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1.
Urologiia ; (6): 47-50, 2021 Dec.
Artigo em Russo | MEDLINE | ID: mdl-34967164

RESUMO

INTRODUCTION: The high prevalence of kidney diseases caused by urinary tract obstruction has led to the need for experimental studies of the dynamics of pathological processes in their lesions. Despite the fact that the general patterns of development of obstructive uropathy are known, the features of renal tissue damage, in particular structural and molecular biological changes in this pathology, remain insufficiently studied. OBJECTIVE: to study the dynamics of changes in the phenotype of epithelial cells of the nephron of an obstructive kidney with unilateral ureteral obstruction using an experimental model. MATERIALS AND METHODS: The experimental study was carried out on the basis of the Rostov State Medical University. The model of unilateral ureteral obstruction was reproduced in adult rabbits. The studies were carried out on the 7th, 14th and 21st days of complete obstruction of the left ureter. Immunophenotyping of obstructive kidney tissue samples was performed for markers of epithelial phenotype (cytokeratin 7, E-cadherin) and mesenchymal phenotype (vimentin, - smooth muscle actin). RESULTS: The sequence of changes in the phenotype of nephron epithelial cells during ureteral obstruction has been established. The first signs of an epithelial-mesenchymal transition (EMT) appear by day 7 in the form of a decrease in visualization of markers of the epithelial phenotype. On the 14th day, the expression of both epithelial and mesenchymal markers is noted. Significant changes in the phenotype of nephron epithelial cells: loss of epithelial markers (cytokeratin 7, E-cadherin) and the acquisition of mesenchymal markers (vimentin, - smooth muscle actin), are noted by the 21st day of the experiment. CONCLUSION: An experimental model of unilateral ureteral obstruction revealed the transformation of the nephron tubule cell phenotype from epithelial to mesenchymal.


Assuntos
Nefropatias , Obstrução Ureteral , Animais , Transição Epitelial-Mesenquimal , Fibrose , Rim/patologia , Nefropatias/patologia , Néfrons , Coelhos , Obstrução Ureteral/complicações
2.
Urologiia ; (4): 27-31, 2017 Sep.
Artigo em Russo | MEDLINE | ID: mdl-28952688

RESUMO

RELEVANCE: Acute pyelonephritis is known to be the most complicated and severe urinary tract infection occurring in all age groups and accounting for 14% of all kidney diseases. The generally recognized standard antibiotic therapy cannot completely prevent the progression of the disease to its chronic form after relief of its acute manifestations thus leading to a high incidence of relapses. The aim of our study was to investigate the spectrum of uropathogens and their antibiotic sensitivity in acute obstructive pyelonephritis. MATERIALS AND METHODS: The study comprised 72 patients who underwent semi-rigid ureteroscopy and ultrasonic lithotripsy for ureteral stones. In all patients, bladder urine samples collected by a transurethral catheter were tested bacteriologically using an extended set of culture media within 3 hours after hospital admission. Antibiotics used in antibiotic sensitivity testing for all uropathogens, were grouped into 4 classes (carbapenems, fluoroquinolones, cephalosporins, penicillins). Etiotropic treatment was started upon the availability of the spectrum of microbial patterns, the level of bacteriuria and antibioticogram of uropathogens, 5-6 days after administering initial empirical antibiotic therapy. RESULTS: The study patients had a high detection rate (83.3%) of canonical uropathogens in the bladder urine identified due to using an extended set of culture media, with a bacteriuria of more or equal 103 CFU/mL. Given the results of local antibiograms, a rational antimicrobial therapy should include carbapenems, namely ertapenem or meropenem as initial empirical antibiotics. Using fluoroquinolones as the first line treatment can lead to an inadequate effect in 15.0 to 67.0% of the cases. The findings of the antibiotic resistance testing of uropathogens to cephalosporins and semisynthetic penicillins showed that they should not be used as initial empirical antibiotic therapy for acute obstructive pyelonephritis in the given department of urology.


Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/microbiologia , Pielonefrite/microbiologia , Obstrução Ureteral/microbiologia , Infecções Urinárias/microbiologia , Doença Aguda , Adolescente , Adulto , Idoso , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Prospectivos , Pielonefrite/complicações , Cálculos Ureterais/complicações , Cálculos Ureterais/microbiologia , Obstrução Ureteral/etiologia , Adulto Jovem
3.
Arkh Patol ; 79(2): 3-9, 2017.
Artigo em Russo | MEDLINE | ID: mdl-28418351

RESUMO

AIM: Тo conduct an immunohistochemical (IHC) study of the expression of connexin 43 in the samples of glial tumors of various grades: gemistocytic astrocytomas (Grade 2), oligodendrogliomas (Grade 2) and glioblastomas (Grade 4). MATERIAL AND METHODS: The material investigated was fragments of human brain glial tumors (grade 2 gemistocytic astrocytomas (n=2), grade 2 oligodendrogliomas (n=2), and grade 4 glioblastomas (n=14) and those of tumor-surrounding tissue (n=4). The material was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin according to the standard technique. IHC studies of the slices applied primary rabbit polyclonal antibodies against connexin 43 ('Spring Bioscience', USA) and the Dako EnVision + Peroxidase (DAB) visualization system ('Dako', Denmark). After the immunohistochemical reaction, the cell nuclei were stained with Mayer's hematoxylin. RESULTS: Immunohistochemistry showed the changing pattern of connexin 43 expression as compared with intact tissue in the glial tumors. Instead of the fine-granular expression in the thin cellular processes in the neuropil, the tumors mainly displayed a coarse-grained cytoplasmic and even nuclear reaction. The morphology and localization of positive structures depended on the variant of an examined tumor. In addition, the most malignant brain gliomas generally exhibited a reduction in the expression of connexin 43, i.e. its quantity is inversely proportional to the degree of malignancy of the tumor. CONCLUSION: The low connexin 43 expression levels may reflect both a reduction in astroglial functional gap junctions and semicanals and a decrease in the amount of the protein itself that has independently antioncogenic properties. The observed cytoplasmic and nuclear expression of connexin 43 is most likely to be associated with the aberrant activity of a number of kinases, such as proto-oncogene tyrosine-kinase Src or protein kinase C (PKC).


Assuntos
Astrocitoma/genética , Conexina 43/genética , Glioblastoma/genética , Oligodendroglioma/genética , Astrocitoma/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Estadiamento de Neoplasias , Oligodendroglioma/patologia , Proto-Oncogene Mas
4.
Dev Biol ; 417(1): 91-103, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27196393

RESUMO

The fin-to-limb transition is an important evolutionary step in the colonization of land and diversification of all terrestrial vertebrates. We previously identified a gene family in zebrafish, termed actinodin, which codes for structural proteins crucial for the formation of actinotrichia, rigid fibrils of the teleost fin. Interestingly, this gene family is absent from all tetrapod genomes examined to date, suggesting that it was lost during limb evolution. To shed light on the disappearance of this gene family, and the consequences on fin-to-limb transition, we characterized actinodin regulatory elements. Using fluorescent reporters in transgenic zebrafish, we identified tissue-specific cis-acting regulatory elements responsible for actinodin1 (and1) expression in the ectodermal and mesenchymal cell populations of the fins, respectively. Mutagenesis of potential transcription factor binding sites led to the identification of one binding site crucial for and1 expression in ectodermal cells. We show that these regulatory elements are partially functional in mouse limb buds in a tissue-specific manner. Indeed, the zebrafish regulatory elements target expression to the dorsal and ventral ectoderm of mouse limb buds. Absence of expression in the apical ectodermal ridge is observed in both mouse and zebrafish. However, cells of the mouse limb bud mesoderm do not express the transgene, in contrast to zebrafish. Altogether these results hint for a change in regulation of and1 during evolution that led to the downregulation and eventual loss of this gene from tetrapod genomes.


Assuntos
Nadadeiras de Animais/embriologia , Extremidades/embriologia , Mesoderma/citologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Nadadeiras de Animais/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Evolução Biológica , Extremidades/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/crescimento & desenvolvimento , Botões de Extremidades/metabolismo , Camundongos , Morfogênese/fisiologia , Regiões Promotoras Genéticas/genética
5.
Morfologiia ; 150(4): 24-9, 2016.
Artigo em Russo | MEDLINE | ID: mdl-30136819

