RESUMO
AIM: Microsomal triglyceride transfer protein (MTP) takes part in the mobilization and secretion of triglyceride-rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT-130, a novel intestine-specific MTP inhibitor, on high fat diet-induced obesity and glucose intolerance. METHODS: Male Sprague-Dawley rats were fed a 3.1% fat diet or a 35% fat diet with or without JTT-130 as a food admixture (0.029%). Food intake, body weight, abdominal fat, hepatic triglyceride, faecal free fatty acids and plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were assessed. Plasma levels of glucose and insulin were measured during intraperitoneal glucose tolerance tests. In addition, indirect calorimetry was performed on rats fed with a 35% fat diet. RESULTS: JTT-130 treatment decreased body weights, abdominal fat and hepatic triglyceride with suppression of food intake and elevation of faecal free fatty acids and plasma GLP-1 and PYY levels in rats fed with the 35% fat diet, whereas no significant effects on these parameters except for increased faecal free fatty acids were observed in rats fed with the 3.1% fat diet. JTT-130 treatment decreased plasma levels of glucose and insulin during intraperitoneal glucose tolerance tests on rats fed with the 35% fat diet, but not on rats fed with the 3.1% fat diet. JTT-130-treated rats showed increased O(2) consumption and CO(2) production on a 35% fat diet. CONCLUSIONS: JTT-130 suppresses high fat diet-induced obesity and glucose intolerance with suppression of food intake and fat absorption and could be useful for prevention and treatment of obesity and obesity-related insulin resistance.
Assuntos
Benzamidas/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Intolerância à Glucose/tratamento farmacológico , Hipoglicemiantes/farmacologia , Malonatos/farmacologia , Obesidade/tratamento farmacológico , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Gorduras na Dieta/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Ácidos Graxos/metabolismo , Fezes/química , Peptídeo 1 Semelhante ao Glucagon/sangue , Intolerância à Glucose/prevenção & controle , Teste de Tolerância a Glucose , Insulina/sangue , Resistência à Insulina , Fígado/metabolismo , Masculino , Obesidade/induzido quimicamente , Obesidade/prevenção & controle , Peptídeo YY/sangue , Ratos , Ratos Sprague-Dawley , Triglicerídeos/metabolismoRESUMO
AIM: Microsomal triglyceride transfer protein (MTP) takes part in the mobilization of triglyceride-rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT-130, a novel intestine-specific MTP inhibitor, on impaired glucose and lipid metabolism in Zucker diabetic fatty (ZDF) rats. METHODS: Male ZDF rats were fed a regular powdered diet with or without JTT-130 as a food admixture (0.01-0.02%) for 6 weeks. Food intake, body weight, blood biochemical parameters, fecal lipid contents, hepatic lipid contents, tissue mRNA levels and glucose utilization in adipose tissues were assessed. An intraperitoneal glucose tolerance test (IPGTT) and histological analysis of the pancreas were performed. RESULTS: JTT-130 treatment decreased food intake, glycated hemoglobin, plasma levels of glucose, triglycerides and total cholesterol, hepatic levels of triglycerides and cholesterol and hepatic mRNA levels of glucose-6-phosphatase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase. JTT-130 treatment increased fecal levels of free fatty acids and cholesterol, plasma levels of glucagon-like peptide-1 and peptide YY, mRNA levels of glucose transporter 4 (GLUT4) and lipoprotein lipase in adipose tissues and GLUT4 in muscle and glucose utilization in adipose tissues. Plasma insulin decreased after 2 weeks and increased after 4 weeks of JTT-130 treatment. Plasma glucose in the JTT-130-treated rats was lower with higher plasma insulin than in the control rats during the IPGTT. The islets of the JTT-130-treated rats were larger and contained more insulin than those of the control rats. CONCLUSIONS: JTT-130 ameliorates impaired glucose and lipid metabolism in the ZDF rats thereby suggesting that JTT-130 could be useful for prevention and treatment of type 2 diabetes.
