RESUMO
OBJECTIVE: To assess and risk-stratify the medium-term clinical outcomes after infrainguinal bypass grafting (IBG) to treat critical limb ischaemia (CLI) in patients with end-stage renal disease. METHODS: This was a retrospective single-centre study. Between April 2007 and March 2011, 112 limbs from 89 patients were studied. In particular, amputation-free survival (AFS), 30 day mortality, freedom from major adverse limb events (MALE), limb salvage, and overall survival were examined. The aim was to identify outcome predictors. RESULTS: Eight patients (9%) died within 30 days of IBG. The only positive predictor of 30-day mortality was an ejection fraction (EF) < 40% (hazard ratio [HR] 5.57, 95% confidence interval [CI] 1.16-26.83; p = .03). The mean follow-up duration was 14 months. The 1- and 2-year AFS rates were 64% and 43%, respectively, and the rates of freedom from MALE were 81% and 77%, respectively. In addition, the 1- and 2-year limb salvage rates were 89% and 85%, and the survival rates were 68% and 50%, respectively. Non-ambulatory status was negatively associated with AFS (HR 3.04, 95% CI 1.59-5.82; p < .01), freedom from MALE (HR 4.98, 95% CI 1.91-12.96; p < .01), and limb salvage (HR 5.18, 95% CI 1.47-18.30; p = .01). The other negative predictors of overall survival were a serum albumin level <3.0 g/dL (HR 2.26, 95% CI 1.12-4.58; p = .02) and an EF <40% (HR 2.24, 95% CI 1.05-4.79; p = .04). CONCLUSION: Patients with CLI on dialysis enjoyed satisfactory freedom from MALE and limb salvage, but survival and AFS were significantly less than reported for IBG in patients with CLI who did not receive dialysis. In addition, patients with an EF <40%, lower serum albumin (<3.0 g/dL), or non-ambulatory status experienced particularly poor clinical outcomes after IBG.
Assuntos
Isquemia/cirurgia , Falência Renal Crônica/terapia , Diálise Renal , Enxerto Vascular , Idoso , Idoso de 80 Anos ou mais , Amputação Cirúrgica , Biomarcadores/sangue , Estado Terminal , Intervalo Livre de Doença , Feminino , Humanos , Isquemia/complicações , Isquemia/diagnóstico , Isquemia/mortalidade , Isquemia/fisiopatologia , Japão , Estimativa de Kaplan-Meier , Falência Renal Crônica/complicações , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/mortalidade , Salvamento de Membro , Masculino , Modelos de Riscos Proporcionais , Diálise Renal/efeitos adversos , Diálise Renal/mortalidade , Reoperação , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Albumina Sérica/metabolismo , Albumina Sérica Humana , Volume Sistólico , Fatores de Tempo , Resultado do Tratamento , Enxerto Vascular/efeitos adversos , Enxerto Vascular/mortalidadeRESUMO
To investigate whether anti-apoptotic factors play a role in the malignant growth of canine haemangiosarcomas (HSAs), 83 HSAs and 22 haemangiomas were examined immunohistochemically for bcl-2 and survivin expression. Additionally, bcl-2 and survivin mRNA expression was quantified by semiquantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunolabelling for bcl-2 was observed in 50 of the 83 HSA samples (60.2%) but in none of the haemangiomas. The average survivin positive index was 24.7% in the HSAs and 0.6% in the haemangiomas. In contrast to the high average value for survivin mRNA expression, which was approximately six times that for the haemangiomas, no significant difference was observed between HSAs and haemangiomas for the average bcl-2 mRNA expression level. The discrepancy between bcl-2 mRNA and bcl-2 protein expression requires further investigation, but the results suggest that malignant proliferation in canine HSAs is associated with bcl-2 and survivin expression.
