Assuntos
Hirudinas/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Bacillus subtilis/genética , Sítios de Ligação , DNA/genética , Escherichia coli/genética , Hirudinas/análogos & derivados , Hirudinas/biossíntese , Hirudinas/metabolismo , Sanguessugas/genética , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
One week after treatment of a urinary infection with co-trimoxazole (twice daily 160 mg trimethoprim and 800 mg sulphamethoxazole) a 21-year-old man suddenly started to vomit, accompanied by watery diarrhoea, abdominal swelling and weight loss of 5 kg. Plain X-ray film of the abdomen while standing showed multiple fluid levels in the small intestine of the upper and lower abdomen. Serum IgE concentration was elevated to 325 U/ml. There was a leukocytosis of 25,800/microliters, with a differential count of 45% eosinophils. Protein-rich ascites contained numerous eosinophils and the mucosa of the terminal ileus and the duodenum was infiltrated with eosinophils, findings which indicated eosinophilic gastroenteritis. All symptoms regressed completely within 10 days of stopping co-trimoxazole and administering prednisolone (50 mg/day). Four years later a similar episode of eosinophilic gastroenteritis developed after the patient had taken trimethoprim with a sulphonamide (once daily 180 mg trimethoprim and 820 mg sulphadiazine). It again quickly responded to short-term administration of glucocorticoids.
Assuntos
Hipersensibilidade a Drogas/complicações , Eosinofilia/induzido quimicamente , Gastroenterite/induzido quimicamente , Combinação Trimetoprima e Sulfametoxazol/efeitos adversos , Adulto , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/tratamento farmacológico , Hipersensibilidade a Drogas/etiologia , Eosinofilia/diagnóstico , Eosinofilia/tratamento farmacológico , Gastroenterite/diagnóstico , Gastroenterite/tratamento farmacológico , Humanos , Masculino , Prednisolona/administração & dosagem , Recidiva , Fatores de Tempo , Infecções Urinárias/complicações , Infecções Urinárias/tratamento farmacológicoRESUMO
Human leukocyte elastase has been crystallized in complex with recombinant Pro44-eglin c in the orthorhombic space group P2(1)2(1)2(1). The cell constants are a = 126.1 A, b = 127.8 A, c = 69.4 A, alpha = beta = gamma = 90 degrees. The crystals diffract to at least 2.5 A resolution and are suitable for crystallographic structure analysis.
Assuntos
Leucócitos/enzimologia , Proteínas , Cristalização , Humanos , Elastase Pancreática/antagonistas & inibidores , Serpinas , Difração de Raios XAssuntos
Elastase Pancreática/sangue , Inibidores de Proteases/farmacologia , Proteínas/farmacologia , Serpinas , Animais , Proteínas Sanguíneas , Ensaio de Imunoadsorção Enzimática , Humanos , Sanguessugas , Leucócitos/enzimologia , Elastase Pancreática/antagonistas & inibidores , Proteínas/uso terapêuticoRESUMO
Eglin c is an elastase/cathepsin G inhibitor from leech Hirudo medicinalis. The gene for this 70 aminoacid peptide was synthesized chemically, cloned and expressed by E. coli. Here we report biochemical and pharmacological studies. The rate of complex formation between Eglin c and human leukocyte elastase (HLE) or human cathepsin G (H. Cat. G) was determined and compared to those of a number of other proteinase/proteinase-inhibitor interactions (alpha 1 PI and alpha 2M). The association rate constants of Eglin c with the leukocyte enzymes are of the same order of magnitude as those with the naturally occurring inhibitors alpha 1 PI and alpha 2M. The association rate constant of Eglin c (extracted from leech) and Eglin c (biotechnology product) with HLE was found to be identical. The equilibrium constants Ki of the Eglin c/HLE and the Eglin c/H. Cat. G interactions are in the order of 10(-10) M. In an experiment with the hamster emphysema model, 0.5 mg or 2 mg of Eglin c applied intratracheally one hour before an HLE-insult completely protected the animals against emphysema and no signs of toxicity due to Eglin c were observed.
Assuntos
Inibidores de Proteases/farmacologia , Proteínas/farmacologia , Serpinas , Animais , Cricetinae , DNA Recombinante , Humanos , Cinética , Inibidores de Proteases/genética , Proteínas/genética , Enfisema Pulmonar/prevenção & controleRESUMO
A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech.
Assuntos
DNA/síntese química , Escherichia coli/genética , Genes , Sanguessugas/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Inibidores de Proteases/genética , Proteínas/genética , Serpinas , Sequência de Aminoácidos , Animais , Composição de Bases , Sequência de Bases , Clonagem Molecular , DNA Recombinante/metabolismo , Indicadores e Reagentes , PlasmídeosRESUMO
The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 micrograms of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.