Assuntos
Tecido Adiposo/metabolismo , Benzamidas/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Diabetes Mellitus Experimental/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Malonatos/farmacologia , Animais , Benzamidas/uso terapêutico , Peso Corporal , Ingestão de Alimentos , Hemoglobinas Glicadas , Masculino , Malonatos/uso terapêutico , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
OBJECTIVE: Injection of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) causes acute swelling around the joint and subsequent morphological alterations in the condyle. We aimed to evaluate changes in the three-dimensional architecture of the condyle induced with CFA. MATERIALS AND METHODS: The CFA was injected into the unilateral TMJ of rats and morphological changes in the condyle were assessed repeatedly for 14 days by in vivo micro-CT. RESULTS: Osseous abnormalities of condyle were first observed at 3-5 days after CFA injection on the tomographic images, and the condylar deformation became more obvious thereafter. Among 12 condyles examined at 14 days postinjection, osteophytosis was observed in all of the specimens and bone erosion coexisted in five condyles. None of the saline-treated condyles showed architectural changes. Significant changes were detected in the mesiolateral and rostrocaudal widths of the CFA-treated condyles at 10-14 days postinjection (P < 0.01). The extent of both condylar bone formation and resorption was greater in the CFA-injected TMJs than in saline-injected TMJs (P < 0.05). CONCLUSION: These results indicate that CFA causes dynamic morphological changes in the condyle and that our experimental approach will provide new insights into the subacute inflammatory processes in the TMJ.
Assuntos
Artrite Experimental/patologia , Côndilo Mandibular/patologia , Transtornos da Articulação Temporomandibular/patologia , Articulação Temporomandibular/patologia , Adjuvantes Imunológicos/efeitos adversos , Animais , Artrite Experimental/etiologia , Temperatura Corporal/fisiologia , Peso Corporal , Reabsorção Óssea/etiologia , Reabsorção Óssea/patologia , Cefalometria , Fluoroscopia , Adjuvante de Freund/efeitos adversos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Masculino , Osteogênese , Osteófito/etiologia , Osteófito/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio , Transtornos da Articulação Temporomandibular/etiologia , Termografia , Fatores de Tempo , Microtomografia por Raio-X/métodosRESUMO
Collectins contribute to host defence through interactions with glycoconjugates on pathogen surfaces. We have prepared recombinant trimeric neck and carbohydrate recognition domains (NCRD) of collectins, and we now show that the NCRD of bovine conglutinin and CL-46 (like that of CL-43) have greater intrinsic antiviral activity for influenza A virus (IAV) than the human SP-D NCRD (hSP-D-NCRD). The three serum collectins differ from SP-D by having insertions adjacent to amino acid 325 and substitution of hydrophobic residues for arginine 343. We previously showed that a three amino acid (RAK) insertion, as found in CL-43, increases antiviral activity and mannan-binding activity of the hSP-D-NCRD, while the substitution of valine at 343, as in conglutinin, more strongly increased these activities. Mannan-binding activity of collectins has been considered to predict for ability to bind to high mannose glycans on viruses or other pathogens. We now show, however, that combined mutants containing the RAK insertion and R343V or R343I substitutions have greatly increased mannan-binding ability, but lower IAV binding or inhibiting activity than mutants containing R343V or R343I substitutions only. These findings indicate differences in the recognition of glycan structures of mannan and IAV by the NCRD and emphasize the importance of the flanking sequences in determining the differing interactions of human SP-D and bovine serum collectins with mannose-rich glycoconjugates on IAV and other pathogens. Of interest, we show conservation of some monoclonal antibody-binding epitopes between bovine collectin NCRD and hSP-D, suggesting shared structural motifs.