RESUMO

The aim of this work was an immunohistochemical study of the expression of neuronal and glial proteins, and of gap junctions proteins (connexin 36, connexin 43) in ventral posteromedial (VPMN), ventral posterolateral (VPLN) and reticular (RТN) nuclei of the thalamus in rats. It was found that VPMN and VPLN of the thalamus were characterized by a homogeneous distribution of synaptophysin, grouped arrangement of astrocytes, horizontal orientation of somatostatincontaining myelinated and unmyelinated nerve fibers, forming the bundles, and running through the barreloid septum, expression of connexin 36 and 43 as well as of parvalbumin revealing barreloids in 4 µm-thick sections. In RTN the content of myelin basic protein, neurofilaments, parvalbumin, and somatostatin was increased, while the amount of glial fibrillary acidic protein and connexin 43 was moderate, and synaptophysin and connexin 36 were absent.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Tálamo/citologia , Tálamo/metabolismo , Animais , Feminino , Imuno-Histoquímica , Masculino , Ratos
6.
Dev Dyn ; 237(2): 417-25, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18163531

RESUMO

The caudal fin of adult zebrafish is used to study the molecular mechanisms that govern regeneration processes. Most reports of gene expression in regenerating caudal fins rely on in situ hybridization (ISH) on whole-mount samples followed by sectioning of the samples. In such reports, expression is mostly confined to cells other than those located between the dense collagenous structures that are the actinotrichia and lepidotrichia. Here, we re-examined the expression of genes by performing ISH directly on cryo-sections of regenerates. We detected expression of some of these genes in cell types that appeared to be non-expressing when ISH was performed on whole-mount samples. These results demonstrate that ISH reagents have a limited capacity to penetrate between the regenerating skeletal matrices and suggest that ISH performed directly on fin sections is a preferable method to study gene expression in fin regenerates.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ/métodos , Regeneração/fisiologia , Cauda/fisiologia , Peixe-Zebra/fisiologia , Animais , Compostos Azo , Criopreservação
7.
Dev Biol ; 312(1): 272-83, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17977526

RESUMO

The fins of actinopterygian can regenerate following amputation. Classical papers have shown that the ray, a structural unit of these fins, might regenerate independent of this appendage. Each fin ray is formed by two apposed contralateral hemirays. A hemiray may autonomously regenerate and segmentate in a position-independent manner. This is observed when heterotopically grafted into an interray space, after amputation following extirpation of the contralateral hemiray or when simply ablated. During this process, a proliferating hemiblastema is formed, as shown by bromodeoxyuridine incorporation, from which the complete structure will regenerate. This hemiblastema shows a patterning of gene expression domain similar to half ray blastema. Interactions between contralateral hemiblastema have been studied by recombinant rays composed of hemirays from different origins on the proximo-distal or dorso-ventral axis of the caudal fin. Dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianine perchlorate labeling of grafted tissues was used as tissular marker. Our results suggest both that there are contralateral interactions between hemiblastema of each ray, and that hemiblastema may vary its morphogenesis, always differentiating as their host region. These non-autonomous, position-dependent interactions control coordinated bifurcations, segment joints and ray length independently. A morphological study of the developing and regenerating fin of another long fin mutant zebrafish suggests that contralateral hemiblastema interactions are perturbed in this mutant.


Assuntos
Estruturas Animais/crescimento & desenvolvimento , Padronização Corporal , Regeneração , Cauda/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Estruturas Animais/citologia , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Mutação/genética , Fenótipo , Cauda/anatomia & histologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
8.
Urologiia ; (4): 15-6, 18-20, 2007.
Artigo em Russo | MEDLINE | ID: mdl-17915441

RESUMO

The article presents results of retrograde endoscopic pyelolithotripsy (REP) in 75 patients with large and coral nephroliths. Retrograde contact pyelolithotripsy was made with application of Swiss Lithoclast unit, semirigid ureteroscopes R. Wolf with a cone 8-9 Ch and working channel 5.3 Ch. This enables maximal removal of the destroyed concrement by hydraulic litholapaxia with consequent insertion of the internal stent. Extracorporeal lithotripsy with low-energy shock-wave impulses was performed on demand (1-3 sessions). The stent was removed in 3-5 days in cases of complete one-stage sanation of the kidney and in 2-3 weeks in the presence of residual fragments of the nephrolith. One-stage complete sanation in coral nephrolithiasis (K1-K2) up to 5 cm3 and large concrements was achieved in 69.3% patients. Three months later residual fragments of the nephroliths were observed in 8% patients, 6 months later--in 5.3%. One year after treatment a complete sanation was achieved in 96%. Complications of the method consisted in exacerbation of chronic pyelonephritis observed in 12% cases. The control examination revealed improvement of renal function early and late after surgery. As shown by 6-year follow-up, recurrent nephroliths occurred in 6% patients. Endoscopic retrograde pyelolithotripsy can be used as a method of choice in the treatment of compound forms of nephrolithiasis in intrarenal type of the pelvis, the absence of marked extension of the caliceal-pelvic system of the kidney, in other stones resistant to extracorporeal lithotripsy.


