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1.
Nat Methods ; 15(5): 347-350, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29578537

RESUMO

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.


Assuntos
Nicotina/farmacologia , Processos Fotoquímicos , Receptores Nicotínicos/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cálcio , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
2.
Mol Pharmacol ; 83(1): 167-78, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23071105

RESUMO

Gram-negative bacterial endotoxin lipopolysaccharide (LPS) triggers the production of inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins (PGs) by pulmonary macrophages. Here, we investigated if ROS influenced PGs production in response to LPS treatment in mouse bone marrow-derived macrophages (BMDM). We observed that pretreatment of BMDM with two structurally unrelated ROS scavengers, MnTMPyP and EUK-134, not only prevented LPS-induced ROS accumulation, but also attenuated the LPS-induced PGD(2), but not PGE(2), production. Conversely LPS-induced PGD(2), but not PGE(2), production, was potentiated with the cotreatment of BMDM with H(2)O(2). These data suggest that ROS differentially regulate PGD(2) and PGE(2) production in BMDM. In addition, selective inhibition of the ROS generator NADPH oxidase (NOX) using either pharmacologic inhibitors or its p47(phox) subunit deficient mouse BMDM also attenuated LPS-induced PGD(2), but not PGE(2) production, suggesting the critical role of NOX-generated ROS in LPS-induced PGD(2) production in BMDM. We further found that both hematopoietic PGD synthase (H-PGDS) siRNA and its inhibitor HQL-79, but not lipocalin PGDS (L-PGDS) siRNA and its inhibitor AT-56, significantly attenuated LPS-induced PGD(2) production, suggesting that H-PGDS, but not L-PGDS, mediates LPS-induced PGD(2) production in BMDM. Furthermore, data from our in vitro cell-free enzymatic studies showed that coincubation of the recombinant H-PGDS with either MnTMPyP, EUK-134, or catalase significantly decreased PGD(2) production, whereas coincubation with H(2)O(2) significantly increased PGD(2) production. Taken together, our results show that LPS-induced NOX-generated ROS production differentially and specifically regulates the H-PGDS-mediated production of PGD(2), but not PGE(2), in mouse BMDM.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Prostaglandina D2/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Animais , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Transdução de Sinais , Zimosan/farmacologia
3.
J AOAC Int ; 96(1): 67-76, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23513960

RESUMO

Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H2 (PGH2), a variety of prostanoids can be generated with diverse physiologic effects on pain, inflammation, allergy, cardiovascular system, cancer, etc. To facilitate the quantitative analysis of prostanoids in human serum of cell culture, an ultra-high pressure LC (UHPLC)/MS/MS method was developed and validated for the measurement of six eicosanoids belonging to the cyclooxygenase pathway: PGE2, PGD2, 8-iso-PGF2alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2). Selectivity, matrix effects, calibration model, precision, and accuracy (intraday and interday), lower limit of quantitation (LLOQ), recovery, stability, and sample dilution were evaluated. Fast UHPLC separation was carried out in only 0.5 min with isocratic elution, and each prostanoid was measured using negative electrospray ionization MS with collision-induced dissociation and selected reaction monitoring. UHPLC/MS/MS provided high throughput with peak widths of approximately 3 s and an LLOQ of 0.020 ng/mL for PGE2, 0.027 ng/mL for PGD2, 0.152 ng/mL for 8-iso-PGF2alpha, 0.179 ng/mL for PGF2alpha and 6-keto-PGF1alpha, and 0.013 ng/mL for TXB2.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Prostaglandinas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Calibragem , Estabilidade de Medicamentos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/química
4.
Zhongguo Zhong Yao Za Zhi ; 37(6): 728-30, 2012 Mar.
Artigo em Zh | MEDLINE | ID: mdl-22715709