Assuntos
Doenças do Cão/metabolismo , Hemangioma/veterinária , Hemangiossarcoma/veterinária , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Vasculares/veterinária , Animais , Apoptose , Proliferação de Células , Doenças do Cão/patologia , Cães , Regulação Neoplásica da Expressão Gênica , Hemangioma/metabolismo , Hemangioma/patologia , Hemangiossarcoma/metabolismo , Hemangiossarcoma/patologia , RNA Mensageiro/metabolismo , Neoplasias Vasculares/metabolismo , Neoplasias Vasculares/patologiaRESUMO
Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.
Assuntos
Toxinas Botulínicas , Proteínas de Ciclo Celular , Fator de Crescimento de Hepatócito/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , ADP Ribose Transferases/farmacologia , Amidas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Cães , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Confocal , Modelos Biológicos , Mutação , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-vav , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteínas rho de Ligação ao GTP/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rhoRESUMO
The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas rab de Ligação ao GTP , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Cães , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab5 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteína rhoA de Ligação ao GTPRESUMO
Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.
Assuntos
Actinas/ultraestrutura , Proteínas de Transporte/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Rim/citologia , Proteínas Serina-Treonina Quinases/metabolismo , ADP Ribose Transferases/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Proteínas de Ligação ao GTP/genética , Genes Dominantes , Peptídeos e Proteínas de Sinalização Intracelular , Rim/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Acetato de Tetradecanoilforbol/farmacologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTPRESUMO
AIMS: We assessed the long-term (more than ten-year) outcomes of the Kudo type-5 elbow prosthesis in patients with rheumatoid arthritis (RA). MATERIALS AND METHODS: We reviewed 41 elbows (Larsen Grade IV, n = 21; Grade V, n = 20) in 31 patients with RA who had undergone a Kudo type-5 total elbow arthroplasty (TEA) between 1994 and 2003, and had been followed up for more than ten years. The humeral component was cementless and the all-polyethylene ulnar component cemented in every patient. Clinical outcome was assessed using the Mayo elbow performance score. We calculated the revision rate and evaluated potential risk factors for revision. The duration of follow-up was a mean 141 months (120 to 203). RESULTS: Aseptic loosening of the ulnar component occurred in 11 elbows. There was no radiolucency around any humeral component. There was one deep infection. The survival rate according to Kaplan-Meier survivorship analysis was 87.8% after five years and 70.7% after ten years. The range of extension/flexion was a mean -38° (-80° to 0°)/105° (30° to 150°) before surgery and -40° (-70° to -20°)/132° (100° to 150°) at the final follow-up, while the mean Mayo elbow performance score was 43 before surgery and 80 at final follow-up. Disease duration of RA up to the TEA of < 15 years and a pre-operative range of movement (ROM) of > 85° were significant risk factors for revision or aseptic loosening. CONCLUSION: Although Kudo type-5 prostheses gave satisfactory results in the short-term, aseptic loosening increased after five years. In most cases, elbow function was maintained in the long-term without loosening of the implant. A short duration from the onset of RA to TEA and a large pre-operative ROM were significant risk factors for revision or aseptic loosening. Cite this article: Bone Joint J 2017;99-B:818-23.
Assuntos
Artrite Reumatoide/cirurgia , Artroplastia de Substituição do Cotovelo/instrumentação , Prótese de Cotovelo , Adulto , Idoso , Artroplastia de Substituição do Cotovelo/efeitos adversos , Artroplastia de Substituição do Cotovelo/métodos , Cimentação , Articulação do Cotovelo/diagnóstico por imagem , Articulação do Cotovelo/fisiopatologia , Articulação do Cotovelo/cirurgia , Prótese de Cotovelo/efeitos adversos , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Falha de Prótese/etiologia , Radiografia , Recuperação de Função Fisiológica , Reoperação , Fatores de RiscoRESUMO
OBJECTIVE: To evaluate the masking ability and translucency of monolithic and bilayer CAD-CAM ceramic structures. METHODS: Discs of high translucency (HT) and low translucency (LT) lithium disilicate-based ceramic (IPS e.max CAD) with different thicknesses (0.7, 1, 1.5, and 2 mm) were evaluated as a monolithic structure or combined (bilayer) with a 0.5-mm-thick zirconia framework (IPS e.max ZirCAD). The masking ability and translucency were calculated based on CIE L*a*b* color coordinates measured with a spectrophotometer (SP60, X-Rite). The translucency parameter (TP) was calculated using color coordinates measured over standard white-and-black backgrounds. The masking ability was calculated by CIEDE2000 color difference metric (ΔE00) for each specimen measured over a tooth-colored substrate (shade A2) compared to three darker backgrounds (shade C4 and two metal substrates). Confidence intervals (CI) for the means (95% CI) were calculated for TP and ΔE00. The Pearson correlation between ΔE00 and TP was investigated for monolithic and bilayer structures over all backgrounds. RESULTS: The thinner the lithium disilicate layer, the greater the translucency and the higher the ΔE00 values. The effect of ceramic thickness on both translucency and masking ability was more pronounced for the monolithic structures. In addition, monolayers always presented a greater color variation than their bilayer counterparts. The metallic background produced greater ΔE00 than the C4-shaded substrate. CONCLUSION: Monolithic veneers were able to mask C4-shaded background but did not mask metallic backgrounds. Bilayer structures showed greater shade masking ability than monolithic structures.