Assuntos
Litotripsia , Nefrolitíase/terapia , Ureteroscopia , Adulto , Idoso , Feminino , Seguimentos , Humanos , Pelve Renal/patologia , Litotripsia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Nefrolitíase/complicações , Nefrolitíase/patologia , Pielonefrite/etiologia , Pielonefrite/patologia , Estudos Retrospectivos , Ureteroscopia/efeitos adversos
9.
J Hist Neurosci ; 16(1-2): 90-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17365555

RESUMO

This article expounds the history of the formation and development of neurology in St. Petersburg and emphasizes the original character of St. Petersburg school of neurology. The authors state that many prominent neurologists of St. Petersburg dedicated their work to the development of neurological concepts and have made an important contribution to different areas of neurology, including vascular and demyelinating diseases, diseases of the peripheral nervous system, neuroinfections, epilepsy, etc.


Assuntos
Doenças do Sistema Nervoso/história , Neurologia/história , Faculdades de Medicina/história , História do Século XIX , História do Século XX , Humanos , Federação Russa
10.
J Hist Neurosci ; 16(1-2): 100-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17365556

RESUMO

V.M. Bekhterev (1857-1927) was an outstanding Russian neurologist, psychiatrist, psychologist, morphologist, physiologist, and public figure, who authored over 1000 scientific publications and speeches. At the beginning of the twentieth century he created a new multidimensional multidisciplinary scientific branch - psychoneurology, which included the objective knowledge of the anatomy and physiology of the nervous system, psychology, psychiatry, neurology, philosophy, sociology, pedagogy, and other disciplines. Psychoneurology in V.M. Bekhterev's understanding has furthered the introduction into the idea of a "biosocial" essence of man of a third - psychological - component, thus having created a "biopsychosocial" model in the interpretation of human diseases.


Assuntos
Neurologia/história , Neuropsicologia/história , Psiquiatria/história , História do Século XIX , História do Século XX , Humanos , Federação Russa
11.
Dev Biol ; 299(2): 438-54, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16959242

RESUMO

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular , Osteoblastos/citologia , Regeneração , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteína Morfogenética Óssea 6 , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Condrócitos/citologia , Regulação para Baixo , Glicoproteínas/biossíntese , Proteínas HMGB/biossíntese , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Osteogênese , Fatores de Transcrição SOX9 , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Dyn ; 235(2): 478-89, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16292774

RESUMO

We have characterized two new members of the Hedgehog (Hh) family in zebrafish, ihha and dhh, encoding for orthologues of the tetrapod Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) genes, respectively. Comparison of ihha and Type X collagen (col10a1) expression during skeletal development show that ihha transcripts are located in hypertrophic chondrocytes of cartilaginous elements of the craniofacial and fin endoskeleton. Surprisingly, col10a1 expression was also detected in cells forming intramembranous bones of the head and in flat cells surrounding cartilaginous structures. The expression of col10a1 in both endochondral and intramembranous bones reflects an atypical composition of the extracellular matrix of the zebrafish craniofacial skeleton. In addition, during fin ray regeneration, both ihha and col10a1 are detected in scleroblasts, osteoblast-like cells secreting the matrix of the dermal bone fin ray. The presence of cartilage markers suggests that the dermal fin ray possesses an intermediate phenotype between cartilage and bone.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/classificação , Proteínas Hedgehog/metabolismo , Sistema Musculoesquelético/embriologia , Sistema Musculoesquelético/metabolismo , Transativadores/classificação , Transativadores/metabolismo , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Colágeno Tipo X/metabolismo , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Larva/genética , Larva/metabolismo , Dados de Sequência Molecular , Sistema Musculoesquelético/química , Filogenia , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Peixe-Zebra/classificação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
13.
Voen Med Zh ; 325(6): 26-32, 80, 2004 Jun.
Artigo em Russo | MEDLINE | ID: mdl-15327137

RESUMO

The experience of urolithiasis treatment with the use of modern diagnostic methods (the computer tomography with concrement density detection, the DV-Med program which permits to obtain the calculus volumetric image and to study its physical characteristics), and therapy (distant lithotripsy with the use of different types of lithotriptors as well as retrograde contact lithotripsy with "Lithoclast") permits to minimize the percent of open surgical interventions. For the last 3 years 780 patients with different clinical disease forms were treated with favorable clinical outcome and only in 0.5% cases the open surgical intervention was conducted. These patients underwent the pyelolithotomy for the secondary nephrolithiasis conditioned by the pelvis-and-ureteral segment stricture when the endoscopic method failed to eliminate this problem.