RESUMO

OBJECTIVE: To observe dynamic changes of blood lead concentration in rats with long-term toxicity test with Goupi Gao by the flame atomic absorption spectrometry, in order to provide reference for safe administration of Goupi Gao. METHOD: The rats were administered with Goupi Gao by high-dose (7 g x kg(-1)), medium-dose (3.5 g x kg(-1)), low-dose (1.75 g x kg(-1)) by external use for consecutively 90 days. Then, the blood samples were collected from the rats before the administration and at 10, 30, 45, 52, 60, 90 d after the administration respectively, as well as 16 d and 28 d after the drug withdrawal. The samples were dispelled with microwave digestion system and then were determined by graphite furnace atomic absorption spectrometry for blood lead levels. RESULT: According to methodological study, the standard curve regression equation in this experiment was A = 0.004 9X + 0. 017, r = 0.999 5, with the detection limit up to 0. 380 microg x L(-1). The RSD was 1.4% by precision checks. Blood lead level of mixed blood samples was 175.77 microg x L(-1), whose RSD was 6. 0%. Blood lead concentration gradually increased after low-dose and medium-dose administration to rats and became stable at the 10th day and the 30th day by high-dose. Dose is directly related to blood lead concentration. Meanwhile, the blood lead concentration decreases after the drug withdrawal. CONCLUSION: The method of determination in this experiment is so accurate and reliable that it can be used for the determination of blood lead. Long-term and high-dose external use of Goupi Gao can increase blood lead.


Assuntos
Medicamentos de Ervas Chinesas/toxicidade , Chumbo/sangue , Espectrofotometria Atômica/métodos , Animais , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Testes de Toxicidade
5.
J Pharmacol Exp Ther ; 339(2): 716-25, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21865441

RESUMO

Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI(2) and PGF(2α) after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH(2) metabolism might be redistributed when the PGE(2) pathway is blocked. To address the determinants that govern the relative amounts of PGs, we developed an in vitro cell-free method, based on liquid chromatography-tandem mass spectrometry, to measure the exact amounts of these PGs formed in response to the addition of recombinant isomerases and their selective inhibitors. Our in vitro cell-free assay results were confirmed in cells using bone marrow-derived macrophage. Initially, we determined the in vitro stability of PGH(2) and noted that there was spontaneous nonenzymatic conversion to PGD(2) and PGE(2). mPGES-1 markedly increased the conversion to PGE(2) and decreased conversion to PGD(2). Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD(2) and decrease of PGE(2). A detailed titration study showed that the ratio of PGE(2)/PGD(2) was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH(2) metabolism is redistributed by the presence of distal isomerases or by blocking the major metabolic outlet, which could determine the relative benefits and risks resulting from interdiction in nonrated-limiting components of PG synthesis pathways.


Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Macrófagos/fisiologia , Prostaglandina D2/metabolismo , Prostaglandina H2/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Animais , Células Cultivadas , Dibenzocicloeptenos/farmacologia , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Humanos , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/metabolismo , Isomerases/antagonistas & inibidores , Isomerases/metabolismo , Isomerases/fisiologia , Lipocalinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Prostaglandina-E Sintases , Ovinos , Tiofenos/farmacologia
6.
PLoS One ; 12(7): e0182142, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28759616

RESUMO

Nicotinic acetylcholine receptors containing α4 subunits (α4ß2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 µg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.


Assuntos
Nicotina/metabolismo , Receptores Nicotínicos/metabolismo , Recompensa , Área Tegmentar Ventral/metabolismo , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Comportamento de Procura de Droga , Feminino , Deleção de Genes , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nicotínicos/genética , Potenciais Sinápticos , Área Tegmentar Ventral/fisiologia
7.
PLoS One ; 7(11): e50244, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226252