Assuntos
Silicatos de Alumínio/química , Cor , Desenho Assistido por Computador , Porcelana Dentária/química , Facetas Dentárias , Zircônio/química , Luz , Teste de Materiais , Espectrofotometria , Propriedades de SuperfícieRESUMO
We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Junções Íntimas/fisiologia , Quinases da Família src/metabolismo , Animais , Células COS , Caderinas/fisiologia , Linhagem Celular , Meios de Cultura Livres de Soro , Células Epiteliais/metabolismo , Feminino , Humanos , Cinesinas , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Miosinas , Nectinas , Fosforilação , Fosfotirosina/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The Rho small G protein family consists of the Rho, Rac, and Cdc42 subfamilies and regulates various cell functions through reorganization of the actin cytoskeleton. We previously showed that the Rho subfamily regulates the formation of stress fibers and focal adhesions whereas the Rac subfamily regulates the E-cadherin-based cell-cell adhesion in MDCK cells. We studied here the function of the Cdc42 subfamily, consisting of two members, Cdc42Hs and G25k, in cell adhesion, migration, and morphology of MDCK cells. For this purpose, we made and used MDCK cell lines stably expressing each of dominant active mutants of Cdc42Hs (sMDCK-Cdc42HsDA) and G25K (sMDCK-G25KDA). Actin filaments at the cell-cell adhesion sites increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Electron microscopic analysis revealed that sMDCK-Cdc42HsDA cells tightly contacted with each other throughout the lateral membranes. Moreover, both the HGF- and TPA-induced disruption of the cadherin-based cell-cell adhesion and the subsequent cell migration were inhibited in both sMDCK-Cdc42HsDA and -G25KDA cells. Co-expression of the dominant negative mutant of Rac1, a member of the Rac subfamily, with the dominant active mutant of Cdc42Hs did not inhibit the increased accumulation of actin filaments at the cell-cell adhesion sites. These results suggest that the Cdc42 subfamily is involved in the cadherin-based cell-cell adhesion in a manner independent of the Rac subfamily. Furthermore, the cells were frequently enveloped by the large multinuclear cells in both sMDCK-Cdc42HsDA and -G25KDA cells. Video microscopic analysis revealed that the cells were engulfed by the large cells during cytokinesis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Transativadores , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular/patologia , Linhagem Celular/ultraestrutura , Movimento Celular/genética , Tamanho Celular/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Rim/citologia , Rim/patologia , Rim/ultraestrutura , Mutagênese Sítio-Dirigida , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional , beta Catenina , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTPRESUMO
Both E-cadherin, a cell-cell adhesion molecule, and c-Met, the hepatocyte growth factor (HGF)/scatter factor (SF) receptor, were colocalized at cell-cell adhesion sites of MDCK cells. HGF/SF or a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced disruption of cell-cell adhesion, which was accompanied by endocytosis of both E-cadherin and c-Met. Reduction of medium Ca2+ to a micromolar range showed the same effects. Re-increase in medium Ca2+ to a millimolar range formed cell-cell adhesion, which was accompanied by exocytosis of E-cadherin and c-Met, followed by their re-colocalization at the cell-cell adhesion sites. These results suggest that E-cadherin and c-Met are colocalized at cell-cell adhesion sites and undergo co-endo-exocytosis. We have previously shown that TPA does not induce disruption of cell-cell adhesion and subsequent scattering of MDCK cells stably expressing a dominant active mutant of RhoA or Rac1 small G protein or a dominant negative mutant of Rab5 small G protein. In these cell lines, the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met was inhibited, but the coendocytosis of E-cadherin and c-Met in response to reduction of medium Ca2+ was not affected. Wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, inhibited the HGF-induced disruption of cell-cell junction and endocytosis of E-cadherin and c-Met, but not the TPA-induced ones. These results suggest that disruption of cell-cell adhesion is involved in the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met in MDCK cells, and that the Rho and Rab family members indirectly regulate this coendocytosis. In addition, coendocytosis of E-cadherin and c-Met in response to HGF is partly mediated by PI 3-kinase. The cross-talk between cell-cell and cell-matrix adherens junctions is discussed.
