Enantioselective aminolysis of an alpha-chloroester catalyzed by Candida cylindracea lipase encapsulated in sol-gel silica glass.

[reaction: see text] Lipase from Candida cylindracea (CcL) encapsulated in porous silica glass by a sol-gel method catalyzes enantioselective aminolysis of ethyl 2-chloropropionate. A silica matrix enhances the enzyme activity, i.e., improves the yield. The scope and limitations of the aminolysis reaction were investigated, and dynamic kinetic resolution of the ester was achieved. Encapsulated lipase remains active when used repeatedly. Encapsulation much improves the chiral discrimination by lipase and makes this enzyme even more useful in organic chemistry.

Disproportionation involving an organomercurial and a sulfhydryl-containing protein. Reaction between chloromercuryferrocene and bakers'-yeast cytochrome c.

Reaction between iso-1 cytochrome c from bakers' yeast and chloromercuryferrocene, FcHgCl, does not result in simple replacement of the sulfhydryl hydrogen atom in Cys 102 with the ferrocenylmercury group, FcHg. Instead, this reaction yields the protein monomer modified at Cys 102 with an HgCl+ group and the protein dimer in which the thiolate groups of Cys 102 are bridged by a mercury(II) atom. These proteins and other organometallic products are identified by chromatographic, spectroscopic, and electrochemical methods. Organomercurials of the type RHgX and biological thiols can undergo not only substitution reactions, but disproportionation reactions as well.

Characterization of zinc-substituted cytochrome c by circular dichroism and resonance Raman spectroscopic methods.

Iron(III) in cytochrome c is replaced with zinc(II) by a modification of a method published by others, and the procedure is described in full detail. Three forms of cytochrome c-those containing iron(III), iron(II), and zinc(II)-are examined by circular dichroism spectroscopy and resonance Raman spectroscopy. Spectra of both kinds show that introduction of zinc(II) ions does not appreciably alter the overall structure and conformation of cytochrome c. Resonance Raman spectra indicate the size of the porphyrin "core" that is inconsistent with six-coordination and consistent with five-coordination. Unlike the iron(III) and iron(II) ions, which are bound to two axial ligands (His 18 and Met 80), the zinc(II) ion in cytochrome c seems to be bound to only one, most probably His 18. Evidence pertaining to the question of axial coordination is discussed.

Epidemiology of end-stage renal disease and current status of hemodialysis in Yugoslavia.

Maintenance hemodialysis (HD) in Yugoslavia started in the sixties and followed the dialysis trends in the Western Europe. However, in the last decade the development of renal replacement therapy (RRT) slowed down. In this report the epidemiology of ESRD from 1997-1999 and the survey of the status of HD treatment in Yugoslavia in 1999 are presented. Epidemiological data are obtained by the annual center questionnaires (response rate: 92.6 -94.2%). The survey of HD status is based on a specific questionnaire and covered 2108 patients (65%). At the end of 1999 there were 56 RRT centers in Yugoslavia treating 3939 patients: 3232 (82%) patients by HD, 248 (6.3%) by peritoneal dialysis, and 459 (11.7%) living with transplanted kidney. In a three year period, incidence of ESRD ranged from 108-128 pmp, point prevalence from 435-463 pmp and mortality rate from 20.7-17.9. Numerous refugee patients were treated over the last 10 years. Main causes of ESRD were glomerulonephritis (30%); Balkan nephropathy represented 11% and diabetic nephropathy 7% of all primary renal diseases. Cardiovascular and cerebrovascular diseases were the most common causes of death of RRT patients. Most centers are overcrowded and HD machines are worn out. Mean Kt/V was 1.19+/-0.08, mean URR% 58.8+/-7.4. The shortage of drugs prevented adequate management: 83% of HD patients had hemoglobin level less than 100 g/L but only 10.3 -17.8% were treated with rHuEpo; 64.5% of patients had phosphate levels higher than 1.7 mmol/L but only 33.5% used phosphate binders; 47% of patients had hypertension despite the antihypertensive therapy. The prevalence of hepatitis B remained unchanged (about 14%) in HD population during the last three years, but the prevalence of anti-HCV positive patients decreased (31-23%). In conclusion, there is a well developed dialysis service in Yugoslavia but insufficient conditions for adequate treatment.

Systematical approach in evaluation of LC method for determination of raloxifene hydrochloride and its impurities employing experimental design.