Assuntos
Colectinas/farmacologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Mananas/imunologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Motivos de Aminoácidos , Linhagem Celular , Colectinas/genética , Colectinas/imunologia , Ensaio de Imunoadsorção Enzimática , Testes de Inibição da Hemaglutinação , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
SUMMARY: This prospective study, in the very early phase after initiation of glucocorticoid (GC) treatment, showed that alendronate was effective in suppressing accelerated bone resorption and subsequent decrease in bone mineral density (BMD) at the lumbar spine of patients with high-dose GC treatment. INTRODUCTION: How bisphosphonates affect bone metabolism and BMD of patients with high-dose GC in the early phase, especially within 1 month is unclear. METHODS: We examined the prospective effects of daily 5 mg alendronate on bone metabolism and BMD in 20 patients with high-dose GC (at least 40 mg prednisolone/day) and compared them to 34 high-dose GC-treated patients without alendronate. RESULTS: Serum levels of calcium decreased at day 28 in the alendronate group. Urinary calcium excretion significantly increased after day 7 in both groups. The increase in serum parathyroid hormone (PTH) level at day 7 in the control group was not observed in the alendronate group, but PTH levels increased at day 28 and month 3 in the alendronate group. As for the bone turnover markers, the serum osteocalcin level decreased in both alendronate and control groups, but serum bone-type alkaline phosphatase levels did not show significant changes. Although the urinary type I collagen cross-linked N-telopeptide (NTX) level showed significant increases on days 7 and 28 in the control group; such early increases in urinary NTX were not observed in the alendronate group. Thereafter, the urinary NTX levels fell slowly in the alendronate group significantly. BMD at the lumbar spine significantly decreased from month 1 in the control group, whereas in the alendronate group, BMD at the lumbar spine maintained almost the same level at all time points observed. CONCLUSION: Alendronate was effective in suppressing bone resorption and subsequent BMD decrease at the lumbar spine in patients with high-dose GC treatment.
Assuntos
Alendronato/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Glucocorticoides/efeitos adversos , Osteoporose/prevenção & controle , Adulto , Idoso , Biomarcadores/metabolismo , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/metabolismo , Reabsorção Óssea/prevenção & controle , Cálcio/metabolismo , Esquema de Medicação , Feminino , Glucocorticoides/administração & dosagem , Humanos , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Osteoporose/induzido quimicamente , Osteoporose/metabolismo , Hormônio Paratireóideo/sangue , Prednisolona/administração & dosagem , Prednisolona/efeitos adversos , Estudos Prospectivos , Resultado do TratamentoRESUMO
Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.
Assuntos
Cromossomos de Mamíferos/genética , Evolução Molecular , Pan troglodytes/genética , Mapeamento Físico do Cromossomo , Animais , Cromossomos Humanos Par 21/genética , Perfilação da Expressão Gênica , Genes/genética , Genômica , Humanos , Mutagênese/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Análise de Sequência de DNARESUMO
Tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant mutations of epidermal growth factor receptor (EGFR) are associated with lung adenocarcinoma. EGFR mutants were previously shown to exhibit ligand-independent activation. We have previously demonstrated that pulmonary surfactant protein D (SP-D, SFTPD) suppressed wild-type EGFR signaling by blocking ligand binding to EGFR. We herein demonstrate that SFTPD downregulates ligand-independent signaling in cells harboring EGFR mutations such as TKI-sensitive exon 19 deletion (Ex19del) and L858R mutation as well as TKI-resistant T790M mutation, subsequently suppressing cellular growth and motility. Lectin blotting and ligand blotting in lung cancer cell lines suggested that EGFR mutants express oligomannose-type N-glycans and interact with SFTPD directly. Cross-linking assay indicated that SFTPD inhibits ligand-independent dimerization of EGFR mutants. We also demonstrated that SFTPD reduced dimerization-independent phosphorylation of Ex19del and T790M EGFR mutants using point mutations that disrupted the asymmetric dimer interface. It was confirmed that SFTPD augmented the viability-suppressing effects of EGFR-TKIs. Furthermore, retrospective analysis of 121 patients with lung adenocarcinoma to examine associations between serum SFTPD levels and clinical outcome indicated that in TKI-treated patients with lung cancer harboring EGFR mutations, including Ex19del or L858R, high serum SFTPD levels correlated with a lower number of distant metastases and prolonged overall survival and progression-free survival. These findings suggest that SFTPD downregulates both TKI-sensitive and -resistant EGFR