RESUMO
BACKGROUND AND AIMS: Advanced glycation end products (AGEs)-receptor RAGE interaction evokes oxidative stress and inflammatory reactions, thereby being involved in endothelial cell (EC) damage in diabetes. Sulforaphane is generated from glucoraphanin, a naturally occurring isothiocyanate found in widely consumed cruciferous vegetables, by myrosinase. Sulforaphane has been reported to protect against oxidative stress-mediated cell and tissue injury. However, effects of sulforaphane on AGEs-induced vascular damage remain unclear. METHODS AND RESULTS: In this study, we investigated whether and how sulforaphane could inhibit inflammation in AGEs-exposed human umbilical vein ECs (HUVECs) and AGEs-injected rat aorta. Sulforaphane treatment for 4 or 24 h dose-dependently inhibited the AGEs-induced increase in RAGE, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecular-1 (VCAM-1) gene expression in HUVECs. AGEs significantly stimulated MCP-1 production by, and THP-1 cell adhesion to, HUVECs, both of which were prevented by 1.6 µM sulforaphane. Sulforaphane significantly suppressed oxidative stress generation and NADPH oxidase activation evoked by AGEs in HUVECs. Furthermore, aortic RAGE, ICAM-1 and VCAM-1 expression in AGEs-injected rats were increased, which were suppressed by simultaneous infusion of sulforaphane. CONCLUSION: The present study demonstrated for the first time that sulforaphane could inhibit inflammation in AGEs-exposed HUVECs and AGEs-infused rat aorta partly by suppressing RAGE expression through its anti-oxidative properties. Inhibition of the AGEs-RAGE axis by sulforaphane might be a novel therapeutic target for vascular injury in diabetes.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Aortite/prevenção & controle , Células Endoteliais/efeitos dos fármacos , Produtos Finais de Glicação Avançada , Isotiocianatos/farmacologia , Animais , Aorta/metabolismo , Aortite/induzido quimicamente , Aortite/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Receptor para Produtos Finais de Glicação Avançada/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Sulfóxidos , Fatores de TempoRESUMO
Advanced glycation end products (AGEs) and receptor RAGE play a role in diabetic nephropathy. We have previously shown that increased glucose uptake into proximal tubular cells via sodium-glucose cotransporter 2 (SGLT2) stimulates oxidative stress generation and RAGE expression, thereby exacerbating the AGE-induced apoptosis in this cell type. However, the protective role of SGLT2 inhibition against the AGE-RAGE-induced renal damage in diabetic animals remains unclear. In this study, we investigated the effects of empagliflozin, SGLT2 inhibitor on AGE-RAGE axis, inflammatory and fibrotic reactions, and tubular injury in the kidney of streptozotocin-induced diabetic rats.Administration of empagliflozin for 4 weeks significantly improved hyperglycemia and HbA1c, and decreased expression levels of AGEs, RAGE, 8-hydroxydeoxyguanosine (8-OHdG), and F4/80, markers of oxidative stress and macrophages, respectively, in the diabetic kidney. Although empagliflozin did not reduce albuminuria, it significantly decreased urinary excretion levels of 8-OHdG and L-fatty acid binding protein, a marker of tubular injury. Moreover, inflammatory and fibrotic gene expression such as monocyte chemoattractant protein-1, intercellular adhesion molecule-1, plasminogen activator inhibitor-1, transforming growth factor-ß, and connective tissue growth factor was enhanced in the diabetic kidney, all of which were prevented by empagliflozin. The present study suggests that empagliflozin could inhibit oxidative, inflammatory and fibrotic reactions in the kidney of diabetic rats partly via suppression of the AGE-RAGE axis. Blockade of the increased glucose uptake into renal proximal tubular cells by empagliflozin might be a novel therapeutic target for tubulointerstitial damage in diabetic nephropathy.