Method validation presents a detailed investigation of analytical method and provision of the evidence that the method, when correctly applied, produces results that fit to the purpose. In order to achieve the method validation scope efficiently, experimental design presents a very useful tool. The greatest benefits of such approach could be seen in robustness testing through the provision of very useful data about the control of the chromatographic system during the routine application. In this paper, robustness testing of the LC method proposed for the determination of raloxifene hydrochloride and its four impurities was done employing Plackett-Burman design. Applying this design, the effect of five real factors (acetonitrile content, sodium dodecyl sulfate content, column temperature, pH of the mobile phase and flow rate) on the corresponding resolution factors was investigated through twelve experiments. Furthermore, the insignificance intervals for significant factors were calculated and the parameters for system suitability tests were defined. Eventually, the other validation parameters were tested and the effectiveness of the proposed analytical method with a high degree of accuracy was confirmed.

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities.

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.

[Circadian rhythm of blood pressure and daily hormonal variations]. / Cirkadijalni ritam krvnog pritiska i dnevne hormonalne varijacije.

As the mortality rate due to cardiovascular complications has increased, knowing the importance of the diurnal cycle of blood pressure, ambulatory blood pressure was recorded in 50 noninsulin-dependent diabetics aged 45 to 65 years. The diurnal cycle of the serum levels of cortisol, prolactin and growth hormone were determined by RIA (radioimmunoassay) method and urinary catechlamines by radioenzymatic method. The obtained results showed significantly higher blood pressure values during sleeping periods in patients with severe hypertension and consequently significantly lower night/day ratio of cortisol and urinary catecholamines as well as increased prolactin. In diabetic patients with autonomic neuropathy blood pressure values were significantly higher during night, but without changes in regard to hormones. It can be concluded that glucocorticoid hormones and catecholamines might be associated with the control of diurnal cycle of blood pressure in patients with severe hypertension.

[Functional, organic and histologic changes in the stomach in diabetic patients in relation to the presence of diabetic neuropathy]. / Fukcionalne, organske i histoloske promene zeluca u dijabetesnih bolesnika u odnosu na prisustvo dijabetesne neuropatije.

Functional, organic and histological gastric changes were investigated in relation to the presence of autonomic neuropathy in 105 noninsulin-dependent diabetics (20 with autonomic neuropathy, 35 with peripheral neuropathy, 30 with both peripheral and autonomic neuropathy and 20 without neuropathy) and 40 control persons. The diagnosis of autonomic neuropathy was assessed by parasympathetic test of cardiovascular reflexes and peripheral neuropathy by classical neurological examination and EMG (Electromyogram). All subjects were examined by radiologic and endoscopic methods with gastric biopsy specimens for histologic and immunocytochemical analysis. The results of investigation showed that functional abnormalities, hiatus hernia (55%) and pylorospasm (50%) occur more frequently in patients with autonomic neuropathy. Gastritis, duodenal ulcer (5%) and polyps (25%) are significantly more frequent. Atrophic gastritis type A (20%) was more frequent in diabetics with autonomic neuropathy than in other diabetics, as well as other types of gastritis. Immunocytochemical analysis showed the presence of rare, pale ECL (Extensor Caudae Lateralis) cells in corpus and pale, hypogranulated G cells in antrum and hyperplasia and metaplasia of EC (Embryonal carcinoma) cells in antrum of the stomach. Functional, organic and histologic changes in patients with diabetic autonomic neuropathy suggest that the systemic investigation of the Gut is very important in such patients.

[Response of pancreatic polypeptide to a protein test meal in the evaluation of diabetic autonomic neuropathy]. / Odgovor pankreaticnog polipeptida na proteinski test obrok u proceni dijabetesne autonomne neuropatije.

In normal subjects, the early human pancreatic polypeptide increase induced by food is mainly dependent on vagal activity. Parasympathetic function and plasma human pancreatic polypeptide response to a protein rich meal were evaluated in 105 insulin nondependent diabetic patients: 20 only with autonomic neuropathy (group A), diagnosed by clonidin test and tests of cardiovascular reflexes, 35 patients with neurophysiological evidence of polyneuropathy (group B), 30 patients with autonomic neuropathy and polyneuropathy (group C) and 20 patients without any sign of neuropathy (group D). Plasma human pancreatic polypeptide levels were determined by radioimmunoassay using an anti-human pancreatic polypeptide antiserum. Blood was taken at 0, 45 and 60 minutes after the beginning of the meal. In groups A and C, the meal induced human pancreatic polypeptide increase was significantly lower than in group D (45. min:, 65.8 + 16.2(A), 54.0 + 19.2 pg/ml (C) in regard to 130.0 + 27.6 pg/ml (D); 60, min: 55.2 + 15.6 (A), 37.0 + 11.2 pg/ml (C) in regard to 121.7 + 15.4 pg/ml (D). In group B patients had a marked increase of peptide, similar to that in diabetics without neuropathy. These results suggest that diabetic autonomic neuropathy is associated with the dysfunction of human pancreatic polypeptide secretion and that evaluation of his response to test meal may be a sensitive and simple method for the assessment of parasympathetic impairment in diabetics.