mutant signaling, and SFTPD level is correlated with clinical outcome. These findings illustrate the use of serum SFTPD level as a potential marker to estimate the efficacy of EGFR-TKIs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação , Proteína D Associada a Surfactante Pulmonar/farmacologia , Animais , Células CHO , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cricetinae , Cricetulus , Receptores ErbB/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Inibidores de Proteínas Quinases/uso terapêutico , Proteína D Associada a Surfactante Pulmonar/sangue , Estudos Retrospectivos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions to retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. In our previous study (H. Ohguro et al., Invest. Ophthalmol. Vis. Sci., 40: 82-89, 1999), recoverin, a retina-specific calcium-binding protein, and heat shock cognate protein 70 (hsc 70) were identified as autoantigens recognized by sera from patients with CAR. Therefore, we suggested that autoimmune reactions against both recoverin and hsc 70 might be involved in the pathogenesis of CAR. To elucidate the initial step of the molecular pathology of CAR, we examined the expression of recoverin and hsc 70 by reverse transcription-PCR and Western blot using cell lines of several kinds of cancers, including lung small cell carcinoma, lung adenocarcinoma, gastric cancer, pancreatic cancer, breast cancer, uterine cervical cancer, endometrial cancer, and leukemia. Recoverin was expressed in 21 of the 31 cancer cell lines. The expression levels of hsc 70 were significantly higher in cancer cell lines than in noncancerous cell lines. However, no difference in the expression levels of hsc 70 was observed between recoverin-positive and -negative cell lines. Immunofluorescence labeling by the affinity-purified recoverin antibody revealed the immunoreactivity to recoverin as a granular pattern within the cancer cells. Lung adenocarcinoma A549 cells, which did not express recoverin, exhibited a significant reduction in cell proliferation upon transfection with human recoverin cDNA. Taken together, our present data suggest that the retina-specific calcium-binding protein recoverin is expressed in more than 50% of a variety of cancer cells and may play a significant role in the cell proliferation of these tumor cells.
Assuntos
Antígenos de Neoplasias/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Olho , Regulação Neoplásica da Expressão Gênica , Lipoproteínas , Proteínas do Tecido Nervoso , Sequência de Bases , Biomarcadores Tumorais/genética , Linhagem Celular , Linhagem Celular Transformada , Feminino , Proteínas de Choque Térmico HSP70/genética , Hipocalcina , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Recoverina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais CultivadasRESUMO
Hydrophobic pulmonary surfactant proteins (SP-B and SP-C) promote the adsorption of phospholipids at the air/liquid interface and the addition of surfactant protein A (SP-A) enhances this function. The developmental profiles of phospholipids and SP-A in the lung have been reported, but that of SP-B and SP-C remain unknown. We recently developed an enzyme-linked immunosorbent assay (ELISA) that measures SP-B in the rat. Using ELISA for SP-A and SP-B, we measured the contents of SP-A and SP-B in lung homogenates. The developmental profiles of SP-A and SP-B during the late gestational and postnatal periods were found to be distinctly different from each other. SP-A increased during late gestation and reached its maximum on day 1 after birth. This developmental profile of SP-A in the lungs was very similar to that of disaturated phosphatidylcholine (DSPC). In contrast, the SP-B contents in fetal lungs were low and increased after birth, reaching its maximum on day 4 after birth. In vivo dexamethasone treatment resulted in significant increases of SP-A content in rat lung homogenate on day 19 and day 21 of gestation, and day 5 after birth, whereas SP-B content increased significantly only on day 19 of gestation by dexamethasone administration. SP-A synthesis may be enhanced both pre- and postnatally, but SP-B synthesis may be stimulated only during the late gestational period by in vivo dexamethasone treatment. The difference in developmental profiles and the different responses to dexamethasone treatment between SP-A and SP-B indicate that the expression of SP-A and SP-B may be regulated independently at least in developing rat lungs.
Assuntos
Dexametasona/farmacologia , Pulmão/crescimento & desenvolvimento , Proteolipídeos/biossíntese , Surfactantes Pulmonares/biossíntese , Envelhecimento , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Idade Gestacional , Glicoproteínas/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Dados de Sequência Molecular , Fosfatidilcolinas/metabolismo , Proteolipídeos/isolamento & purificação , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/isolamento & purificação , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.