Assuntos
Compostos Benzidrílicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Fibromialgia/prevenção & controle , Glucosídeos/farmacologia , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Inflamação/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Nefropatias Diabéticas/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Advanced glycation end products (AGEs) decrease adiponectin expression and suppress insulin signaling in cultured adipocytes through the interaction with a receptor for AGEs (RAGE) via oxidative stress generation. We have recently found that high-affinity DNA aptamer directed against AGE (AGE-aptamer) prevents the progression of experimental diabetic nephropathy by blocking the harmful actions of AGEs in the kidney. This study examined the effects of AGE-aptamer on adipocyte remodeling, AGE-RAGE-oxidative stress axis, and adiponectin expression in fructose-fed rats. Although AGE-aptamer treatment by an osmotic mini pump for 8 weeks did not affect serum insulin levels, it significantly decreased average fasting blood glucose and had a tendency to inhibit body weight gain in fructose-fed rats. Furthermore, AGE-aptamer significantly suppressed the increase in adipocyte size and prevented the elevation in AGEs, RAGE, and an oxidative stress marker, 8-hydroxydeoxyguanosine (8-OHdG), levels in adipose tissues of fructose-fed rats at 14-week-old, while it restored the decrease in adiponectin mRNA levels. Our present study suggests that AGE-aptamer could improve glycemic control and prevent adipocyte remodeling in fructose-fed rats partly by suppressing the AGE-RAGE-mediated oxidative stress generation. AGE-aptamer might be a novel therapeutic strategy for fructose-induced metabolic derangements.
Assuntos
Adipócitos/patologia , Aptâmeros de Nucleotídeos/administração & dosagem , Glicemia/análise , Frutose/administração & dosagem , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adiponectina/genética , Animais , Tamanho Celular/efeitos dos fármacos , Dieta , Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/genética , Resistência à Insulina , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Aumento de Peso/efeitos dos fármacosRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, a gut hormone secreted from K cells in the intestine in response to food intake. It could be a potential therapeutic target for the treatment of patients with type 2 diabetes. However, effects of GIP on vascular injury remain unknown. Since interaction of advanced glycation end products (AGEs) with their receptor RAGE has been shown to play a crucial role in vascular damage in diabetes, this study investigated whether and how GIP blocked the deleterious effects of AGEs on human umbilical vein endothelial cells (HUVECs). GIP receptor was expressed in HUVECs. GIP, an analogue of cyclic AMP or inhibitors of NADPH oxidase inhibited the AGE-induced reactive oxygen species (ROS) generation in HUVECs. Furthermore, GIP reduced both RAGE mRNA and protein levels in HUVECs. GLP-1 also blocked the AGE-induced increase in mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 in HUVECs. In addition, an antioxidant N-acetylcysteine mimicked the effects of GIP on RAGE and VCAM-1 gene expression in HUVECs. Our present study suggests that GIP could block the signal pathways of AGEs in HUVECs by reducing ROS generation and subsequent RAGE expression probably via GIP receptor-cyclic AMP axis.
Assuntos
Antioxidantes/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Ehrlich ascites cells were cultured with 1-O-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) or 1-O-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC) to reveal the selective retention of polyunsaturated fatty acids at second position of ether-containing phospholipids. Although small percentages of the lysophospholipids were degraded into long-chain alcohol, both alkyllyso-GPE and -GPC were acylated at the rate of approximately 2 nmol/30 min per 10(7) cells. Alkylacylacetylglycerols were prepared from the acylated products by phospholipase C treatment, acetylation and TLC, and fractionated according to the degree of unsaturation by AgNO3-TLC. The distribution of the radioactivity among the subfractions indicated that both alkyllysophospholipids were mainly esterified by docosahexaenoic acid and to a somewhat lesser extent by arachidonic acid. The selectivity for docosahexaenoic acid in the esterification of 1-alkyl-GPE was much stronger than in that of 1-alkyl-GPC. Although acyl-CoA: 1-alkyl-glycerophosphoethanolamine acyltransferase activity of Ehrlich cell microsomes with arachidonoyl-CoA and docosahexaenoyl-CoA as acyl donors was negligible compared with the acyl-CoA:1-alkyl-glycerophosphocholine acyltransferase activity, a significant amount of 1-alkyl-GPE was acylated in the microsomes without exogenously added acyl-CoA. HPLC analysis revealed that docosahexaenoic acid and arachidonic acid were mainly esterified by the microsomal transferase. Acylation of 1-alkyl-GPC with docosahexaenoic acid and arachidonic acid was also observed in the absence of added acyl-CoA, but the activity was lower than that for 1-alkyl-GPE. Although the source of the acyl donor in the acylation has not been determined, the acylation is probably due to the direct transfer of acyl groups between intact phospholipids. The above results provided the first evidence that the lysophospholipid acyltransferase system including the transacylase activity participates in the selective retention of docosahexaenoic acid in intact cells and a cell free system.