[Effect of autogenic training on glucose regulation and lipid status in non-insulin dependent diabetics]. / Uticaj autogenog treninga na glikoregulaciju i lipidni status kod insulinneza visnih dijabeticara.

INTRODUCTION: The objective of this study was to examine the benefits of autogenic training in patients with type 2 diabetes and 40 diabetics treated with oral antidiabetic agents were assigned to receive autogenic training. Treatment effects on GHb levels, glycemia, lipids and lipid peroxidases were evaluated after 12 weeks. RESULTS: Subjects demonstrated significant improvements of GHb level (8.94 +/- 2.21% vs. 7.9 +/- 2.395) (p < 0.005). Fasting glucose was significantly lower after treatment (11.6 +/- 6.1 mmol/l vs. 8.32 +/- 4.53 mmol/l) (p < 0.005). The serum level of HDL cholesterol was significantly higher after autogenic training (1.21 +/- 0.11 vs. 1.36 +/- 1.42) (p < 0.005). Cholesterol level was significantly lower after training (6.63 +/- 1.66 mmol/l vs. 6.10 +/- 1.12 mmol/l) (p < 0.05). Lipid peroxidase was significantly lower after treatment (4.05 +/- 0.58 vs. 3.69 +/- 1.26) (p < 0.005). CONCLUSION: Autogenic training in selected patients, especially those who are most responsive to stress would provide benefits for glucosE control and lipid metabolism that are not always achieved by conventional treatment.

[Effect of hyperlipoproteinemia on blood fibrinolytic activity and thrombocyte aggregation]. / Uticaj hiperlipoproteinemije na fibrinoliticku aktivnost krvi i agregaciju trombocita.

BACKGROUND AND AIM: An impaired lipid metabolism and macrovascular disease are often associated with diabetes mellitus. Effects of hyperlipoproteinemia on fibrinolytic blood activity and platelet aggregation were investigated in noninsulin-dependent diabetes mellitus. MATERIAL AND METHODS: The study sample consisted of 50 patients with noninsulin-dependent diabetes mellitus, only on dietetic regime. The following was studied: lipids (total cholesterol, HDL cholesterol and total triglycerides) fibrinolytic blood activity and platelet aggregation in patients with increased levels of cholesterol and triglycerides. We used enzymatic methods for lipids, Buckell's method for fibrinolysis (> 180 min) and Crean's method for platelet aggregation (6-8 s). RESULTS: Statistical analysis of the obtained results shows significant changes in lipids. Triglycerides were significantly higher in diabetics with poor control (p < 0.01). Total cholesterol was significantly higher in patients with coronary disease, hypertension and diabetics with occlusive disease of low extremities (p < 0.01). HDL cholesterol was significantly lower in diabetics with coronary heart disease (p < 0.05). Statistical analysis showed significant changes in blood coagulability. Decreased fibrinolytic blood activity (158.10 + 31.3 min v.s. 169.82 + 21.05 min) (p < 0.05), as well as faster platelet aggregation (6.7 + 0.48 sec v.s. 6.8 + 0.93 sec) (p < 0.1) in patients with hypertriglyceridemia compared with the diabetics with normal triglycerides. Other lipids did not influence significantly the fibrinolysis and platelet aggregation in noninsulin-dependent diabetics. CONCLUSION: All of this indicates the importance of the studied factors in the onset of atherosclerosis with patients with noninsulin-dependent diabetes; the latter is relevant to the importance of planned diabetic regime and moderate physical activity in preventing vascular complications of diabetes.

[Serum cholesterol and triglycerides in patients with benign and malignant tumors of the colon and rectum]. / Holesterol i trigliceridi u serumu bolesnika sa benignim i malignim tumorima kolona i rektuma.

There are many epidemiological papers on the relation of lipids especially of cholesterol, and the incidence of colorectal adenoma and cancer. In the studies of pathogenesis of the cancer of the large intestine attention is paid to the role of certain lipoproteins of bile acids and certain gastrointestinal hormones. We examined 30 patients: 10 were healthy subjects (the control group), 9 with colorectal adenoma, 2 with villous adenoma and 9 with adenocarcinoma of the colon and the rectum. The study concerned: total lipids, total cholesterol, total triglycerides, LDL cholesterol, HDL cholesterol, phospholipids, gastrinaemia and insulinaemia. Statistical analysis of the obtained results showed that hypocholesterolemia followed the incidence of adenocarcinoma. This is in accordance with the most frequently quoted literature. The incidence of hypergastrinaemia, however, was found primarily in the patients with villous adenoma, which indicates possible secretory function of the studied tumours.

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction.

Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.

Redox reactivity and reorganization energy of zinc cytochrome c cation radical.