Assuntos
Caderinas/metabolismo , Adesão Celular/fisiologia , Endocitose/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Meios de Cultura , Cães , Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions. The Rho subfamily members regulate ERM (ezrin, radixin and moesin)-dependent association of the actin cytoskeleton with the plasma membrane. Moreover, the N-terminal regions of ERM interact with Rho GDI, an inhibitory regulator of all the Rho family members, and reduce its inhibitory action, finally initiating the activation of the Rho family members. We show here that the N-terminal region of radixin furthermore interacts with Dbl, a stimulatory GDP/GTP exchange protein of the Rho family members. This interaction does not affect the Dbl activity to stimulate the GDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbl does not interact with radixin which is precomplexed with Rho GDI, and Rho GDI displaces Dbl from radixin. Thus, radixin plays an important role in activation of the Rho family members by recruiting their positive and negative regulators.
Assuntos
Proteínas Sanguíneas/química , Proteínas do Citoesqueleto , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas de Membrana/química , Proteínas Oncogênicas de Retroviridae/química , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Oncogênicas de Retroviridae/metabolismo , Fator Rho/química , Fator Rho/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The alpha catalytic subunits of Na+/K(+)-ATPase were isolated from the kidney and brain of rats (alpha 1 and alpha 2, respectively). The antisera raised against these subunits were used as probes to analyze the isoform of catalytic subunits of Na+/K(+)-ATPase in various tissues of rats. Of 27 rat tissues examined, most had a catalytic subunit identical to alpha 1 but some, such as the nervous and muscle tissues, had both alpha 1 and alpha 2 isoforms as judged by their reactivities to antisera and their electrophoretic mobility. We found that the submandibular gland contained a new electrophoretic variant of immunoreactive alpha subunit (designated alpha(S) in this report) in addition to alpha 1 identical to those found in kidney and brain. The new variant, alpha(S), strongly cross-reacted with anti-alpha 1 antiserum, but to a lesser extent with anti-alpha 2 antiserum. The alpha(S) had a molecular mass which was found to be slightly less (approx. 90 kDa) than brain and kidney alpha 1. We examined whether or not the alpha(S) is formed by proteolytic cleavage of alpha subunits during preparation and concluded that this is not the case. The alpha(S) reacted with [gamma-32P]ATP, resulting in the formation of radioactive alpha subunit which was stabilized by 2 mM ouabain but which was labile in the presence of 70 mM potassium chloride. Since N-terminal amino acid sequence of alpha(S) protein [G()DKY()PAAVS] corresponds exactly and uniquely with the sequence of the alpha 1 chain between residues 1 and 11, it is very probable that alpha(S) protein originated from alpha 1 protein following the post-translational processing.