Assuntos
Carcinoma de Ehrlich/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/metabolismo , Acilação , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ácidos Docosa-Hexaenoicos , Lisofosfolipídeos , Masculino , Camundongos , Microssomos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fatores de TempoRESUMO
In order to characterize nephrotoxic serum nephritis accelerated with rabbit gamma-globulin in mice, histopathological studies were carried out 15 days after NTS injection, the time when increases in urinary protein and serum cholesterol and a decrease in serum albumin were apparent. Characteristic changes were widespread thickening of glomerular capillary walls and widening of mesangial areas, owing to deposits of mesangial matrixlike substances. The mesangial interposition into subendothelial areas and the resultant narrowing of the capillary lumen were shown ultrastructurally. In severely affected glomeruli, a hyaline nodular lesion was observed. Visceral epithelial cells demonstrated fusion of the foot processes, microvilli formation, occasional proliferation, and enlargement. Parietal epithelial cells proliferated, forming a cellular crescent. Based on these characteristics, it appears this nephritic model shares a common pathology with human membranoproliferative glomerulonephritis type 1 and crescentic glomerulonephritis and can be considered an appropriate model for producing severe nephritis for short periods.
Assuntos
Glomerulonefrite/patologia , Animais , Nitrogênio da Ureia Sanguínea , Colesterol/sangue , Glomerulonefrite/fisiopatologia , Imunização Passiva , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Proteinúria/metabolismo , Albumina Sérica/metabolismo , gama-Globulinas/imunologiaRESUMO
Lobular carcinoma of the male breast is very rare, because lobules do not exist in the male mammary gland. Seven cases of lobular carcinoma of the male breast have been reported in Europe and U.S.A., although no case in Japan. We encountered a very rare case of the lobular carcinoma of 74-year-old male breast. Histopathological examinations of both primary tumor and recurrent tumors of the skin led to the diagnosis of lobular carcinoma.
Assuntos
Neoplasias da Mama/cirurgia , Carcinoma/cirurgia , Idoso , Neoplasias da Mama/patologia , Carcinoma/secundário , Humanos , Masculino , Neoplasias Cutâneas/secundário , Neoplasias Cutâneas/cirurgiaRESUMO
BACKGROUND AND OBJECTIVES: The growth factors derived from platelets and plasma proteins mediate the wound-healing process that is characterized by the sequential migration and differentiation of several cell populations that give rise to angiogenesis, collagen synthesis, wound contraction, and re-epithelialization. To evaluate the efficacy of the blood-derived factors in wound healing, we examined a novel wound dressing consisting of concentrated human plasma proteins and platelet releasate (CPPP). MATERIALS AND METHODS: To generate CPPP, plasma proteins and platelets in the peripheral blood (n = 5) were concentrated with the cold ethanol precipitation method. The thrombin obtained from the same blood unit and calcium chloride (CaCl(2)) were mixed to a concentrate. The CPPP has enough strength to dress cutaneous wounds and contains large amounts of cytokines and fibronectin. We applied the CPPP to excisional skin wounds in genetically healing-impaired model mice (n= 5) and the wounds were evaluated 10 days after the operation. RESULTS: The area of CPPP-treated wounds decreased significantly compared with that of the control wounds (65% vs. 94% of the original size, respectively, P= 0.032). The immunostained section revealed a striking effect of CPPP on vascularization compared with the control wounds (13.2 vs. 2.7 vessels per mm(2) as mean vascular density observed in the sections, respectively, P= 0.013). CONCLUSIONS: Our results suggest that CPPP is a promising biologically active dressing for full-thickness skin wounds. CPPP can be an entirely autologous biological dressing, suggesting that it is free from the risk of transmission of pathogens through blood products.