Assuntos
Aminoácidos/análise , Fragmentos de Peptídeos/isolamento & purificação , Inibidores de Proteases , Proteínas , Serpinas , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , MicroquímicaAssuntos
Hiperlipidemias/complicações , Nefropatias/sangue , Lipídeos/sangue , Tecido Adiposo/metabolismo , Animais , Colesterol/sangue , Insulina/sangue , Nefropatias/complicações , Metabolismo dos Lipídeos , Lipoproteínas VLDL/sangue , Masculino , Ratos , Triglicerídeos/sangue , Uremia/sangue , Uremia/dietoterapiaRESUMO
Lipid metabolism was studied in experimental uremia. Uremic (U) rats were compared with sham-operated, pair-fed (PF) controls and with ad-lib-fed (AL) controls. In U animals, fasting glucose concentrations were normal, immunoreactive serum insulin (IRI) levels were decreased, and immunoreactive glucagon levels were increased. A significant increase in the serum concentration of all lipid classes was observed: triglycerides were elevated 10-fold above the values in PF and AL controls; phospholipids, twofold; total cholesterol, threefold; and free cholesterol, sixfold. Cholesterol concentration was increased in beta- and pre-beta-lipoproteins and even more so in alpha- and pre-alpha-lipoproteins. There was an increase in the ratio of free cholesterol/total cholesterol. The fatty acid composition of serum lipoproteins was unchanged. Concomitantly, in liver tissue, there was no change in lipid content (triglyceride, cholesterol) and fatty acid composition. These findings argue against glucose- or insulin-mediated changes in hepatic de novo fatty acid synthesis, chain elongation, or poly-desaturation. In U animals, the HMG-CoA-reductase activity of liver microsomes was slightly, but not significantly, reduced as was tritiated water incorporation into cholesterol in isolated perfused liver preparations. In adipose tissue, there was a decrease in triglyceride content. The results provide evidence against insulin-mediated hepatic overproduction as a major cause of hyperlipoproteinemia in this model of experimental renal insufficiency and point to peripheral under-utilization of lipoproteins.
Assuntos
Hiperlipidemias/complicações , Falência Renal Crônica/complicações , Lipoproteínas/metabolismo , Animais , Glicemia , Modelos Animais de Doenças , Glucagon/sangue , Hiperlipidemias/metabolismo , Insulina/sangue , Falência Renal Crônica/metabolismo , Lipídeos/sangue , Masculino , Nefrectomia , RatosRESUMO
The effect of different lipoproteins (lipoprotein-X and lipoprotein-B; LP-X and LP-B) on hepatic cholesterol synthesis was studied in vivo in rats. Lipoproteins were continuously infused into rats for 16 hours so that 24 mg cholesterol/100 g body weight were applied. Serum cholesterol level was nearly doubled after the infusion period. Lipoprotein electrophoresis revealed the predominance of the infused lipoprotein in the serum. LP-B infusion caused a reduction of cholesterol synthesis (42% of control values) and reduced the increased cholesterol synthesis of bile fistula rats to values below normal. LP-X did not reduce hepatic cholesterol synthesis significantly nor did it normalize the enhanced synthesis following biliary diversion. However, hepatic free cholesterol concentration increased after LP-X infusion. The effect of LP-X on liver cholesterol synthesis is similar to that of lecithin: cholesterol dispersions. The failure of LP-X to exert a feedback inhibition on cholesterol synthesis may therefore contribute to the mechanism of hypercholesterolemia in obstructive jaundice.
Assuntos
Colesterol/biossíntese , Lipoproteínas LDL/farmacologia , Fígado/efeitos dos fármacos , Animais , Peso Corporal , Colesterol/sangue , Fígado/metabolismo , Masculino , RatosRESUMO
The isolated liver of male Sprague-Dawley rats was perfused by means of media containing lithocholic acid, taurolithocholic acid, lithocholic acid sulfate and taurolithocholic acid sulfate. 150 minutes later the tissue was being examined light- and electrone microscopically. After LC and TLC perfusion considerable alterations were found in the bile capillaries, in the ergastoplasm and minor ones in mitochondria. After perfusion with sulfate esters the tissue was unchanged. Our investigations have shown that sulfation provides a highly effective mechanism of detoxication in rats; but detoxication results not only in a decrease of reabsorption of excreted lithocholic acid sulfate esters but sulfation tenders the very lithocholic acid untoxic for the liver cell. The primary point of action of lithocholic acid seems to be the lipoprotein membrane.
Assuntos
Ácidos Cólicos/farmacologia , Ácido Litocólico/farmacologia , Fígado/efeitos dos fármacos , Perfusão , Sulfatos/farmacologia , Animais , Lipoproteínas/metabolismo , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos , Ácido Taurocólico/farmacologiaAssuntos
Ácidos e Sais Biliares/metabolismo , Fígado/metabolismo , Animais , Masculino , Perfusão , RatosAssuntos
Colesterol/biossíntese , Lipoproteínas/metabolismo , Fígado/metabolismo , Animais , Humanos , RatosRESUMO
Morphologic alterations in liver cells after bile duct ligation are well known and documented in numerous reports. Biochemical studies concerning metabolic changes in cholestatic liver are rare. Therefore, in this study, liver cell metabolites and the capacity of the perfused cholestatic rat liver to produce glucose, urea and ketone bodies were measured. In addition the influence of dihydroxy bile acids on normal and bile duct ligated rat livers was studied. Concentrations of adenine nucleotides, lactate, pyruvate, 3-hydroxybutyrate, acetoacetate, glucose and UDP-glucose were found to be identical in cholestatic and normal livers. Glycogen content, however, was significantly lowered in cholestatic livers. Gluconeogenesis from lactate and urea production from ammonium chloride were only slightly reduced in bile duct obstructed rat livers. Dihydroxy bile acids did not affect the metabolism of normal or cholestatic livers. Ketone body production from oleate was reduced to 66% in bile duct obstructed livers, taurochenodeoxycholate further reduced this value to the normal value. In contrast to earlier reports (Fisher and co-workers, 1971 Lab. Invest. 21; 88-91; Gastroenterology 60: 742) chenodeoxycholate induced neither cholestasis nor a marked fall in ATP content or rat liver in our experiments with female Wistar rats. In conclusion, dihydroxy bile salts did exert toxic short term effects on rat livers.