RESUMO
The main aim of this work was to stimulate bone-forming cells to produce three-dimensional networks of mineralized proteins such as those occurring in bones. This was achieved by a novel approach using a specific type of mesenchymal progenitor cells (i.e., primary fibroblast cells from human hair roots) seeded on to polymer scaffolds. We wrote polymer microstructures with one or more levels of quadratic pores on to a flexible substrate by means of two-photon polymerization using a Ti-sapphire femtosecond laser focused into a liquid acrylate-based resin containing a photoinitiator. Progenitor cells, differentiated into an osteogenic lineage by the use of medium supplemented with biochemical stimuli, can be seeded on to the hydrophilic three-dimensional scaffolds. Due to confinement to the microstructures and/or mechanical interaction with the scaffold, the cells are stimulated to produce high amounts of calcium-binding proteins, such as collagen type I, and show an increased activation of the actin cytoskeleton. The best results were obtained for quadratic pore sizes of 35 µm: the pore volumes become almost filled with both cells in close contact with the walls of the structure and with extracellular matrix material produced by the cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 891-899, 2017.
Assuntos
Diferenciação Celular , Matriz Extracelular/química , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Osteoblastos/metabolismo , Alicerces Teciduais/química , Feminino , Fibroblastos/citologia , Folículo Piloso/citologia , Humanos , Masculino , Osteoblastos/citologia , Processos FotoquímicosRESUMO
AIM: Our aim was to determine whether the benefits of autologous skeletal-muscle-derived cell injection to treat obstetric anal incontinence are sustained at 5 years. METHOD: An observational study was performed of 10 women suffering from obstetric anal incontinence refractory to non-surgical therapy. Autologous skeletal-muscle-derived cells were injected into the external sphincter defect under ultrasound guidance. Incontinence diaries and quality of life questionnaires were obtained pre-implantation and annually after implantation for 5 years. Anal physiology testing was performed before implantation and at 1, 2 and 5 years after implantation. The end-points included were adverse events, Wexner incontinence scores, incontinence episodes, anal squeeze pressures and quality of life over 5 years. An independent statistician used multilevel linear regression to analyse changes in repeated measures over time. Any skewed distributions were log transformed prior to analysis. RESULTS: No procedure-related adverse events occurred and haematological and biochemical parameters were normal during the 5-year period. There were sustained significant improvements in the Wexner incontinence score and reduced frequency of defaecation and number of incontinence episodes (all comparisons P < 0.001). Anal resting and squeeze pressures showed sustained improvement (all P < 0.001) and quality of life improved overall (P < 0.001), including all submeasures studied (P < 0.001). CONCLUSION: Autologous skeletal-muscle-derived cells to treat obstetric anal incontinence resulted in sustained improvement in incontinence episodes, physiological measurements of anal function and quality of life at 5 years.
Assuntos
Canal Anal/lesões , Canal Anal/fisiopatologia , Parto Obstétrico/efeitos adversos , Incontinência Fecal/etiologia , Incontinência Fecal/terapia , Mioblastos Esqueléticos/transplante , Adulto , Idoso , Defecação , Feminino , Seguimentos , Inquéritos Epidemiológicos , Humanos , Injeções Intramusculares , Manometria , Pessoa de Meia-Idade , Qualidade de Vida , Índice de Gravidade de Doença , Transplante AutólogoRESUMO
RATIONALE: Stress urinary incontinence is still a "battlefield" for many minimally invasive therapies, but, unfortunately, few can restore the anatomical and functional background of this disorder. OBJECTIVE: Assessing the latest minimally invasive procedures of intra and perisphincterian injection of autologous stem cells. METHOD AND RESULT: The first stem cell implantation (myoblasts and /or mature fibroblasts grown and multiplied in the laboratory from biopsy samples taken from the pectoralis muscle) in the urethral sphincter was performed on October 18, 2010, in "Fundeni" Clinic of Urology and Renal Transplantation, in Romania. DISCUSSION: The follow-up at six weeks with the quality of life questionnaires, micturition diary and clinical examination revealed a decrease of urine loss from six pads/ day at one per day, which significantly improved the patient's quality of life according to visual analogue scale. Clinical and urodynamic evaluations will continue and will be future scientific topics.
