RESUMEN
Early detection of diabetic retinopathy (DR) is an urgent ophthalmological problem in Russia and globally. PURPOSE: This study assesses the prevalence of asymptomatic retinopathy and attempts to identify risk groups for its development in patients with type 1 and 2 diabetes mellitus (T1DM and T2DM). MATERIAL AND METHODS: The study involved clinics from 5 cities in the Russian Federation and it included 367 patients with DM, 34.88% men and 65.12% women, aged 50.88±20.55 years. 34.88% of patients suffered from T1DM, 65.12% suffered from T2DM, the average duration of the disease was 9.02±7.22 years. 58.31% of patients had a history of arterial hypertension, 13.08% had a history of smoking. The primary endpoint was the frequency of detection of diabetic changes in the eye fundus of patients with T1DM and T2DM in general; the secondary endpoint - same but separately, and for T2DM patients depending on the duration of the disease. The exploratory endpoint was the assessment of the influence of various factors on the development of DR. The patients underwent visometry (modified ETDRS table), biomicroscopy, mydriatic fundus photography according to the «2 fields¼ protocol. RESULTS: The average detection rate of DR was 12.26%, primarily observed in patients with T2DM (13.81%), women (9.26%), in both eyes (8.17%). Among patients with DR, 26 (19.55%) had glycated hemoglobin (HbA1c) level exceeding 7.5% (p=0.002), indicating a direct relationship between this indicator and the incidence of DR. Logistic regression analysis showed that the duration of diabetes of more than 10 years has a statistically significant effect on the development of DR. In the modified model for odds estimation, the likelihood of developing DR is increased by the duration of DM for more than 10 years; increased blood pressure; HbA1c level >7.5%. CONCLUSION: The obtained results, some of which will be presented in subsequent publications, highlight the effectiveness of using two-field mydriatic fundus photography as a screening for DR.
Asunto(s)
Diabetes Mellitus Tipo 2 , Retinopatía Diabética , Fondo de Ojo , Fotograbar , Humanos , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/epidemiología , Femenino , Masculino , Persona de Mediana Edad , Federación de Rusia/epidemiología , Prevalencia , Fotograbar/métodos , Adulto , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Anciano , Factores de Riesgo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/diagnóstico , Diagnóstico PrecozRESUMEN
The behavior of subtilisin 72 in some aprotic solvents (acetonitrile, dioxane, and tetrahydrofurane) was studied. The enzyme was shown to be partially soluble in tetrahydrofurane, but it is rendered profoundly inactive in this solution. In acetonitrile and dioxane, subtilisin formed dilute suspensions whose activities were measured after dilution with water. Under these conditions, subtilisin suspended in acetonitrile manifested an activity that was an order of magnitude higher than that of its dioxane suspension, and this activity continued for a long time. Z-Ala-Ala-Leu-pNA was synthesized from Z-Ala-Ala-OCH3 and Leu-pNA under the catalysis by dilute suspension of subtilisin in acetonitrile. p-Nitroanilides of tetrapeptides, Z-Ala-Ala-P1-P'1-pNA, where P1 and P'1 were either Leu or Phe, were similarly synthesized in acetonitrile under catalysis by dilute subtilisin suspension at [S]:[E] = 10(5):1. p-Nitroanilides of tripeptides, Z-Ala-Ala-Leu-pNA, Z-Ala-Ala-Phe-pNA, and Z-Ala-Ala-Phe-NH2, were also synthesized in the presence of a concentrated subtilisin suspension at [S]:[E] = 10(3):1. It was shown that the increase in enzyme concentration resulted in the double coupling of nucleophile, and Z-Ala-Ala-Leu-Leu-pNA, Z-Ala-Ala-Phe-Phe-pNA, and Z-Ala-Ala-Phe-Phe-NH2 were obtained with 13, 33, and 40% yields, respectively. Therefore, such reaction systems can be used for creating long hydrophobic peptides whose synthesis in water-organic mixtures is difficult due to the poor solubility of starting components in aqueous buffer solutions.
Asunto(s)
Oligopéptidos/síntesis química , Solventes/química , Subtilisinas/química , Acetonitrilos/química , Tampones (Química) , Catálisis , Cromatografía Líquida de Alta Presión , Dioxanos/química , Furanos/química , Solubilidad , Espectrofotometría Ultravioleta , SuspensionesRESUMEN
A convenient procedure for thiolsubtilisin purification from an admixture of subtilisin involving affinity chromatography on bacitracin-Sepharose is presented. Thiolsubtilisin activity was measured by hydrolysis of p-nitrophenyl acetate, p-nitroanilide-peptide (Glp-Ala-Ala-Leu-pNA), and azocasein. The thiolenzyme catalyzes peptide synthesis. Under these conditions only activated peptide esters, e.g., p-chlorophenyl, N-hydroxysuccinimide, or p-nitrophenyl esters form peptide bonds during interaction with appropriate nucleophiles such as peptides and their derivatives and amino acid amides.