Assuntos
Isoenzimas/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , Glândula Submandibular/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/enzimologia , Variação Genética , Soros Imunes , Isoenzimas/genética , Rim/enzimologia , Substâncias Macromoleculares , Masculino , Microssomos/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Homologia de Sequência do Ácido Nucleico , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
9-cis-Retro-gamma-rhodopsin (lambda max = 420 nm) was prepared from 9-cis-retro-gamma-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-gamma-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-gamma-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of batho-rhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.
Assuntos
Pigmentos da Retina , Rodopsina , Animais , Bovinos , Congelamento , Isomerismo , Fotoquímica , EspectrofotometriaRESUMO
The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/biossíntese , Trifosfato de Adenosina/farmacologia , Calcimicina/farmacologia , Meios de Cultura , Ácido Egtázico/farmacologia , Humanos , Cinética , Células Tumorais CultivadasRESUMO
We describe a patient with a t(7;11)(p15;p15) acute myeloid leukemia who was subsequently found to harbor the Philadelphia (Ph) translocation, in addition to the t(7;11), at the second relapse. A BCR/ABL transcript was detected at the second relapse by reverse transcription-polymerase chain reaction assay; the leukemic cells had a BCR/ABL fusion gene involving the minor breakpoint cluster region (minor-BCR; situated in intron 1 of the BCR gene). Although the Ph translocation is commonly detected in de novo acute leukemia and chronic myeloid leukemia as the primary leukemia-specific chromosomal translocation, our case suggests that this cytogenetic change might occur as an additional chromosomal change in neoplastic cells. Moreover, minor-BCR/ABL rearrangements may also occur as a late appearance of Ph translocation.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/genética , Sequência de Bases , Aberrações Cromossômicas/patologia , Transtornos Cromossômicos , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 7 , Primers do DNA/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mieloide Aguda/patologia , Dados de Sequência Molecular , RNA Neoplásico/genética , Fatores de TempoRESUMO
A proform of high molecular weight type IV collagenase was isolated and purified 1230-fold from human metastatic carcinoma tissue. Like matrix metalloproteinases (MMPs), the enzyme was activated by trypsin and degraded type IV collagen and gelatin at a neutral pH, the activity was inhibited by EDTA and o-phenanthroline. However, the molecular weight was much higher than MMPs which degraded type IV collagen, gelatinase A (MMP-2; 72 kDa gelatinase/type IV collagenase) (EC 3.4.24.24), gelatinase B (MMP-9; 92 kDa gelatinase/type IV collagenase) (EC 3.4.24.35), stromelysin-1 (MMP-3; 57 kDa) (EC 3.4.24.17) and stromelysin-2 (MMP-10; 57 kDa) (EC 3.4.24.22). The other significant difference from MMPs was that the enzyme was not activated by 4-aminophenylmercuric acetate nor inhibited by TIMP. Taking together these results, this high molecular weight type IV collagenase might be a newly found enzyme different from MMPs or might have the same configuration as MMPs already reported.