Assuntos
Curativos Biológicos , Plaquetas , Proteínas Sanguíneas/uso terapêutico , Extratos Celulares/uso terapêutico , Complicações do Diabetes/terapia , Diabetes Mellitus Tipo 2/terapia , Membranas Artificiais , Dermatopatias/terapia , Pele/lesões , Animais , Plaquetas/química , Extratos Celulares/química , Complicações do Diabetes/patologia , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Elasticidade , Humanos , Camundongos , Pele/patologia , Dermatopatias/patologia , Cicatrização , Ferimentos e Lesões/patologia , Ferimentos e Lesões/terapiaRESUMO
Various kinds of epithelial and non-epithelial tumors, and tumor-like lesions spontaneously developed in long-lived mice of strains A/St, C57BL/6 and CBA, and of (C57BL/6 X CBA) F1 hybrids, all of which had been fed in the 2nd Department of Pathology, Gifu University School of Medicine. Some tumors were successfully transplanted into the same strain of mice. An interesting tumor line showing phagocytic activity and growing only in the liver even when inoculated subcutaneously, which was obtained from a reticulum cell neoplasm, Type A by Dunn, originated in the liver of a (C57BL/6 X CBA) F1 hybrid male mouse.
Assuntos
Envelhecimento , Camundongos Endogâmicos , Neoplasias/veterinária , Doenças dos Roedores/epidemiologia , Animais , Feminino , Hibridização Genética , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Transplante de Neoplasias , Neoplasias/epidemiologia , Neoplasias/patologia , Doenças dos Roedores/patologiaRESUMO
A 20-year-old woman had the complaints of a tumor in the lower abdominal region and irregular menstruation. A spherical tumor measuring 7 X 6 X 3.5 cm in the great dimension was removed from the left ovary. The cut surface showed a lobular pattern and cystic degeneration in some areas. Histological examination revealed edematous areas and highly cellular areas. The cells were variable in size and shape. Round cells resembled signet-ring cells in places. By PAS reaction and alcian blue stain, these cells were found to be negative, but by Sudan III stain, positive. Postoperatively, the patient complained of genital bleeding for five days. Subsequently, her menstrual pattern returned to normal.
Assuntos
Neoplasias Ovarianas/patologia , Adulto , Cistos/patologia , Feminino , Humanos , Ovário/patologiaRESUMO
A transplantable osteogenic sarcoma originally arising in the right femur of an AKR/Ms male mouse is described. The original tumor showed a conspicuous bone and cartilage formation, but the capability of forming the bone was lost in the 4th transplant generation and that of cartilage formation was lost in the 7th generation. Alkaline phosphatase activity was histochemically demonstrated in only a few tumor cells, and the activity did not rise in the host serum. Intracisternal type-A particles of an average diameter of 70 nm were abundant in the rough endoplasmic reticulum, while type-C particles were rarely found to be budding from the cell surface or free in the extracellular spaces by electron microscopy. Three of the 27 thymectomized AKR mice that had been neonatally injected with cell-free material of the transplanted tumor developed osteomas, but no osteogenic sarcoma was found.
Assuntos
Neoplasias Femorais/veterinária , Camundongos Endogâmicos AKR , Osteossarcoma/veterinária , Doenças dos Roedores/patologia , Fosfatase Alcalina/sangue , Animais , Retículo Endoplasmático/ultraestrutura , Neoplasias Femorais/ultraestrutura , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais , Ossificação Heterotópica , Osteoma/patologia , Osteossarcoma/ultraestrutura , Retroviridae/ultraestrutura , Timectomia , Transplante HomólogoRESUMO
Characteristic pulmonary findings in an autopsy case of a 46-year-old female who presented heavy dyspnea as her chief complaint after 3 months of busulfan therapy for chronic myeloid leukemia were reported. The pulmonary findings were classified into four types: I. alveolar proteinosis type, II. intra alveolar fibrosis type, III. interstitial fibrosis type, and IV. lipid pneumonia type with cholesterol granuloma. No other case with various findings like this case has been previously reported. It was considered that type I is the basic type, type II is a type that developed from type I, type III is a type with interstitial cell infiltration and fibrosis and type IV is a lipidrich variant of type I. A large lamellar body was first found in the granular material of type I. It is supposed that such a body consists of osmiophilic body which originated from type B alveolar epithelial cells and blood plasma.
