RESUMO
It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.
Assuntos
Núcleosídeo-Fosfato Quinase/isolamento & purificação , Fosfotransferases/isolamento & purificação , Placenta/enzimologia , Timidina Quinase/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Fracionamento Celular , Cromatografia de Afinidade , Citosol/enzimologia , Humanos , Mitocôndrias/enzimologia , Peso Molecular , Nucleotídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Heat-shock protein 47 (HSP47) is a collagen-binding stress protein that is thought to act as a collagen-specific molecular chaperon during the biosynthesis and secretion of procollagen. In this study we examined the expression of HSP47 mRNA and protein in systemic sclerosis (SSc) skin fibroblasts. HSP47 mRNA and protein levels were significantly higher in fibroblast cultures from SSc patient-involved skin samples than in fibroblasts from normal skin from healthy individuals, as assessed by northern blot and immunoblot analyses, respectively. SSc cultured fibroblasts with increased levels of HSP47 mRNA and protein showed high expression of type I procollagen. By in situ hybridization, SSc skin had a higher number of fibroblasts with high HSP47 and procollagen alpha1(I) mRNA levels than normal skin, and the distribution of HSP47 mRNA was similar to that of procollagen alpha1(I) mRNA. We also investigated the effects of cytokines on the expression of HSP47 in normal cultured fibroblasts. Transforming growth factor-beta1 and interleukin-4 increased HSP47 mRNA and protein levels, whereas interferon-gamma reduced HSP47 expression. The same pattern of cytokine-regulated expression was observed for type I procollagen levels. These results indicate that HSP47 expression is closely associated with that of type I procollagen in skin fibroblasts, and that increased expression of HSP47 may be involved in the abundant production of type I procollagen by SSc fibroblasts.
Assuntos
Fibroblastos/química , Fibroblastos/metabolismo , Proteínas de Choque Térmico/biossíntese , Pró-Colágeno/biossíntese , Escleroderma Sistêmico/metabolismo , Pele/citologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP47 , Proteínas de Choque Térmico/genética , Humanos , Immunoblotting , Hibridização In Situ , Interferon gama/farmacologia , Interleucina-4/farmacologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Psoralen plus UVA (320-400 nm radiation; PUVA) is a highly effective therapy for cutaneous diseases caused by skin infiltration with normal or neoplastic T-lymphocytes. In comparing the effects of pharmacologically relevant, low-dose PUVA treatment on growth of human keratinocytes, peripheral blood leukocytes (PBMC), and T-lymphocyte cell lines, we determined that PBMC or T-lymphocytes were > 50-fold more sensitive to cytotoxic effects of PUVA, while antiproliferative effects were produced by similar PUVA levels in all cell types. Low doses of PUVA (10 ng/mL 8-methoxypsoralen and 1-2 J/cm2) were highly cytotoxic for phytohemagglutinin-activated normal lymphocytes or transformed T-lymphocytes as assessed by two viability assays and by flow cytofluorometry. Altered lymphocyte morphology, nuclear fragmentation, TUNEL+ nuclei or nuclear fragments, and the appearance of a sub-G1 DNA peak indicated that cell death occurred by apoptosis, beginning about 1 day after PUVA treatment and continuing for several days thereafter. From assessment of cell cycle progression in mimosine-synchronized cells, PUVA treatment markedly slowed cell cycle progression, eventually producing cell cycle arrest and apoptotic entry. We propose that the probable basis for disease remissions (psoriasis, cutaneous T-cell lymphoma) produced by PUVA treatment is through selective cytotoxic effects on clonal T-lymphocyte populations that are concentrated in diseased skin.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Metoxaleno/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Linfócitos T/efeitos dos fármacos , Raios Ultravioleta , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Linfócitos T/citologia , Linfócitos T/efeitos da radiaçãoRESUMO
The myxomatous materials in cutis from a patient with pretibial myxedema (PTM) with Graves' disease were found to be mainly hyaluronic acid (HA), which had accumulated extensively in the upper dermis. Fibroblasts were increased in number in the mid to lower dermis. To clarify the mechanism of the deposition of HA in the dermis, we employed an antibody against serum-derived hyaluronan-associated protein (SHAP). This immunohistochemical study disclosed that the greater part of the fibroblasts in the mid to lower dermis stained positively, while the fibroblasts surrounded by fairly large amounts of HA in the upper dermis stained faintly or not at all. From these results, we suspect that the accumulation of HA progresses from the upper to the low dermis and that the interaction of fibroblasts and SHAP leads to development of the physiophathological cutaneous changes seen in our case.
