RESUMEN
Neurofibromas are benign tumors that comprise primarily of Schwann cells and fibroblasts. Mast cells have been found scattered in the tumor tissue, and their role in promoting the proliferation of neurofibroma has been suggested. Tranilast (N-[3,4-dimethoxycinnamolyl]anthranilic acid) is an anti-allergic drug that inhibits release of the chemical mediators from mast cells and it used for the treatment of keloids and hypertrophic scars by its inhibition of growth-promoting transforming growth factor (TGF)-beta(1) from fibroblasts. We assumed that tranilast would suppress neurofibroma cell growth. In order to prove this hypothesis, we investigated the effectiveness of tranilast in inhibiting the tumor growth using a new cell culture system obtained from patients with neurofibromas. We called this culture system with the mixture of Schwann cells and fibroblasts "NF1 cells culture". Mast cells were differentiated from CD34(+) peripheral blood mononuclear cells of normal healthy subjects, and were co-cultured with NF1 cells. Three days after tranilast (10 approximately 100 microM) added to the culture dishes, we counted viable cell numbers and measured the concentrations of TGF-beta(1), stem cell factor (SCF) and tryptase, which exists in the histamine granule, in the culture medium. Tranilast significantly suppressed proliferation of the NF1 cells and lowered the levels of TGF-beta(1), SCF and tryptase. These results suggest that tranilast retards tumor proliferation through not only suppression of cell growth factor, but also the inhibition of a chemical mediator released from mast cells. Thus, tranilast can be a potent therapeutic agent to inhibit the growth of neurofibromas.
Asunto(s)
Antialérgicos/farmacología , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Neurofibromatosis 1/fisiopatología , ortoaminobenzoatos/farmacología , Células Cultivadas , Humanos , Mastocitos/citología , Neurofibromatosis 1/tratamiento farmacológico , Factor de Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , TriptasasRESUMEN
In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.
Asunto(s)
Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación , Secuencia de Bases , Western Blotting , Catepsinas/antagonistas & inhibidores , Cisteína Endopeptidasas , Cartilla de ADN , Drosophila/metabolismo , Escherichia coli , Humanos , Hibridación in Situ , Hígado/metabolismo , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Linfocitos T CitotóxicosRESUMEN
Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium glutamicum/enzimología , Fumarato Hidratasa/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/aislamiento & purificación , Benzoatos/química , Estabilidad de Enzimas , Fumarato Hidratasa/antagonistas & inhibidores , Fumarato Hidratasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Peso Molecular , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Especificidad por Sustrato , Compuestos de Sulfhidrilo/química , Tartratos/químicaRESUMEN
We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).
Asunto(s)
Proteasas ATP-Dependientes/genética , Corteza Suprarrenal/enzimología , Proteasas ATP-Dependientes/química , Proteasas ATP-Dependientes/clasificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario/genética , Humanos , Espectrometría de Masas , Mitocondrias/enzimología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Homología de Secuencia de AminoácidoRESUMEN
In the field of dermatology and plastic and reconstructive surgery, fibrin gel is regarded as a material that promotes wound healing. To test the hypothesis that fibrin may promote the growth of the epidermis, we examined its effects on the proliferation of cultured keratinocytes. Human keratinocytes were cultivated in fibrin-coated wells, and the cell numbers and transforming growth factor (TGF)-alpha, secreted into the cultured medium, were measured. We also assessed the capacity of epidermal growth factor receptor (EGF-R) that is responsible for all known actions of TGF-alpha and epidermal growth factor. The keratinocytes increased dramatically in their number, and the TGF-alpha secretion and the binding capacity of EGF-R were also increased dramatically in the presence of fibrin. These findings suggest that fibrin supports the proliferation of keratinocytes in an autocrine fashion via EGF-R; namely, fibrin stimulates keratinocytes to secrete TGF-alpha, which in turn increases cell proliferation and EGF-R capacity. We propose that fibrin can support the wound healing process of the epidermis via the TGF-alpha/EGF-R pathway.
Asunto(s)
Receptores ErbB/metabolismo , Fibrina/fisiología , Queratinocitos/citología , Factor de Crecimiento Transformador alfa/metabolismo , Células 3T3 , Animales , Sitios de Unión , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo/metabolismo , Citocinas/biosíntesis , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Epidermis/metabolismo , Fibrina/química , Fibrina/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , Unión Proteica , Factores de Tiempo , Cicatrización de HeridasRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by microcephaly, syndactyly of toes, ambiguous genitalia, and mental retardation. The underlying DHCR7 gene has been identified and a wide variety of distinct mutations were reported in USA and European SLOS patients. A significant difference has been suggested in the frequency of SLOS among different ethnic populations. Here, we report mutational analysis of seven Japanese SLOS patients. Five mutations, R352Q, R242H, G303R, X476Q, and S192F, were identified, and R352Q appeared most frequent, since nine out of the 13 mutations of Japanese origin were the same R352Q. These results suggest that R352Q is a predominant founder mutation in Japanese SLOS patients.
Asunto(s)
Mutación Missense/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Síndrome de Smith-Lemli-Opitz/epidemiología , Síndrome de Smith-Lemli-Opitz/genética , Línea Celular , Colesterol/sangre , Análisis Mutacional de ADN , Cartilla de ADN , Cromatografía de Gases y Espectrometría de Masas , Humanos , Japón/epidemiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
In cytological examination, human cytomegalovirus (HCMV) infection can not be implied unless typical HCMV-infected cells like owl's-eye cells are present. However, such cells are not always observed in HCMV-infection cases. The aim of our study is to establish the cytopathological features induced by HCMV. In vitro transfection and fluorescence in situ hybridization (FISH) were performed on human embryo lung (HEL) cells. Marked cellular aggregation was observed at 6-hr postinfection (hpi). Multinucleated cells, giant cells, and, particularly, small vacuoles were present in the nuclei or cytoplasm before the appearance of inclusion bodies. However, molding and ground glass in nuclei were absent. Cell clusters displayed round cytoplasm, dispersed later, and showed anisocytosis. All features occurred before 48 hpi when the owl's-eye cell appeared. In FISH, the positive signal highlighted viral particles that became predominant and localized in nuclei. These cytological aspects are dependent on viral replication and contribute to the cytological detection of HCMV infection.
Asunto(s)
Citodiagnóstico/métodos , Infecciones por Citomegalovirus/patología , Citomegalovirus/fisiología , Pulmón/patología , Agregación Celular , Células Cultivadas , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , ADN Viral/análisis , Células Gigantes/patología , Células Gigantes/virología , Humanos , Hibridación Fluorescente in Situ , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/virología , Pulmón/embriología , Pulmón/virología , Microscopía Fluorescente , Factores de Tiempo , TransfecciónRESUMEN
Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.