Assuntos
Bussulfano/efeitos adversos , Pneumopatias/induzido quimicamente , Alvéolos Pulmonares/patologia , Feminino , Humanos , Leucemia Mieloide/tratamento farmacológico , Pneumopatias/patologia , Pessoa de Meia-IdadeRESUMO
A male case of Alagille's syndrome associated with a hamartomatous nodule of the liver is described. The patient developed jaundice soon after birth, and was diagnosed as the syndrome with signs such as paucity of the intrahepatic bile ducts, pulmonary stenosis and embryotoxon in the cornea at 15 years of age. The liver was examined in recurrent biopsies and other tests. However, no evidence of liver cirrhosis was confirmed until his 15th year. The patient died of hepatic dysfunction when he was 17 years old. At autopsy, a large hamartomatous nodule was found in the liver showing biliary cirrhosis. Morphology of the nodule resembled that of focal nodular hyperplasia. Abnormalities of the large vessels were noted around the liver. Vascular abnormalities were also seen in the mass. The relation of these vascular abnormalities to etiological background of the syndrome and occurrence of the nodular lesion is discussed.
Assuntos
Ductos Biliares Intra-Hepáticos/anormalidades , Hamartoma/patologia , Neoplasias Hepáticas/patologia , Adolescente , Autopsia , Face/anormalidades , Humanos , Cirrose Hepática Biliar/patologia , Masculino , Estenose da Valva Pulmonar , SíndromeRESUMO
Although there are many articles describing the detection of complement components in formalin-fixed and paraffin-embedded renal tissues, it is still difficult to obtain reproducible results. The authors determined the optimal conditions for detecting complement components in routine paraffin sections by the avidin-biotin-peroxidase complex method and concluded that proteolytic digestion of the deparaffinized sections was crucial to detect complement and to preserve tissue structures. The optimal proteolytic digestion was a 15-minute incubation at 37 degrees C in trypsin solution, 0.5 mg/ml, pH 7.6. Under these conditions, all of the complement components so far studied (C1q, C1s, C4, C3c, C3b, C5, C6, C9, and properdin) were detected without significant destruction of tissue structures in 52 renal biopsy specimens from patients with various forms of glomerulonephritis. Immunohistochemistry using the avidin-biotin-peroxidase complex method was equivalent to indirect immunofluorescence and superior to direct immunofluorescence in utility and was useful in the diagnosis of glomerular diseases.
Assuntos
Proteínas do Sistema Complemento/análise , Glomerulonefrite/imunologia , Rim/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Imunofluorescência , Glomerulonefrite por IGA/imunologia , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranosa/imunologia , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Valor Preditivo dos TestesRESUMO
The mechanism involved in the enzymic acylation of 1-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) in brain microsomes was investigated in comparison with the acylation of 1-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC). Both the alkyllsophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1-stearoyl-2-[14C]arachidonoyl-GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1-[3H]alkyl-GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1-[3H]alkyl-GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1-[3H]alkyl-GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1-[3H]alkyl-GPC by both docosahexaenoate and arachidonate increased 1.5-2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1-[3H]alkyl-GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aciltransferases/metabolismo , Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Ácidos Graxos Insaturados/metabolismo , Fosfatidiletanolaminas/metabolismo , Acilação , Animais , Ácido Araquidônico , Encéfalo/enzimologia , Ácidos Docosa-Hexaenoicos , Masculino , Microssomos/metabolismo , Ratos , Ratos EndogâmicosRESUMO
An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the interspaces of cell clusters of carrot (Daucus carota L.) callus, as well as in the extracellular spaces of carrot seeds, being located both in the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites for N-linked glycosylation or hydrophobic regions. Since its sequence was found to be homologous to that of inhibitors of cysteine proteinases, namely cystatins, EIP18 was renamed EICC (extracellular insoluble cystatin of carrot). EICC expressed in yeast was also found in an insoluble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteinase extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its transcript was markedly enhanced upon treatment of somatic embryos with abscisic acid.