Assuntos
Apoproteínas/imunologia , Surfactantes Pulmonares/imunologia , Líquido Amniótico/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Proteinose Alveolar Pulmonar/metabolismo , Coelhos , Ratos , Ratos Endogâmicos , SuínosRESUMO
Surfactant protein A (SP-A) is a lung-specific glycoprotein in pulmonary surfactant and has a collagen like sequence on its N-terminal. SP-A has been shown to function as an inhibitor of phospholipid secretion by primary culture of alveolar type II cells via cell surface receptor(s) for SP-A. In a previous report, we showed that the C-terminal non-collagen like domain of human SP-A possessed the biological activities, and that a monoclonal antibody against human SP-A, PE10, abolished the biological activity of SP-A (Murata et al. (1993) Biochem. J. 291, 71-76). In the present study, we investigated an epitope of SP-A for PE10. Western blot analysis with fragmented peptides of human SP-A generated by both lysyl endopeptidase and BrCN showed that PE10 reacted with the peptide corresponding with Glu202 to the C-terminal but that it lacked the ability to bind to the peptide corresponding with Tyr208 to the C-terminal. The antibodies against a synthetic peptide (P1) corresponding with Glu202 to Asn217 of human SP-A inhibited the binding of PE10 to SP-A, suggesting that a similar site was recognized by both PE10 and anti-P1 antibodies. Anti-P1 antibodies as well as PE10 suppressed the biological activity of SP-A. A direct interaction between P1 and rat lung membranes, or between P1 and alveolar type II cell membranes was shown from the measurement of the fluorescence emission spectra of dansyl-labeled P1. These results suggest that an area contiguous to or near the region from Glu202 to Met207 of SP-A is important for expressing the biological activities.
Assuntos
Mapeamento de Epitopos , Glicoproteínas/química , Fosfatidilcolinas/metabolismo , Proteolipídeos/química , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Compostos de Dansil , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Pulmão/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/antagonistas & inibidores , Proteolipídeos/imunologia , Proteolipídeos/farmacologia , Proteolipídeos/fisiologia , Alvéolos Pulmonares/citologia , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/farmacologia , Surfactantes Pulmonares/fisiologia , Ratos , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Espectrometria de FluorescênciaRESUMO
The abundant and restricted expression of surfactant proteins SP-A and SP-D within the lung makes these collectins specific markers for lung diseases. The measurement of SP-A and SP-D in amniotic fluids and tracheal aspirates reflects lung maturity and the production level of the lung surfactant in infants with respiratory distress syndrome (RDS). The SP-A concentrations in bronchoalveolar lavage (BAL) fluids are significantly decreased in patients with acute respiratory distress syndrome (ARDS) and also in patients at risk to develop ARDS. The prominent increase of these proteins in BAL fluids and sputum is diagnostic for pulmonary alveolar proteinosis (PAP). The concentrations of SP-A and SP-D in BAL fluids from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia with collagen vascular diseases (IPCD) are rather lower than those in healthy controls and the SP-A/phospholipid ratio may be a useful marker of survival prediction. SP-A and SP-D appear in the circulation in specific lung diseases. Their serum concentrations significantly increase in patients with PAP, IPF and IPCD. The successive monitoring of serum levels of SP-A and SP-D may predict the disease activity. The serum SP-A levels increase in patients with ARDS. SP-A is also a marker for lung adenocarcinomas and can be used to differentiate lung adenocarcinomas from other types and metastatic cancers from other origins, and to detect metastasis of lung adenocarcinomas.
Assuntos
Glicoproteínas/isolamento & purificação , Pneumopatias/diagnóstico , Proteolipídeos/isolamento & purificação , Surfactantes Pulmonares/isolamento & purificação , Transtornos Respiratórios/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imuno-Histoquímica/métodos , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes PulmonaresRESUMO
Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein synthesized by alveolar type II cells. Antiserum against rat SP-D was raised in rabbits and an enzyme-linked immunosorbant assay (ELISA) has been developed using anti-rat SP-D IgG. In the present study we examined the developmental profile of SP-D in the rat lung compared with that of surfactant protein A (SP-A). SP-A content in the lungs increased during late gestation and reached its maximum on day 1 of neonate, and then gradually decreased until at least day 5. SP-D content during early gestation was less than 10 ng/mg protein until day 18, but on day 19 there was a 4-fold increase in SP-D (compared to that on day 18). It increased twice between day 21 and the day of birth, when it reached the adult level of 250 ng/mg protein, which is about one fourth that of the adult level of SP-A. Unlike SP-A there seemed to be no decrease in SP-D content after birth. These results demonstrate that SP-D is regulated developmentally as are the other components of surfactant, but the inconsistency in the developmental profiles of SP-A and SP-D suggests that these proteins may play different roles in lung maturation.