Assuntos
Doença de Graves/imunologia , Receptores de Hialuronatos/imunologia , Dermatoses da Perna/imunologia , Mixedema/imunologia , Fibroblastos/química , Fibroblastos/patologia , Doença de Graves/complicações , Humanos , Ácido Hialurônico/análise , Técnicas Imunoenzimáticas , Dermatoses da Perna/complicações , Dermatoses da Perna/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mixedema/complicações , Mixedema/fisiopatologia , Pele/química , Pele/patologiaRESUMO
The immunohistochemical localization of metallothioneins (MTs), low-molecular-weight metal-binding proteins, was investigated in a case of annular lichen planus (LP) that enlarged centrifugally with healing in the centre after a month. The annular lesion was found to exhibit the early, developed and late stages of LP, as judged by the clinical and histopathological findings. Immunostaining for MTs was increased around the lesion, discontinuous around the rim of the erythema and undetectable in the centre of the lesion. MT expression was examined in eight specimens of idiopathic LP. Discontinuous immunostaining for MTs was observed in six specimens and increased staining around the lesion was observed in two specimens. These results suggest that MT expression changes depending on the inflammatory stage of LP. Although the role of MTs in dermatological disease is still unknown, their expression might be related to the pathological process of LP.
Assuntos
Líquen Plano/metabolismo , Metalotioneína/metabolismo , Doenças do Pênis/metabolismo , Idoso , Humanos , Imuno-Histoquímica , Líquen Plano/patologia , Masculino , Doenças do Pênis/patologiaRESUMO
The gene expression of matrix metalloproteinase-1 and -3 was examined in basal cell carcinomas by in situ hybridization using digoxigenin-labelled riboprobes. Nodulo-ulcerative basal cell carcinomas demonstrated the gene expression for both metalloproteinases but superficial basal cell carcinomas did not present any transcripts for them. Transcripts for matrix metalloproteinase-1 (interstitial collagenase) were demonstrated densely in stromal cells among tumour masses, and those for matrix metalloproteinase-3 (stromelysin-1) were detected only in more advanced cases. Neither were expressed in tumour cells. The two metalloproteinases were produced by stromal cells according to the tumour invasion process, in which various growth factors, cytokines and inflammatory factors, which could regulate gene expressions of matrix metalloproteinases, were involved. It was also found that hybridization signals were enhanced by treatment with chondroitin ABC lyase, which digested abundant glycosaminoglycans in basal cell carcinoma. The procedure for the digestion is simple, and appears to be of value for in situ hybridization studies on tissues containing large amounts of glycosaminoglycans.
Assuntos
Carcinoma Basocelular/enzimologia , Condroitina Liases/metabolismo , Colagenases/genética , Metaloproteinase 3 da Matriz/genética , Neoplasias Cutâneas/enzimologia , Carcinoma Basocelular/patologia , Colagenases/metabolismo , Digoxigenina/química , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ , Metaloproteinase 1 da Matriz , Metaloproteinase 3 da Matriz/metabolismo , Invasividade Neoplásica , Sondas RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Cutâneas/patologia , Células Estromais/citologia , Células Estromais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Matrix metalloproteinase (MMP) plays an important role in extracellular matrix degradation associated with cancer invasion. An expression of MMP-1 (interstitial collagenase), MMP-2 (72-kDa type IV collagenase) and MMP-3 (stromelysin-1) was investigated in squamous cell carcinoma (SCC) and its precancerous condition, actinic keratosis (AK), using in situ hybridization techniques. MMP-1 mRNA was detected in tumour cells and/or in stromal cells in all cases of SCC, four of six AKs adjacent to SCC and four of 16 AKs. MMP-2 and MMP-3 mRNAs were detected in SCC but not in AK. The expression of MMP-3 correlated to that of MMP-1 (P = 0.03) localized at the tumour mass and stroma of the invasive area, while MMP-2 mRNA was detected widely throughout the stroma independent of MMP-1 expression. Our results indicated that the expression of MMP-1, -2 and -3 showed different localization patterns, suggesting a unique role of each MMP in tumour progression. Moreover, MMP-1 expression could be an early event in the development of SCC, and AK demonstrating MMP-1 mRNA, might be in a more advanced dysplastic state, progressing to SCC.