RESUMO
BACKGROUND AND PURPOSE: Ro 11-1464 is a thienotriazolodiazepine previously described to selectively stimulate apolipoprotein A-I (apoA-I) production and mRNA level in human liver cells. Here, we studied its effects upon oral administration to human apoA-I transgenic (hapoA-I) mice. EXPERIMENTAL APPROACH: HapoA-I mice were treated for 5 days with increasing doses of Ro 11-1464. Macrophage reverse cholesterol transport (mph-RCT) was assessed by following [(3) H]-cholesterol mobilization from pre-labelled i.p. injected J774 macrophages to plasma, liver and faeces. Effects on plasma lipids, apoproteins, lecithin-cholesterol : acyltransferase (LCAT) and liver enzymes, as well as on faecal excretion of cholesterol and bile salts, and on liver lipids and mRNA contents were determined. KEY RESULTS: Treatment with Ro 11-1464 300 mg·kg(-1) ·day(-1) resulted in a nearly 2-fold increase in plasma apoA-I, a 2- to 3-fold increase in the level of large sized-pre-ß high-density lipoprotein and a 3-fold selective up-regulation of hepatic apoA-I mRNA, but a marked decrease in all plasma lipids and LCAT activity. Mpm-RCT was decreased in blood but markedly increased in faecal sterols (4-fold) and bile acids (1.7-fold). However, liver weight and liver enzymes in plasma were also increased, in parallel with an increase in liver cholesterol ester content (all these effect being significant). CONCLUSION AND IMPLICATIONS: In this model Ro 11-1464 causes increased hepatic expression and plasma levels of apoA-I and a suppression of LCAT, and a marked enhancement of reverse cholesterol transport, but also some symptoms of liver toxicity. The compound may therefore be a prototype for a next generation of anti-atherosclerotic medicines.
Assuntos
Apolipoproteína A-I/biossíntese , Azepinas/farmacologia , Colesterol/metabolismo , Lipídeos/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Tiofenos/farmacologia , Alanina Transaminase/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/genética , Aterosclerose/metabolismo , Colesterol/sangue , Fezes/química , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacosRESUMO
Comparative gene expression studies are often limited by low availability of tissue and poor quality of extractable mRNA. Collective PCR amplification of minute quantities of mRNA has great potential for overcoming these limitations. However, there remains significant concern about the effects of amplification on the absolute and relative abundance of individual mRNAs that could complicate subsequent gene expression studies. To address this problem, we systematically compared the relative abundance of many specific mRNAs from complex cDNA preparations (from tissue and cultured cells) both before and after amplification by PCR. Our results demonstrated that, as expected, the absolute abundance of different mRNAs in a cDNA library is altered in an unpredictable manner by PCR amplification. However, we found that the concentration ratios of specific mRNAs among different cDNA preparations were routinely well conserved after PCR amplification. Thus, for the purpose of comparative expression studies for specific mRNAs in two (or more) complex cDNAs, PCR-amplified cDNA is equally useful as unamplified cDNA. These results provide a rigorous experimental validation and offer a theoretical treatment to support the utility of PCR amplified cDNA for differential gene expression studies. We conclude that the inherent difficulties in performing differential screening studies such as gene chip and array analyses on limited amounts of biological materials can be overcome by a PCR amplification step without compromising data quality.
Assuntos
DNA Complementar/genética , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Sequência de Bases , Primers do DNA , Técnicas Genéticas , RNA Mensageiro/genéticaRESUMO
Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-1, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Elementos Facilitadores Genéticos , HIV-1/genética , Regiões Operadoras Genéticas , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Virais/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Viral/genética , Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV , Dados de Sequência Molecular , Relação Estrutura-Atividade , Fatores de TranscriçãoRESUMO
An artificial 24-residue DDT-binding polypeptide (Moser, R., Thomas, R.M., and Gutte, B. (1983) FEBS Lett. 157, 247-251) and several analogues of this peptide were characterized by ligand binding, spectroscopic, and immunological studies. Comparison of dissociation constants showed that Phe14 and His16 were important for DDT binding and that the designed peptide had noticeable ligand specificity. Measurement of the circular dichroism of the artificial DDT-binding peptide revealed a high proportion of beta-structure which was abolished only partly by 8 M urea. When Tyr15, Tyr17, and Phe3 whose side chains were on the same side of the proposed beta-sheet were replaced by non-aromatic amino acids, the cross-reactivity with antibodies against the original DDT-binding peptide decreased stepwise. In summary, the results of this study support essential features of our structural model of the designed 24-residue DDT-binding peptide.
Assuntos
DDT , Peptídeos/síntese química , Sequência de Aminoácidos , Dicroísmo Circular , Reações Cruzadas , Desenho de Fármacos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Conformação Proteica , Radioimunoensaio , Relação Estrutura-AtividadeRESUMO
The antigenic determinant recognized by the monoclonal antibody that had been raised against synthetic human interferon-alpha 1 (IFN-alpha 1) fragment 111-166 [Arnheiter, H., Thomas, R.M., Leist, T., Fountoulakis, M., and Gutte, B. (1981) Nature (Lond.) 294, 278-280] and that cross-reacted with human IFN-alpha 1, IFN-alpha 2, and IFN-alpha A made in Escherichia coli, was localized to the region between residues 151 and 166 using synthetic COOH-terminal interferon fragments. In solid-phase radioimmunoassays neither the strongly hydrophilic COOH-terminal nonapeptide IFN 158-166 nor its mixtures with IFN 151-162 or IFN 149-158 showed any measurable interaction with the antigen binding site of the monoclonal antibody. For antibody binding, the full covalent structure of IFN 151-166 was required. Quantitatively very similar results were obtained with IFN 149-166 and IFN 143-166. The synthetic COOH-terminal hexadecapeptide of human IFN-alpha 1 (IFN 151-166) could be crystallized.