Assuntos
Litotripsia/métodos , Cálculos Urinários/terapia , Unidade Hospitalar de Urologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Resultado do Tratamento , Cálculos Urinários/cirurgia
14.
Dev Biol ; 252(2): 214-24, 2002 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12482711

RESUMO

Teleost fin ray bifurcations are characteristic of each ray in each fin of the fishes. Control of the positioning of such morphological markers is not well understood. We present evidence suggesting that the interray blastema is necessary for a proper bifurcation of each ray during regeneration in Danio rerio (Hamilton-Buchanan) (Cyprinidae, Teleostei). We performed single ray ablations, heterotopical graftings of ray fragments and small holes in lateral rays which do not normally bifurcate, to generate recombinants in which the lateral rays are surrounded with ectopic interrays originating from different positions within the tail fin. These ray-interray recombinants do now bifurcate. Furthermore, we show that the interray tissue and surrounding epidermis can modulate the length of the ray. These results stress the role of the interray in inducing bifurcations of the ray blastema as well as modulating ray morphogenesis in general. In addition, gene expression analysis under these experimental conditions suggests that msxA and msxD expression in the ray and interray epidermis is controlled by the ray blastema and that bmp4 could be a candidate signal involved in these inductions.


Assuntos
Regeneração , Peixe-Zebra/fisiologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/fisiologia , Hibridização In Situ , Peixe-Zebra/anatomia & histologia , Proteínas de Peixe-Zebra
15.
Dev Dyn ; 221(4): 380-90, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11500975

RESUMO

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di-I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU-injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di-I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.


Assuntos
Regeneração , Peixe-Zebra/fisiologia , Animais , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Carbocianinas/farmacologia , Divisão Celular , Movimento Celular , Epiderme/metabolismo , Extremidades , Corantes Fluorescentes/farmacologia , Fatores de Tempo
16.
J Neurochem ; 75(4): 1600-7, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10987841

RESUMO

We report here the structural and functional characterization of an ionotropic P2X ATP receptor from the lower vertebrate zebrafish (Danio rerio). The full-length cDNA encodes a 410-amino acid-long channel subunit zP2X(3), which shares only 54% identity with closest mammalian P2X subunits. When expressed in Xenopus oocytes in homomeric form, ATP-gated zP2X(3) channels evoked a unique nonselective cationic current with faster rise time, faster kinetics of desensitization, and slower recovery than any other known P2X channel. Interestingly, the order of agonist potency for this P2X receptor was found similar to that of distantly related P2X(7) receptors, with benzoylbenzoyl ATP (EC(50) = 5 microM) >> ATP (EC(50) = 350 microM) = ADP > alpha,beta-methylene ATP (EC(50) = 480 microM). zP2X(3) receptors are highly sensitive to blockade by the antagonist trinitrophenyl ATP (IC(50) < 5 nM) but are weakly sensitive to the noncompetitive antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. zP2X(3) subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon-Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.


Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Ativação do Canal Iônico/efeitos dos fármacos , Dados de Sequência Molecular , Neurônios Aferentes/metabolismo , Especificidade de Órgãos , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X3 , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra
17.
Mol Cell Biol ; 19(10): 7106-22, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10490647

RESUMO

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras , Animais , Sítios de Ligação/genética , Padronização Corporal , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteína Goosecoid , Proteínas de Homeodomínio/isolamento & purificação , Fator C1 de Célula Hospedeira , Leucina/genética , Mesoderma , Mutação , Fator 1 de Transcrição de Octâmero , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Prolina/genética , Ligação Proteica/genética , Receptores de Glucocorticoides/genética , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra
18.
Dev Dyn ; 214(3): 207-15, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10090147