Assuntos
Apoproteínas/biossíntese , Pulmão/metabolismo , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/biossíntese , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Apoproteínas/imunologia , Apoproteínas/isolamento & purificação , Líquido da Lavagem Broncoalveolar , Eletroforese em Gel de Poliacrilamida , Desenvolvimento Embrionário e Fetal , Ensaio de Imunoadsorção Enzimática , Soros Imunes/imunologia , Immunoblotting , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/isolamento & purificação , Ratos , Ratos EndogâmicosRESUMO
Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Glicoproteínas/isolamento & purificação , Fosfolipídeos/isolamento & purificação , Surfactantes Pulmonares/isolamento & purificação , Animais , Ligação Competitiva , Centrifugação , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/metabolismo , Masculino , Fosfatidilcolinas/isolamento & purificação , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Proteolipídeos/isolamento & purificação , Proteolipídeos/metabolismo , Alvéolos Pulmonares/metabolismo , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.
Assuntos
Anexina A4/genética , Lipídeos de Membrana/química , Fosfolipídeos/química , Alanina/química , Anexina A4/química , Ácido Aspártico/química , Sítios de Ligação , Cálcio , Ácido Glutâmico/química , Lipossomos/química , Mutação , Cloreto de SódioRESUMO
Calcium-dependent conformational changes of surfactant protein A (SP-A) and the collagenase resistant fragment (CRF) of SP-A were studied by measuring fluorescence spectra. The emission peaks of both SP-A and CRF in the absence of Ca2+ appeared at 343 nm when they were excited at 280 nm. In the presence of Ca2+, the peaks appeared at 340 nm and were accompanied by an increase in the fluorescence intensity. The magnitude of the fluorescence intensity change induced by Ca2+ was amplified by the addition of dithiothreitol (DTT) in both SP-A and CRF. The Ca2+ binding of CRF was measured by a flow dialysis method with 45CaCl2 in the Ca2+ concentration range where the Ca(2+)-induced fluorescence changes occurred. The maximum binding number of Ca2+ to CRF was about 2 mol per mol of CRF, and the value was independent of the presence of DTT.
Assuntos
Cálcio/química , Ditiotreitol/química , Proteolipídeos/química , Surfactantes Pulmonares/química , Cátions Bivalentes , Colagenases/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Espectrometria de FluorescênciaRESUMO
Pulmonary surfactant protein A (SP-A) augments the uptake of phospholipid liposomes containing dipalmitoylphosphatidylcholine (DPPC) by alveolar type II cells. The SP-A-mediated uptake process of lipids by type II cells have not been well understood. In the present study we investigated the SP-A-mediated interaction of phospholipids with plasma membrane isolated from alveolar type II cells. SP-A increased the amount of liposomes containing radiolabeled DPPC associated with type II cell plasma membrane by 4-fold compared to the control without SP-A when analyzed by sucrose density gradient centrifugation. This effect is dependent upon the SP-A concentration. The enhancement was inhibited by anti-SP-A antibody and EGTA. When type II cell plasma membrane and liposomes containing [14C]DPPC and [3H]triolein were coincubated with or without SP-A, analysis on sucrose density gradients revealed that the profiles of [14C]DPPC and [3H]triolein in each fraction were almost identical with or without SP-A, indicating that SP-A mediates the binding of liposomes to plasma membrane but not transfer of DPPC. SP-A increased the association of liposomes containing DPPC with the membrane by 2-fold more than that containing 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC). SP-A induced aggregation of phospholipid liposomes containing PLPC as well as those containing DPPC, but the final turbidity of DPPC liposomes aggregated by SP-A was only by 15% greater than that of PLPC liposomes. The amount of DPPC liposomes associated with the plasma membrane derived from type II cells was 2-fold greater than that from liver. We speculate that the SP-A-mediated interaction of lipids with type II cell plasma membrane may contribute, in part, to the lipid uptake process by type II cells.