Asunto(s)
Bacillus subtilis/enzimología , Subtilisinas/aislamiento & purificación , Subtilisinas/metabolismo , Aminoácidos/análisis , Bacitracina , Caseínas/metabolismo , Cromatografía de Afinidad/métodos , Cromatografía en Gel , Hidrólisis , Nitrofenoles , Biosíntesis de Péptidos , Péptidos/química , Sefarosa , Especificidad por SustratoRESUMEN
Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases--porcine pepsin, human pepsin, gastricsin, and cathepsin D--were prepared. These peptide derivatives, o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide and N-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N'-2,4-dinitrophenyl ethylenediamine, contain a fluorescent o-aminobenzoyl moiety as well as p-nitroaniline or N-2,4-dinitrophenyl ethylenediamine--the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity of o-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.
Asunto(s)
Aminobenzoatos/química , Catepsina D/análisis , Oligopéptidos/química , Pepsina A/análisis , Secuencia de Aminoácidos , Animales , Fluorometría , Humanos , Datos de Secuencia Molecular , Estructura Molecular , PorcinosRESUMEN
Pepsin successfully catalyzed the synthesis of several hydrophobic octa- and decapeptides in dimethylformamide-water solutions containing concentrated urea at pH 4.65. The factors that influence peptide synthesis in the presence of urea were studied using condensation of the tripeptides Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 as a model. The dependence of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 yield on pepsin concentration and pH, as well as the behavior of pepsin during peptide synthesis were studied. It was shown that pepsin catalyzed the synthesis of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 in guanidine hydrochloride and sodium dodecyl sulfate solutions. Other proteinases, subtilisin and thermolysin, were applied for the synthesis of p-nitroanilides of tri- and tetrapeptides in urea solutions. Proteinase-catalyzed peptide synthesis in the presence of denaturing agents might help to overcome the limitations caused by poor solubility of the starting peptide derivatives, although this effect is sometimes counterbalanced by the product solubility.
Asunto(s)
Oligopéptidos/síntesis química , Pepsina A/química , Secuencia de Aminoácidos , Catálisis , Datos de Secuencia Molecular , Desnaturalización Proteica , Soluciones , Subtilisinas/química , Termolisina/química , UreaRESUMEN
The porcine pepsin immobilized on inorganic supports catalyzes the peptide bond formation in organic solvents. Dependence of the peptide bond formation between Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 upon the porous material, organic solvent, reaction time, enzyme concentration, ionic strength and pH was studied. Syntheses of peptides of the general formula Z-Ala-Ala-Xaa-Yaa-Ala-Ala-OCH3, where Xaa = Phe, Tyr, Trp; Yaa = Leu, Phe, Tyr, Trp, were carried out.
Asunto(s)
Enzimas Inmovilizadas/química , Oligopéptidos/síntesis química , Pepsina A/química , Acetatos , Acetonitrilos , Secuencia de Aminoácidos , Animales , Catálisis , Etanol , Concentración de Iones de Hidrógeno , Cinética , Cloruro de Metileno , Datos de Secuencia Molecular , Concentración Osmolar , Solventes , PorcinosRESUMEN
Porcine pepsin behaviour during the synthesis of peptide p-nitroanilides and esters has been studied. In many cases, especially when long-chain peptides, such as Z-Ala-Ala-Phe-Leu-Ala-Ala-OMe, were synthesized, pepsin disappeared from the solution, being entrapped by the product precipitate rather than inactivated. Sorption of the enzyme on the product might be partially responsible for this effect. The active enzyme could be eluted from the precipitate by NaCl and isopropanol. Non-proteolytic proteins (lysozyme, bovine albumin, carbonic anhydrase) could also co-precipitate with pepsin.
Asunto(s)
Pepsina A/metabolismo , Péptidos/síntesis química , Secuencia de Aminoácidos , Animales , Precipitación Química , Datos de Secuencia Molecular , PorcinosRESUMEN
Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri-, tetra-, penta- and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide-water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved--up to 1:3.10(5) for relatively long peptides.
Asunto(s)
Anilidas/química , Oligopéptidos/síntesis química , Pepsina A/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Animales , Catálisis , Ésteres/química , Datos de Secuencia Molecular , PorcinosRESUMEN
Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.
Asunto(s)
Oligopéptidos/biosíntesis , Pepsina A/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Precipitación Química , Técnicas In Vitro , Datos de Secuencia Molecular , Oligopéptidos/químicaRESUMEN
It has been shown that in the course of equilibrium peptide synthesis pepsin gradually disappeared from the liquid phase due to its entrapment within a gel formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin so far as inert proteins-lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin-an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.