positive blue cells were found in the wall of the gut in three locations. Most were found at the peripheral edge of the developing circular muscle and within the developing submucosa, sites characteristic of developing ganglia. LacZ-positive cells in these ganglionic sites were always surrounded by HNK-1 immunostained cells, confirming their neural crest origin. LacZ-positive cells were also seen in a third location, the circular muscle layer of the esophagus and crop, and were separated from the HNK-1 positive ganglionic elements. These cells in the circular muscle are probably muscle cells derived from labeled mesodermal cells of the somite. Injection of somites 3, 4, 5, and 6 resulted in the largest percentage of preparations with lacZ-positive crest-derived cells and in the largest number of positive cells in the gut. After injection of these somites, lacZ-positive crest-derived cells were found in all regions of the gut from the proventriculus to the rectum. Very few positive crest-derived cells were found in the esophagus.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Embrião de Galinha/inervação , Sistema Nervoso Entérico/embriologia , Animais , Movimento Celular , Embrião de Galinha/citologia , Vírus Defeituosos/genética , Sistema Nervoso Entérico/citologia , Gânglios/citologia , Gânglios/embriologia , Marcadores Genéticos , Moela das Aves/citologia , Moela das Aves/embriologia , Moela das Aves/inervação , Óperon Lac , Crista Neural/citologia , Crista Neural/embriologia , Retroviridae/genética , Fatores de Tempo
17.
J Cell Biol ; 126(1): 127-38, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8027172

RESUMO

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.


Assuntos
Caveolinas , Compartimento Celular , Membrana Celular/química , Proteínas de Membrana/química , Músculo Liso/química , Animais , Caveolina 1 , Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Galinhas , Proteínas de Ligação ao GTP/isolamento & purificação , Moela das Aves/citologia , Glicosilfosfatidilinositóis , Imuno-Histoquímica , Microscopia Imunoeletrônica , Músculo Liso/ultraestrutura , Frações Subcelulares/química , Frações Subcelulares/ultraestrutura
18.
Eur J Histochem ; 38(4): 319-26, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7535129

RESUMO

The time of appearance, morphology, and topographic distribution of somatostatin, neurotensin, bombesin, gastrin/CCK and serotonin immunoreactive cells during embryonic development were studied in the duck gastrointestinal tract by immunohistochemical methods. Somatostatin immunoreactive cells first appeared in the duodenum of duck embryos at 9 days of incubation (d.i.). They progressively appeared in the other segments at 19 d.i., and at hatching they were present in all gastrointestinal segments except for the caecum. At hatching, the antrum was the richest region in somatostatin endocrine cells, and the gizzard the poorest. Neurotensin immunoreactive cells were detected at 21 d.i. in the proventriculus, antrum, duodenum, and rectum; at 23 d.i. they were present in all the other segments. Bombesin immunoreactive cells were observed in the proventriculus at 17 d.i., and in the gizzard and antrum at 23 d.i. No cells were detected in the intestinal segments. Gastrin/CCK immunoreactive cells first appeared at 17 d.i. in the antrum region; at 21 d.i. they appeared in the small intestine and around hatching they were found in the other intestinal segments except for the proventriculus and gizzard. Serotonin immunoreactive cells appeared at 21 d.i. in the proventriculus, duodenum, and jejunum-ileum. At 23 d.i., they were present in all other segments. Our results show that the time of appearance of immunoreactive cells may be related to the general development of the gut wall in the duck and may reflect cell differentiation in the mucosa.


Assuntos
Sistema Digestório/citologia , Sistema Digestório/crescimento & desenvolvimento , Patos/crescimento & desenvolvimento , Glândulas Endócrinas/citologia , Glândulas Endócrinas/crescimento & desenvolvimento , Animais , Bombesina/metabolismo , Diferenciação Celular/fisiologia , Sistema Digestório/metabolismo , Glândulas Endócrinas/metabolismo , Moela das Aves/citologia , Moela das Aves/crescimento & desenvolvimento , Imuno-Histoquímica , Neurotensina/metabolismo , Inclusão em Parafina , Serotonina/metabolismo , Somatostatina/metabolismo
20.
Cell Regul ; 2(11): 927-38, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1725601

RESUMO

In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibronectinas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/imunologia , Embrião de Galinha , Galinhas , Ácido Edético/farmacologia , Epitopos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/imunologia , Imunofluorescência , Moela das Aves/citologia , Técnicas In Vitro , Ligação Proteica/efeitos dos fármacos , Splicing de RNA , Cloreto de Sódio/farmacologia , Relação Estrutura-Atividade , Tenascina , Ureia/farmacologia
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