RESUMO

Cyclooxygenase (COX)-2 expression and release of prostaglandins (PGs) by macrophages are consistent features of lipopolysaccharide (LPS)-induced macrophage inflammation. The two major PGs, PGE(2) and PGD(2), are synthesized by the prostanoid isomerases, PGE synthases (PGES) and PGD synthases (PGDS), respectively. Since the expression profile and the individual role of these prostanoid isomerases-mediated inflammation in macrophages has not been defined, we examined the LPS-stimulated PGs production pattern and the expression profile of their synthases in the primary cultured mouse bone marrow derived macrophages (BMDM). Our data show that LPS induced both PGE(2) and PGD(2) production, which was evident by ∼8 hrs and remained at a similar ratio (∼1∶1) in the early phase (≤12 hrs) of LPS treatment. However, PGE(2) production continued increase further in the late phase (16-24 hrs); whereas the production of PGD(2) remained at a stable level from 12 to 24 hrs post-treatment. In response to LPS-treatment, the expression of both COX-2 and inducible nitric oxide synthase (iNOS) was detected within 2 to 4 hrs; whereas the increased expression of microsomal PGES (mPGES)-1 and a myeloid cell transcription factor PU.1 did not appear until later phase (≥12 hrs). In contrast, the expression of COX-1, hematopoietic-PGDS (H-PGDS), cytosolic-PGES (c-PGES), or mPGES-2 in BMDM was not affected by LPS treatment. Selective inhibition of mPGES-1 with either siRNA or isoform-selective inhibitor CAY10526, but not mPGES-2, c-PGES or PU.1, attenuated LPS-induced burst of PGE(2) production indicating that mPGES-1 mediates LPS-induced PGE(2) production in BMDM. Interestingly, selective inhibition of mPGES-1 was also associated with a decrease in LPS-induced iNOS expression. In summary, our data show that mPGES-1, but not mPGES-2 or c-PGES isomerase, mediates LPS-induced late-phase burst of PGE(2) generation, and regulates LPS-induced iNOS expression in BMDM.


Assuntos
Dinoprostona/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Oxirredutases Intramoleculares/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Animais , Ciclo-Oxigenase 1 , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/antagonistas & inibidores , Lipocalinas/genética , Lipocalinas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Cultura Primária de Células , Prostaglandina D2/biossíntese , Prostaglandina-E Sintases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Tiofenos/farmacologia , Transativadores/genética , Transativadores/metabolismo
8.
Tissue Eng Part A ; 16(5): 1633-43, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19995150

RESUMO

This study aimed to investigate the chondrogenic transdifferentiation potential of human dermal fibroblasts (DFs) by stimulation with cartilage-derived morphogenetic protein 1 (CDMP1). Using CDMP1 (100 ng/mL) we induced human DFs at passage 5 in both monolayer and micromass culture. Chondrogenic-specific markers were detected via immunochemistry, reverse transcription-polymerase chain reaction, and Western blot analysis in the collected specimens. The expression profile of adhesion molecules including integrin alpha5, beta1, and N-cadherin of DFs accompanying with chondrogenesis was further investigated. After 7 days of induction in monolayer culture, DFs acquired the polygonal chondrocyte-like shape with positive expression of chondrogenic-specific markers. Such a phenotypic transition of DFs was lost at 14 days. However, in micromass culture the chondrogenic transdifferentiation of DFs can be maintained even at 14 days. No chondrogenesis was detected in DFs without CDMP1 treatment under both culture conditions. By neutralization assay with blocking antibodies, it was further revealed that integrin alpha5 expression was in direct proportion to the degree of chondrogenic differentiation. Based on our findings, it can be ascertained that DFs are capable of transdifferentiating into a chondrogenic lineage by stimulation with CDMP1 in vitro. The integrin alpha5 mediating cell-cell and cell-matrix interactions is required for maintaining the chondrogenic phenotype of DFs.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fator 5 de Diferenciação de Crescimento/farmacologia , Adolescente , Antígenos de Superfície/metabolismo , Moléculas de Adesão Celular/metabolismo , Transdiferenciação Celular/genética , Células Cultivadas , Criança , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/genética , Fibroblastos/metabolismo , Imunofluorescência , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Biomaterials ; 30(19): 3241-50, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19286250

RESUMO

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.