Assuntos
Incontinência Urinária por Estresse/terapia , Feminino , Humanos , Qualidade de Vida , Romênia , Transplante de Células-Tronco/métodos , Resultado do TratamentoRESUMO
OBJECTIVE: Treatment of stress urinary incontinence consists of a wide range of options, from conservative therapies like lifestyle changes, medication, pelvic floor muscles exercises, electro-stimulation, to minimally invasive procedures--injection of collagen, suburethral slings TVT/TOT and last but not least, invasive surgical treatment reserved for recurrent and complex cases. Among the latest minimally invasive procedures reported in literature, the injection of intra-and perisphincterian of autologous stem cell (mioblasts and/or mature fibroblasts grown and multiplied in the laboratory from biopsy samples taken from the pectoralis muscles). MATERIAL AND METHOD: On October 18, 2010, in 'Fundeni' Clinical Institute of Uronephrology and Renal Transplantation was performed the first stem cell implantation procedure in the urethral sphincter, in Romania. RESULTS: Assessment at 6 weeks, the quality of life questionnaires, micturition diary and clinical examination revealed a stunning decrease of urine loss from 6 pads/day at one per day, which significantly improved the patient's quality of life. CONCLUSIONS: Stem-cell-mioblasts therapy may represent in the future an every-day intervention in the urologist's armamentarium. The effectiveness of this treatment can change the course of therapy and last but not least, the accessibility to urological evaluation of patients with stress urinary incontinence. Clinical and urodynamic evaluations will continue and will be future scientific topics.
Assuntos
Transplante de Células-Tronco , Incontinência Urinária por Estresse/terapia , Biópsia , Feminino , Humanos , Tampões Absorventes para a Incontinência Urinária , Músculos Peitorais/citologia , Exame Físico , Qualidade de Vida , Romênia , Inquéritos e Questionários , Resultado do Tratamento , Ultrassonografia , Uretra/diagnóstico por imagem , Uretra/cirurgia , Incontinência Urinária por Estresse/cirurgiaRESUMO
In the last years preclinical studies have paved the way for the use of adult muscle derived stem cells for reconstruction of the lower urinary tract. Between September 2002 and October 2004, 42 women and 21 men suffering from urinary stress incontinence (age 36-84 years) were recruited and subsequently treated with transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. In parallel, 7 men and 21 women (age 39-83 years) also diagnosed with urinary stress incontinence were treated with standard transurethral endoscopic injections of collagen. Patients were randomly assigned to both groups. After a follow-up of 12 months incontinence was cured in 39 women and 11 men after injection of autologous myoblasts and fibroblasts. Mean quality of life score (51.38 preoperatively, 104.06 postoperatively), thickness of urethra and rhabdosphincter (2.103 mm preoperatively, 3.303 mm postoperatively) as well as contractility of the rhabdosphincter (0.56 mm preoperatively, 1.462 mm postoperatively) were improved postoperatively. Only in two patients treated with injections of collagen incontinence was cured. The present clinical results demonstrate that, in contrast to injections of collagen, urinary incontinence can be treated effectively with ultrasonography-guided injections of autologous myo- and fibroblasts.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Colágeno/administração & dosagem , Endossonografia/métodos , Implantação de Prótese/métodos , Transplante de Células-Tronco/métodos , Incontinência Urinária/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas/transplante , Cistoscopia , Feminino , Fibroblastos/citologia , Fibroblastos/transplante , Seguimentos , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Mioblastos/citologia , Mioblastos/transplante , Próteses e Implantes , Estudos Retrospectivos , Transplante Autólogo , Resultado do Tratamento , Uretra , Bexiga Urinária , Incontinência Urinária/diagnóstico por imagemRESUMO
Experimental and clinical studies investigated whether urinary incontinence can be effectively treated with transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts.This new therapy was performed in eight female pigs. It could be shown that the injected cells survived well and that new muscle tissue was formed. Next, 42 patients (29 women, 13 men) suffering from urinary stress incontinence were treated. The fibroblasts were mixed with a small amount of collagen as carrier material and injected into the urethral submucosa to treat atrophies of the mucosa. The myoblasts were directly injected into the rhabdosphincter to reconstruct the muscle and to heal morphological and functional defects. In 35 patients urinary incontinence could be completely cured. In seven patients who had undergone multiple surgical procedures and radiotherapy urinary incontinence improved. No side effects or complications were encountered postoperatively. The experimental as well as the clinical data clearly demonstrate that urinary incontinence can be treated effectively with autologous stem cells. The present data support the conclusion that this new therapeutic concept may represent a very promising treatment modality in the future.
Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/transplante , Mioblastos/transplante , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Incontinência Urinária/diagnóstico , Incontinência Urinária/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Fibroblastos/patologia , Rejeição de Enxerto/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mioblastos/patologia , Transplante de Células-Tronco/efeitos adversos , Engenharia Tecidual/efeitos adversos , Resultado do TratamentoRESUMO
We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP(+)-PKH(+) myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.
Assuntos
Canais de Cálcio/metabolismo , Regulação para Baixo/fisiologia , Mioblastos Esqueléticos/metabolismo , Miocárdio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/metabolismo , Animais , Células Cultivadas , Masculino , Potenciais da Membrana/fisiologia , Mioblastos Cardíacos/metabolismo , Mioblastos Esqueléticos/transplante , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/cirurgia , Miocárdio/citologia , Ratos , Ratos Endogâmicos F344RESUMO
OBJECTIVES: To prove whether intramyocardial transplantation of combined skeletal myoblasts (SM) and mononuclear bone marrow stem cells is superior to the isolated transplantation of these cell types after myocardial infarction in rats. METHODS: In 67 male Fischer rats myocardial infarction was induced by direct ligature of the LAD. Seven days postinfarction baseline echocardiography and intramyocardial cell transplantation were performed. Via lateral thoracotomy 200 microl containing either 10(7) SMs or 10(7) bone marrow-derived mononuclear cells (BM-MNC) or a combination of 5x10(6) of both cell types (MB) were injected in 10-15 sites in and around the infarct zone. In controls (C) 200 microl of cell-free medium were injected in the same manner. Before injection both cell types were stained using a fluorescent cell linker kit (PKH, Sigma). In addition, SMs were transfected with green fluorescent protein. Nine weeks postinfarction follow-up echocardiography was performed and animals were sacrificed for further analysis. RESULTS: At baseline echocardiography there was no difference in left ventricular ejection fraction (LVEF; C, SM, BM-MNC, MB: 60.1+/-3.2, 53.3+/-10.2, 53.1+/-8.7, 49+/-9.0%) and left ventricular end diastolic diameter (LVEDD; C, SM, BM-MNC, MB: 6.5+/-0.8, 5.17+/-0.8, 5.77+/-1.4, 6.25+/-0.8 mm) between the different therapeutic groups. Eight weeks after cell transplantation LVEDD was significantly increased in all animals except those that received a combination of myoblasts and bone marrow stem cells (MB; C, SM, BM-MNC, MB: 7.7+/-0.6 mm, P=0.001; 7.7+/-1.5 mm, P<0.001; 7.7+/-1.1 mm, P=0.005; 6.6+/-1.7 mm, P=0.397. At the same time LVEF decreased significantly in the control group (C), stayed unchanged in animals that received bone marrow stem cells (BM-MNC) and increased in animals that received myoblasts (SM) and a combination of both cell types (MB; C, SM, BM-MNC, MB: 45.3+/-7.0%, P=0.05; 63.9+/-15.4%, P=0.044; 54.3+/-6.3%, P=0.607; 63.0+/-11.5%, P=0.039). CONCLUSIONS: The present data show that the concept of combining SMs with bone marrow-derived stem cells may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential.
Assuntos
Transplante de Medula Óssea/métodos , Cardiomioplastia/métodos , Mioblastos Esqueléticos/transplante , Infarto do Miocárdio/terapia , Animais , Modelos Animais de Doenças , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Endogâmicos F344 , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an ectopic non-muscular environment.