Assuntos
Epitopos/análise , Interferon Tipo I , Anticorpos Monoclonais/imunologia , Cromatografia de Afinidade , Cristalização , Humanos , Interferon Tipo I/síntese química , Interferon Tipo I/imunologia , Interferon-alfa , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Radioimunoensaio , Proteínas RecombinantesRESUMO
Collagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen. Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate. Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage. The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Colagenase Microbiana/farmacologia , Neutrófilos/enzimologia , Elastase Pancreática/farmacologia , Colágeno/metabolismo , Humanos , Colagenase Microbiana/análise , Elastase Pancreática/análiseRESUMO
Immunohistochemical studies were performed in synovial tissues from 40 patients with rheumatoid arthritis (RA), 9 with juvenile rheumatoid arthritis (JRA), 7 with psoriatic arthritis, and 4 with various rheumatic diseases. Overall synthesis of IgG- and/or IgM-rheumatoid factor (RF) was found in all patients with seropositive RA and JRA, in 75% of patients with seronegative RA, and in 1 patient with psoriatic arthritis. Agglutinator producing cells were found in 77% of the samples from seropositive RA and in 44% and 56% from seronegative RA and JRA patients, respectively. The percentage of IgG plasma cells synthesizing one or more of the 5 types of agglutinators studied was approximately 10% of plasma cells synthesizing IgG-RF. Intercellular and intracellular immune complex deposits were also found in patients with seropositive and seronegative RA and JRA. These findings suggest that synthesis of agglutinators by synovial tissue plasma cells of RA and JRA patients is a distinct--but definitely less prominent--function than that of RF synthesis.
Assuntos
Aglutininas/imunologia , Plasmócitos/imunologia , Doenças Reumáticas/imunologia , Fator Reumatoide/imunologia , Membrana Sinovial/imunologia , Anticorpos Anti-Idiotípicos , Biópsia por Agulha , Complemento C3/análise , Feminino , Histocitoquímica , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Masculino , Membrana Sinovial/patologiaRESUMO
Using a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C3, alpha 1-PI and rarely alpha 2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Neutrófilos/enzimologia , Elastase Pancreática/sangue , Adulto , Artrite Reumatoide/patologia , Cartilagem Articular/patologia , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Líquido Sinovial/citologia , Líquido Sinovial/enzimologiaRESUMO
Human lysosomal elastase from polymorphonuclear leucocytes is inhibited by the cartilage bone marrow extract Rumalon. Separately, both the cartilage and the bone marrow extracts are able to inhibit the enzymatic activity by 73%, under saturating conditions. The mixture of the two extracts inhibits elastase by 93%. It is suggested that the two partners act as a cumulative inhibition mechanism and this phenomenon is emphasized in a general theoretical model for synergy of proteinase-directed inhibitors.
Assuntos
Elastase Pancreática/metabolismo , Extratos de Tecidos/farmacologia , Artrite Reumatoide/enzimologia , Medula Óssea , Cartilagem Articular , HumanosRESUMO
The synovial fluid (SF) of RA patients contains large amounts of PMN which are well equipped with neutral enzymes to degrade articular cartilage: elastase and cathepsin G, which both destroy proteoglycans and native collagen, as well as 2 types of collagenoases. Indirect evidence suggests that PMN might be important in the destruction of RA articular cartilage. In 19 SF of RA patients no free elastase or collagenase was found. Using immune histochemical methods, we observed that PMN and macrophages of SF contain both elastase and alpha 1-anti-trypsin and alpha 2-macroglobulin. Peripheral PMN - but not monocytes - contain elastase, however both types of cells lack alpha 1-antitrypsin and alpha 2-macroglobulin. Elastase is demonstratable in the superficial layer of pannus free RA articular cartilage. These findings suggest that neutral proteinases from PMN in RA SF are generally neutralized by physiologic inhibitors and removed by phagocytes. The enzyme-inhibitor interaction might be bypassed during "frustrated phagocytosis" so that enzymes like PMN elastase can damage RA articular cartilage.
Assuntos
Artrite Reumatoide/patologia , Cartilagem Articular/patologia , Neutrófilos/enzimologia , Artrite Reumatoide/enzimologia , Catepsinas/metabolismo , Humanos , Colagenase Microbiana/metabolismo , Elastase Pancreática/metabolismoRESUMO
1. An activator of leucocyte latent collagenase has been extracted from rheumatoid synovial fluid by a preparative method consisting of six steps including precipitation by ammonium sulphate and chromatography on Sephadex G-100, QAE-Sephadex and SP-Sephadex C-50. The purification factor was nearly 1000 and the activator isolated could be shown to have a high degree of homogeneity.--2. Gel chromatography indicated a molecular weight of ca. 60 000.--3. Kinetic studies of the activation and inactivation of the activator during incubation at higher temperatures demonstrated its enzymic nature.--4. Activation of latent collagenase was partially inhibited by iPr2P-F and KCN. Soybean trypsin inhibitor, iodoacetamide, TosLysCH2Cl and TosPheCH2Cl had no effect.--5. Leucocyte latent collagenase was also activated by an excess of trypsin and p-hydroxymercuribenzenesulphonic acid, but only to the extent of about 40% of its activation capacity. Purified neutral protease from human leucocyte granules had no effect on latent collagenase.--6. Several typical substrates for proteases, peptidases, esterases and glycosidases were not attacked by the activator. The possibility that the activator is a known enzyme, such as kallikrein, urokinase or cathepsin B1, could be excluded.