RESUMO

Apolipoprotein E (apoE) plays an important role in systemic and local lipid homeostasis. We have examined the expression of apoE during morphogenesis and regeneration of paired and unpaired fins and during scale development in zebrafish (Danio rerio). In situ hybridization analysis revealed that, during embryogenesis, apoE is expressed in the epithelial cells of the median fin fold and of the pectoral fin buds. ApoE remains expressed in the elongating fin folds throughout development of the fins. During the larval to juvenile transition, apoE transcripts were present in the distal, interray and lateral epidermis of developing fins. Furthermore, as scale buds started to form, apoE was expressed in large scale domains which later, became restricted to the external posterior epidermal part of scales. A low level of transcripts could be observed at later developmental stages at these locations probably because fins and scales continue to grow throughout the animal's life. During regeneration of both pectoral and caudal fins, a marked increase in apoE expression is observed as early as 12 hours after amputation in the wound epidermis. High levels of apoE transcripts are then localized primarily in the basal cell layer of the apical epidermis. The levels of apoE expression were maximum between the second to fourth days and then progressively declined to basal level by day 14. ApoE transcripts were also observed in putative macrophages infiltrated in the mesenchymal compartment of regenerating fins a few hours after amputation. In conclusion, apoE is highly expressed in the epidermis of developing fins and scales and during fin regeneration while no expression can be detected in the skin of the trunk. ApoE may play a specific role in fin and scale differentiation at sites where important epidermo-dermal interactions occur for the elaboration of the dermal skeleton and/or for lipid uptake and redistribution within these rapidly growing structures.


Assuntos
Apolipoproteínas E/genética , Epiderme/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regeneração , Peixe-Zebra/embriologia , Animais , Epiderme/metabolismo , Morfogênese , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
19.
Dev Biol ; 206(2): 189-205, 1999 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9986732

RESUMO

DNA methylation constitutes an important epigenetic factor in the control of genetic information. In this study, we analyzed expression of the DNA methyltransferase gene and examined DNA methylation patterns during early development of the zebrafish. Maternal transcripts of the zebrafish DNA methyltransferase gene (MTase) are ubiquitously present at high levels in early embryos with overall levels decreasing after the blastula stage. At 24 h, methyltransferase mRNA is predominantly found in the brain, neural tube, eyes, and differentiating somites. Expression of MTase in the somites is highest in the anterior cells of the somites. Despite the high levels of MTase mRNA in blastula-stage embryos, we observe DNA hypomethylation at the blastula and gastrula stages compared to sperm or older embryos. Zebrafish embryos treated with 5-azacytidine (5-azaC) and 5-aza-2-deoxycytidine (5-azadC), nucleotide analogs known to induce cellular differentiation and DNA hypomethylation in mammalian cells, exhibit DNA hypomethylation and developmental perturbations. These defects are specifically observed in embryos treated at the beginning of the blastula period, just prior to midblastula transition. The most common phenotype is the loss of tail and abnormal patterning of somites. Head development is also affected in some embryos. Histological and in situ hybridization analyses reveal whole or partial loss of a differentiated notochord and midline muscle in treated embryos. When examined during gastrulation, 5-azaC-treated embryos have a shortened and thickened axial mesoderm. We propose that DNA methylation is required for normal gastrulation and subsequent patterning of the dorsal mesoderm.


Assuntos
Padronização Corporal/fisiologia , Metilação de DNA , Gástrula/metabolismo , Somitos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Anormalidades Induzidas por Medicamentos/embriologia , Anormalidades Induzidas por Medicamentos/genética , Sequência de Aminoácidos , Animais , Azacitidina/análogos & derivados , Azacitidina/toxicidade , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Metiltransferases/genética , Dados de Sequência Molecular , Músculos/anormalidades , Músculos/efeitos dos fármacos , Músculos/embriologia , Notocorda/anormalidades , Notocorda/efeitos dos fármacos , Notocorda/embriologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética
20.
Development ; 125(21): 4175-84, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9753672

RESUMO

The signaling molecule encoded by Sonic hedgehog (shh) participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. We have shown that regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA) (Akimenko and Ekker (1995) Dev. Biol. 170, 243-247). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. We have examined the expression of shh in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express shh, and ptc1 which is thought to encode the receptor of the SHH signal. The expression domain of ptc1 is broader than that of shh and adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in patterning of the dermoskeleton of developing and regenerating teleost fins.


Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Membrana/fisiologia , Proteínas/fisiologia , Transativadores , Fator de Crescimento Transformador beta , Peixe-Zebra/fisiologia , Animais , Padronização Corporal/efeitos dos fármacos , Desenvolvimento Ósseo/genética , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores Patched , Receptor Patched-1 , Proteínas/genética , Proteínas/metabolismo , Receptores de Superfície Celular , Regeneração , Tretinoína/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra
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