Assuntos
Implantes Absorvíveis , Reação a Corpo Estranho/patologia , Teste de Materiais/métodos , Mioblastos Esqueléticos/patologia , Mioblastos Esqueléticos/transplante , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Divisão Celular , Transplante de Células/efeitos adversos , Transplante de Células/instrumentação , Transplante de Células/métodos , Células Cultivadas , Reação a Corpo Estranho/etiologia , Modelos Animais , Ratos , Ratos Endogâmicos F344 , Engenharia Tecidual/instrumentaçãoRESUMO
1. Low threshold, T-type, Ca(2+) channels of the Ca(v)3 family display the fastest inactivation kinetics among all voltage-gated Ca(2+) channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Ca(v)3.1 inactivation as observed previously in high voltage-activated Ca(2+) channels. 2. Amino acids that are identical in IIIS6 segments of all Ca(2+) channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine. 3. The kinetic properties of the mutants were analysed with the two-microelectrode voltage-clamp technique after expression in Xenopus oocytes. The time constant for the barium current (I(Ba)) inactivation, tau(inact), of wild-type channels at -20 mV was 9.5 +/- 0.4 ms; the corresponding time constants of the mutants ranged from 9.2 +/- 0.4 ms in V1512A to 45.7 +/- 5.2 ms (4.8-fold slowing) in M1510I. Recovery at -80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4. We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Ca(v)2.1 (Hering et al. 1998) have a significant role in Ca(v)3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca(2+) channels.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Sequência de Aminoácidos/genética , Animais , Canais de Cálcio Tipo T/genética , Cinética , Dados de Sequência Molecular , Mutação/fisiologia , Oócitos , Fatores de Tempo , XenopusRESUMO
Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta-subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha1-beta-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha(1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha(1)2.1/alpha2-delta/beta1a k(inact) = 1.35 +/- 0.25 s(-1) to a 75-fold acceleration in mutant M1811Q (k(inact) = 102 +/- 3 s(-1). Coexpression of mutant alpha(1)2.1-subunits with beta(2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I(Ba) inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta2a-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.
Assuntos
Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/fisiologia , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Canais de Cálcio/genética , Feminino , Humanos , Cinética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Ca(v)2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+-dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming alpha1-subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel alpha1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the alpha1-subunits with auxiliary beta-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.
Assuntos
Canais de Cálcio/metabolismo , Potenciais de Ação , Processamento Alternativo , Animais , Ataxia/genética , Ataxia/metabolismo , Sítios de Ligação , Canais de Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio , Proteínas de Ligação ao GTP/metabolismo , Humanos , Cinética , Proteínas de Membrana/metabolismo , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Subunidades Proteicas , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Isradipino/farmacologia , Animais , Conformação Proteica/efeitos dos fármacos , XenopusRESUMO
A novel molecule expressed by spleen dendritic cells (DC) was isolated using a subtractive hybridization approach. The full-length M1204 clone has 3063 bp, with 1415 bp spanning a single open reading frame, coding for a protein of a predicted size of about 50 kDa. This sequence has strong homology to 2', 5' oligoadenylate synthetase and contains a ubiquitin-like domain. In Northern blot analyses the mRNA is strongly expressed in spleen DC, whereas, in bone marrow-derived DC, the amount of mRNA increases during the maturation process. None of the other leukocytes nor several hemopoietic cell lines tested express this mRNA, but clear expression occurs in many organs, the highest levels being in thymus, lung, and bone marrow. In situ hybridization, combined with immunocytochemical staining of tissue sections of lung and spleen, shows colocalization of M1204 with the 2A1 and NLDC DC markers. In Western blot experiments, an antiserum raised against the recombinant M1204 recognizes a single band in bone marrow-derived DC and in the lung. The expressed oligoadenylate synthetase domain is active in synthesizing 2',5' diadenylate, which by itself may inhibit viral protein synthesis and may also function as a substrate for 2',5' oligoadenylate synthetase. Since the oligoadenylate/RNase L system provides early protection against virus infection, we hypothesize that M1204 prevents virus-induced cell death in DC.
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Células Dendríticas/metabolismo , Fragmentos de Peptídeos/biossíntese , Ubiquitinas/biossíntese , 2',5'-Oligoadenilato Sintetase/química , 2',5'-Oligoadenilato Sintetase/genética , Nucleotídeos de Adenina/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores/química , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/química , Células Dendríticas/enzimologia , Indução Enzimática/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oligorribonucleotídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Baço/citologia , Baço/metabolismo , Ubiquitinas/química , Ubiquitinas/genética , Regulação para Cima/imunologiaRESUMO
Until recently, poly(ADP-ribosyl)ation was supposed to be confined only to polymerizing(ADP-ribosyl)transferase/(ADP-ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP-ribosyl)transferase homologues from mouse and man that lack all of the N-terminal DNA binding and BRCA1 C-terminus domains and will be designated polymerizing(ADP-ribosyl)transferase-2 as distinguished from the classical polymerizing(ADP-ribosyl)transferase (polymerizing(ADP-ribosyl)transferase-1). The murine polymerizing(ADP-ribosyl)transferase-2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue ('APP', GenBank database AF069298). Expression of the murine polymerizing(ADP-ribosyl)transferase-2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP-ribosyl)ation activity might account for most of the residual poly(ADP-ribosyl)ation observed in polymerizing(ADP-ribosyl)transferase-1(-/-) mice.