Assuntos
Derme/citologia , Fibroblastos/metabolismo , Fibrocartilagem/fisiologia , Fator 5 de Diferenciação de Crescimento/metabolismo , Ácido Poliglicólico/metabolismo , Engenharia Tecidual/métodos , Agrecanas/genética , Agrecanas/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Proliferação de Células , Forma Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cães , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibrocartilagem/anatomia & histologia , Fibrocartilagem/patologia , Perfilação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Ácido Poliglicólico/química , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Alicerces Teciduais
10.
J Reconstr Microsurg ; 22(4): 277-80, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16783687

RESUMO

Postoperative wound infection is a rare, but major, complication of replantation. Failure to control infection can lead directly to vascular thrombosis and, in turn, to loss of the replanted extremity. The use of maggots for wound debridement has a long history and has been lately re-introduced for treatment of intractable wounds. In this report, the authors present the experience of successful debridement of a severely infected wound after forearm replantation, using maggot therapy. The results and mechanism of maggot therapy are discussed.


Assuntos
Traumatismos do Antebraço/cirurgia , Larva , Reimplante , Infecção da Ferida Cirúrgica/terapia , Adulto , Amputação Traumática/cirurgia , Animais , Desbridamento/métodos , Dípteros , Drenagem , Seguimentos , Fraturas Cominutivas/cirurgia , Humanos , Masculino , Músculo Esquelético/cirurgia , Fraturas do Rádio/cirurgia , Fraturas da Ulna/cirurgia
11.
J Reconstr Microsurg ; 22(1): 15-20, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16425116

RESUMO

Salvage of the complex amputation of extremities, such as combined with devastating segmental injuries, extensive soft tissue defect, and multiple important organ injuries, continues to be a challenge for plastic surgeons. Temporary ectopic implantation of the amputated part to a healthy recipient site allows the patient to recover from critical combined injuries, radical debridements, and soft tissue repair. In this article, the authors report two cases of temporary ectopic implantation of complexly amputated forearms, followed by successful replantation to their anatomic positions at a second stage. The contralateral upper extremity is an acceptable recipient site for temporary ectopic implantation. In secondary replantation, a cross-arm flap can be designed to carry the vascular pedicle from the ectopic implantation recipient to improve blood supply to the replanted part when the second blood supply is established. The authors validated that temporary ectopic implantation of amputated parts provides an alternative procedure for the salvage of amputated extremities under special circumstances.


Assuntos
Amputação Traumática/cirurgia , Traumatismos do Antebraço/cirurgia , Salvamento de Membro/métodos , Reimplante/métodos , Transplante Heterotópico/métodos , Adulto , Feminino , Antebraço/irrigação sanguínea , Antebraço/cirurgia , Humanos , Masculino , Microcirurgia/métodos , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos/irrigação sanguínea , Resultado do Tratamento
12.
Hypertension ; 45(4): 780-5, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15699473

RESUMO

Neuropeptide Y is a potent inhibitory neurotransmitter expressed in the central neurons that control blood pressure. NO also serves as an inhibitory neurotransmitter, and its deficit causes sympathetic overactivity, which then contributes to hypertension. This study tested the hypothesis that neuropeptide Y functions as a central neurotransmitter to lower blood pressure, therefore its increased signaling ameliorates hypertension induced by NO deficiency. Conscious neuropeptide Y transgenic male rats, overexpressing the peptide under its natural promoter, and nontransgenic littermates (controls) were used in this study. Neuropeptide Y, Y1 receptor antagonist BIBP3226, or vehicle (saline) were administered continuously for 14 days into the cerebral lateral ventricle in unrestrained animals using osmotic pumps. Blood pressure was measured by radiotelemetry. Compared with control animals, transgenic overexpression of neuropeptide Y significantly ameliorated (by 9.7+/-1.5 mm Hg) NO deficiency hypertension (induced by administration of N(omega)-nitro-L-arginine methyl ester in the drinking water). This hypotensive effect of neuropeptide Y upregulation was associated with reduced proteinuria and cardiac hypertrophy and fibrosis. Central administration of neuropeptide Y in nontransgenic rats also reduced (by 10.2+/-1.6 mm Hg) the NO deficiency hypertension, whereas a neuropeptide Y1 receptor antagonist centrally administered in the transgenic subjects during NO deficiency hypertension completely attenuated the depressor effect of neuropeptide Y upregulation. Thus, acting at the level of the central nervous system distinctively via a Y1 receptor-mediated mechanism, endogenous neuropeptide Y exerted a potent antihypertensive function, and its enhanced signaling ameliorated NO deficiency hypertension.