Assuntos
Colagenases/isolamento & purificação , Neoplasias Hepáticas/enzimologia , Colágeno/metabolismo , Colagenases/química , Colagenases/metabolismo , Ácido Edético/farmacologia , Ativação Enzimática/efeitos dos fármacos , Gelatina/metabolismo , Gelatinases , Glicoproteínas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Hepáticas/secundário , Metaloproteinase 9 da Matriz , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Peso Molecular , Pepsina A/isolamento & purificação , Fenantrolinas/farmacologia , Especificidade por Substrato , Inibidores Teciduais de Metaloproteinases , Tripsina/farmacologiaRESUMO
Three hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin were newly derived. The specificity of established monoclonal antibodies to PCNA, TOB7, TO17 and TO30, was compared with that of the previously reported PCNA-specific monoclonal antibodies, 19A2 and 19F4. TOB7, TO17 and TO30 reacted with purified PCNA in an enzyme-linked immunosorbent assay (ELISA). A 34 kDa PCNA polypeptide was noted by immunoblotting, the same polypeptide as recognized by 19A2 and 19F4. Epitopes recognized by all those monoclonal antibodies to PCNA were analyzed by competitive inhibition tests using ELISA. The results of those experiments suggested that the epitope recognized by TO17 and TO30 was almost identical to that of 19A2 and closely related to that of 19F4, but different from that of TOB7. Based on those results, a sandwich type ELISA for detection of PCNA was developed using TO17 and TOB7. This system was applied to measure the concentration of PCNA in normal peripheral blood lymphocytes (PBL) before and after stimulation by phytohemagglutinin (PHA), and in tissue extracts from rabbit kidney and thymus (RKE and RTE, respectively). Before mitogenic stimulation, the PCNA concentration in PBL extracts was less than 6 ng/ml (cell concentration 2.5 x 10(7)/ml), but at 48 h after PHA stimulation, when more than 70% of cells were in S phase, its concentration became 1280 ng/ml. RTE which contained many proliferative lymphocytes had much higher concentration of PCNA than RKE. Those results suggested that sandwich type ELISA using TO17 and TOB7 was useful as the quantitative assay to detect blast-transformation and proliferation of cells in vivo.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Nucleares/análise , Especificidade de Anticorpos , Western Blotting , Divisão Celular , Ciclinas , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos , Imunofluorescência , Humanos , Ativação Linfocitária , Proteínas Nucleares/imunologia , Antígeno Nuclear de Célula em ProliferaçãoRESUMO
We report the second case of post-myelodysplasia acute myeloid leukemia (post-MDS AML) with a sole chromosome change del(15q). This anomaly is rarely seen. To our knowledge, only seven cases so far have been reported in human neoplasias, including one case each of acute myeloid leukemia (AML), acute lymphoid leukemia, post myelodysplasia AML, myelodysplastic syndrome, myelofibrosis, macroglobulinemia, Hodgkin's lymphoma and uterine leiomyoma. This case suggests that del(15q) is related to lympho-myeloproliferative disorders. Moreover, we speculate that certain oncogene(s) located on 15q might have some role in the progression of the disease, since the del(15q) anomaly appeared only in the AML phase in this case.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 15 , Leucemia Mieloide/genética , Doença Aguda , Feminino , Humanos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/complicaçõesRESUMO
We report here a case of refractory anemia with ringed sideroblasts (RARS) with a low risk group by the International Prognostic Scoring System (IPSS) at the time of diagnosis but had a rapid disease progression. Although the patient showed a normal male karyotype at the time of RARS diagnosis, his marrow cells had del(5)(q14) and add(17)(p12) abnormalities 2 months after the diagnosis, and later the marrow cells had multiple abnormalities and the patient expired 6 months after the initial diagnosis of RARS. The patient was diagnosed as having RARS with a low risk group by the IPSS classification, however, one should keep in mind that some patients with myelodysplastic syndromes with low risks by either the French-American-British (FAB) classification or the IPSS classification may have progressive disease and subsequential cytogenetic analysis could predict the disease progression.
Assuntos
Anemia Refratária/genética , Anemia Sideroblástica/genética , Aberrações Cromossômicas , Transtornos Cromossômicos , Leucemia Eritroblástica Aguda/genética , Doença Aguda , Anemia Refratária/fisiopatologia , Anemia Sideroblástica/etiologia , Progressão da Doença , Humanos , Cariotipagem , Leucemia Eritroblástica Aguda/etiologia , Masculino , Pessoa de Meia-IdadeRESUMO
We report two additional patients with acute myeloid leukemia (AML) and a translocation between chromosomes 7 and 11: t(7;11)(p15;p15). One patient was diagnosed as having AML-M2 and the other as AML with myelofibrosis. Both patients had low-level neutrophil alkaline phosphatase (NAP) scores. In the literature, only 15 AML patients with t(7;11)(p15;p15) have been reported; nine of them had an AML-M2 morphology, and all had a decreased NAP score. Moreover, mean survival of the reported AML patients with t(7;11)(p15;p15) was 15 months, although 85% of them obtained complete remission, indicating that this type of leukemia frequently tends to relapse. These findings indicate a strong association between the chromosome abnormality and hematologic manifestations of this disease.