Assuntos
Arabidopsis/enzimologia , Caenorhabditis elegans/enzimologia , Poli(ADP-Ribose) Polimerases/genética , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Sequência de Bases , Caenorhabditis elegans/genética , Sequência Conservada , DNA Complementar , Humanos , Íntrons , Mamíferos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição GênicaRESUMO
The extracellular domains of the TrkA nerve growth factor (NGF) receptor and its homologs harbor a modular mosaic of potential ligand binding motifs, namely two immunoglobulin (Ig)-like modules and an LRM3 cassette consisting of a tandem array of three leucine-rich motifs (LRMs) flanked by cysteine-rich clusters (Schneider, R., and Schweiger, M. (1991) Oncogene 6, 1807-1811). Identification of a structural motif capable of specifically recognizing the various neurotrophins was achieved by assessing their affinities to isolated recombinant modules of TrkA and TrkB. In both receptors the LRM3 cassette alone could mediate the respective neurotrophin selectivities and affinities. Further tracking down of this NGF-binding site in TrkA strikingly revealed that a single LRM of 24 amino acids could bind NGF selectively with nanomolar affinity. Since this is the first example of a single LRM with a highly specific, well defined function, it might serve as a valuable tool to elucidate the general structural requirements of substrate recognition and high affinity binding in the large superfamily of LRM-containing proteins.
Assuntos
Leucina , Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , Clonagem Molecular , Sequência Consenso , Cisteína , Imunoglobulinas , Cinética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Receptor do Fator Neutrófico Ciliar , Receptor trkA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Glândula Submandibular/metabolismoRESUMO
The extracellular domains of the TrkA and TrkB neurotrophin receptors contain defined structural modules such as immunoglobulin-like domains and leucine-rich motifs (LRMs) [Schneider and Schweiger, Oncogene 6 (1991) 1807-1811]. Recently, the second LRM of TrkA was identified as a functional nerve growth factor (NGF) binding site [Windisch et al, J. Biol. chem. (1995) in press]. A peptide corresponding to this region effectively bound NGF and blocked binding of NGF to the recombinant extracellular domain of TrkA. The corresponding TrkB peptide exhibited the same effects with respect to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), indicating that all three TrkB ligands utilize this same binding site. Isolated LRMs therefore embody independent functional entities.
Assuntos
Leucina/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos , Animais , Fator Neurotrófico Derivado do Encéfalo , Humanos , Dados de Sequência Molecular , Neurotrofina 3 , Peptídeos/metabolismo , Ligação Proteica , Receptor do Fator Neutrófico Ciliar , Receptor trkA , Homologia de Sequência de AminoácidosRESUMO
TrkB is a member of the Trk family of neurotrophin receptors. Its extracellular domain exhibits the same modular structure found in its homologs, TrkA and TrkC, consisting of an N-terminal LRM3 cassette and two immunoglobulin-like modules (Ig2 domain) adjacent to the membrane. The LRM3 cassette comprises two cysteine-rich clusters framing a tandem array of three leucine-rich motifs (LRMs). On the basis of the recent identification of a nerve growth factor (NGF) binding site within TrkA, the ability of the different structural entities within the extracellular domain of TrkB to bind the various neurotrophins was determined by using a recombinant receptor approach. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) bound to the LRM3 cassette of TrkB, whereas NGF did not. These binding characteristics evidently reflect in vivo specificities. A more precise mapping of the region(s) responsible for binding BDNF, NT-3, and NT-4 identified the second leucine-rich motif of TrkB as a functional unit capable of binding all three neurotrophins. The affinities and kinetics that this short stretch of amino acids exhibited with respect to the different neurotrophins were clearly akin to those observed for cells ectopically expressing TrkB receptors. With 24 amino acids determining the affinities and kinetics of the interactions with three different partners, the leucine-rich motif is strongly established as one of the most potent and flexible protein--protein interaction motifs.