Assuntos
Sistema Nervoso Central/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , NG-Nitroarginina Metil Éster , Neuropeptídeo Y/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Transdução de Sinais , Animais , Animais Geneticamente Modificados/genética , Pressão Sanguínea , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Coração/fisiopatologia , Frequência Cardíaca , Masculino , NG-Nitroarginina Metil Éster/administração & dosagem , Neuropeptídeo Y/genética , Proteinúria/fisiopatologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima
13.
Microsurgery ; 25(5): 385-9, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16025438

RESUMO

Two cases of temporary ectopic implantation of a complex amputated foot, followed by replantation to its anatomic position, are reported. Both cases of amputated foot were complicated by devastating soft-tissue injuries in the proximal stump of the amputation, fracture of the femur, and hemorrhagic shock, which ruled out the possibility of primary foot replantation. Both feet were temporarily ectopically implanted onto the contralateral legs, with microvascular anastomoses of the vessels to the recipient posterior tibial artery and saphenous vein. When the patient's general condition allowed, and the soft-tissue defects were repaired, the ectopic implanted feet were replanted to their anatomic positions. Both feet survived the temporary ectopic implantation and second-stage replantation. The length of the injured legs was maintained, and the feet regained their function in 4- and 6-month follow-ups. We conclude that temporary ectopic implantation of amputated parts provides an innovative procedure for the salvage of amputated extremities under special circumstances. A contralateral healthy extremity is an ideal recipient site for temporary ectopic implantation. The temporary ectopic implantation and second-stage replantation of an amputated foot and distal leg with indications can obtain satisfactory results.


Assuntos
Amputação Traumática/cirurgia , Traumatismos da Perna/cirurgia , Salvamento de Membro/métodos , Transplante Heterotópico/métodos , Pé/transplante , Humanos , Extremidade Inferior , Masculino , Microcirurgia/métodos , Procedimentos de Cirurgia Plástica/métodos , Reimplante/métodos , Fatores de Tempo , Transplante Autólogo
14.
J Biol Chem ; 278(47): 47062-9, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12970354

RESUMO

The molecular mechanism underlying the transport of G protein-coupled receptors from the endoplasmic reticulum (ER) to the cell surface is poorly understood. This issue was addressed by determining the role of Rab1, a Ras-related small GTPase that coordinates vesicular protein transport in the early secretory pathway, in the subcellular distribution and function of the angiotensin II type 1A receptor (AT1R), beta2-adrenergic receptor (AR), and alpha2B-AR in HEK293T cells. Inhibition of endogenous Rab1 function by transient expression of dominant-negative Rab1 mutants or Rab1 small interfering RNA (siRNA) induced a marked perinuclear accumulation and a significant reduction in cell-surface expression of AT1R and beta2-AR. The accumulated receptors were colocalized with calregulin (an ER marker) and GM130 (a Golgi marker), consistent with Rab1 function in regulating protein transport from the ER to the Golgi. In contrast, dominant-negative Rab1 mutants and siRNA had no effect on the subcellular distribution of alpha2B-AR. Similarly, expression of dominant-negative Rab1 mutants and siRNA depletion of Rab1 significantly attenuated AT1R-mediated inositol phosphate accumulation and ERK1/2 activation and beta2-AR-mediated ERK1/2 activation, but not alpha2B-AR-stimulated ERK1/2 activation. These data indicate that Rab1 GTPase selectively regulates intracellular trafficking and signaling of G protein-coupled receptors and suggest a novel, as yet undefined pathway for movement of G protein-coupled receptors from the ER to the cell surface.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Linhagem Celular , Complexo de Golgi/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transporte Proteico , Ratos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transfecção , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/fisiologia
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