Assuntos
Membrana Celular/metabolismo , Lipossomos/metabolismo , Fosfolipídeos/metabolismo , Proteolipídeos/farmacologia , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Alvéolos Pulmonares/ultraestrutura , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Ratos , Ratos Sprague-Dawley , Trioleína/metabolismo , TrítioRESUMO
Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.
Assuntos
Lipídeos/farmacocinética , Proteolipídeos/química , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Células Cultivadas , Humanos , Lipossomos/farmacocinética , Mutagênese Sítio-Dirigida/genética , Fosfatidilgliceróis/metabolismo , Ligação Proteica/genética , Proteolipídeos/farmacologia , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/farmacologia , Ratos , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Serina Endopeptidases/metabolismoRESUMO
Four cases of late onset clear cell renal cell carcinoma (RCC), a case of gastric cancer, and a case of exocrine pancreatic cancer were identified in a Japanese family. In order to elucidate the underlying mechanism for tumorigenesis in this family, extensive genetic studies were performed including routine and spectral karyotyping (SKY), fluorescence in situ hybridisation (FISH), comparative genomic hybridisation (CGH), loss of heterozygosity studies (LOH), and VHL mutation analysis. A germline translocation t(1;3)(q32-q41;q13-q21) was identified by karyotyping in five members of the family including all three RCC cases tested. The translocation was refined to t(1;3)(q32;q13.3) by FISH analysis using locus specific genomic clones, and the two breakpoints were mapped to a 5 cM region in 3q13.3 and a 3.6 cM region in 1q32. Both CGH and allelotyping using microsatellite markers showed loss of the derivative chromosome 3 carrying a 1q segment in the three familial RCCs analysed. Additional chromosomal imbalances were identified by CGH, including amplifications of chromosomes 5 and 7 and loss of 8p and 9. No germline VHL mutation was found but two different somatic mutations, a splice (IVS1-2A>C) and a frameshift (726delG), were identified in two RCCs from the same patient confirming their distinct origin. Taken together, these results firmly support a three step model for tumorigenesis in this family. A constitutional translocation t(1q;3q) increased the susceptibility to loss of the derivative chromosome 3 which is then followed by somatic mutations of the RCC related tumour suppressor gene VHL located in the remaining copy of chromosome 3.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Ligases , Translocação Genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Sequência de Bases , Carcinoma de Células Renais/patologia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 3/genética , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Neoplasias Renais/patologia , Perda de Heterozigosidade , Masculino , Mutação , Hibridização de Ácido Nucleico , Linhagem , Proteínas/genética , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
OBJECTIVE: The aim was to test the efficacy of the immune system modulator lobenzarit disodium in the treatment of coxsackievirus B3 myocarditis. METHODS: Two week old C3H/He mice were inoculated with 10(3) plaque forming units of coxsackievirus B3. Lobenzarit disodium, 25 mg.kg-1.d-1, was given subcutaneously daily on days 0-14 (experiment I; group 2) and days 14-28 (experiment II; group 4). Both treated groups were compared to infected controls for each experiment (groups 1 and 3). For the analysis of splenic lymphocyte subsets, additional mice in untreated and treated groups were killed on d 7, and the percentages of Thy 1.2 (CD3), L3T4 (CD4), Ly 2 (CD8) subsets were analysed by laser flow cytometry (experiment III). RESULTS: In experiment I, the survival rate in the lobenzarit treated group was significantly lower than in the controls (2/11 v 8/11). Cellular infiltration and myocardial necrosis in the lobenzarit group were more severe. Myocardial virus titres and serum neutralising antibody titres did not differ significantly between the two groups. In experiment II, the survival rate (7/9 v 13/13) and cardiac pathology between the two groups did not differ significantly. In experiment III, the percentage of the Thy 1.2 subset (CD3) in the treated group was significantly lower (p < 0.05) than in the control group, at 36.0(SD 2.9)% v 42.8(5.8)%. CONCLUSIONS: Lobenzarit disodium decreased splenic pan T cells and aggravated both clinical course and cardiac pathology in acute murine coxsackievirus B3 myocarditis.