Little is known about transient intermediates in photoinduced electron-transfer reactions of metalloproteins. Oxidative quenching of the triplet state of zinc cytochrome c, 3Zncyt, is done at 20 degrees C, pH 7.00, and ionic strength of 1.00 M, conditions that suppress the thermal back-reaction and prolong the lifetime of the cation radical, Zncyt+. This species is reduced by [Fe(CN)6]4-, [W(CN)8]4-, [Os(CN)6]4-, [Mo(CN)8]4-, and [Ru(CN)6]4- complexes of similar structures and the same charge. The rate constants and thermodynamic driving forces for these five similar electron-transfer reactions were fitted to Marcus theory. The reorganization energy of Zncyt+ is lambda = 0.38(5) eV, lower than that of native cytochrome c, because the redox orbital of the porphyrin cation radical is delocalized and possibly because Met80 is not an axial ligand to the zinc(II) ion in the reconstituted cytochrome c. The rate constant for electron self-exchange between Zncyt+ and Zncyt, k11 = 1.0(5) x 10(7) M(-1) s(-1), is large owing to the extended electron delocalization and relatively low reorganization energy. These results may be relevant to zinc(II) derivatives of other heme proteins, which are often used in studies of photoinduced electron-transfer reactions.

Electron-transfer reactions of cytochrome f with flavin semiquinones and with plastocyanin. Importance of protein-protein electrostatic interactions and of donor-acceptor coupling.

Reduction of turnip ferricytochrome f by flavin semiquinones and oxidation of this ferrocytochrome f by French bean cupriplastocyanin are studied by laser flash photolysis over a wide range of ionic strengths. Second-order rate constants (+/- 15%) at extreme values of ionic strength, all at pH 7.0 and 22 degrees C, are as follows: with FMN semiquinone at 1.00 and 0.0040 M, 5.0 x 10(7) and 3.9 x 10(8) M-1 s-1; with riboflavin semiquinone at 1.00 and 0.0040 m, 1.7 x 10(8) and 1.9 x 10(8) M-1 s-1; with lumiflavin semiquinone at 1.00 and 0.0045 M, 1.8 x 10(8) and 4.5 x 10(8) M-1 s-1; with cupriplastocyanin at 1.00 and 0.100 M, 1.4 x 10(6) and 2.0 x 10(8) M-1 s-1. These reactions of cytochrome f are governed by the local positive charge of the interaction domain (the exposed heme edge), not by the overall negative charge of the protein. Lumiflavin semiquinone behaves as if it carried a small negative charge, probably because partial localization of the odd electron gives this electroneutral molecule some polarity; local charge seems to be more important than overall charge even for relatively small redox agents. The dependence of the rate constants on ionic strength was fitted to the equation of Watkins; this model recognizes the importance of local charges of the domains through which redox partners interact. There is kinetic evidence that a noncovalent complex between cytochrome f and plastocyanin exists at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic activity of the serine proteases alpha-chymotrypsin and alpha-lytic protease tagged at the active site with a (terpyridine)platinum(II) chromophore.

Incubation of alpha-chymotrypsin and alpha-lytic protease with chloro(2,2':6',2''-terpyridine)platinum(II), [Pt(trpy)Cl]+, results in attachment of Pt(trpy)2+ tags at both His 57 and His 40 in the former and His 57 in the latter. The [Pt(trpy)His]2+ chromophores are readily detected and quantitated owing to their characteristic, strong UV absorption. Although the tagging of His 57 modifies the catalytic triad (Ser 195, His 57, and Asp 102) and disrupts the charge relay, the platinated enzymes retain significant esterase and amidase activity for both specific and nonspecific substrates. Unlike suicide inhibitors, which inactivate the enzymes by filling the active site and imitating the tetrahedral intermediate, [Pt(trpy)Cl]+ reacts with a particular amino acid and permits binding of substrates. The kinetic constants for the following are reported: two esters and two amides with alpha-chymotrypsin and an amide with alpha-lytic protease. The kcat values are between 1 and 25% of, and the Km values are a little higher than, the corresponding values for the native enzymes. The catalytic activity is not due to the native enzymes, trypsin, or some zinc-containing protease. Activities of the native and of the platinated alpha-chymotrypsin depend similarly on pH although the pKa of His 57 is raised to 9.7 upon platination. The platinated enzymes undergo autodigestion slower than do the native enzymes. Because the Pt(trpy)2+ tags are noninvasive, stable, and yet easily removable by thiourea, [Pt(trpy)Cl]+ may be used to retard autodigestion of stored proteolytic enzymes.

New selectivity in peptide hydrolysis by metal complexes. Platinum(II) complexes promote cleavage of peptides next to the tryptophan residue.

Tryptophan-containing N-acetylated peptides AcTrp-Gly, AcTrp-Ala, AcTrp-Val, and AcTrp-ValOMe bind to platinum(II) and undergo selective hydrolytic cleavage of the C-terminal amide bond; the N-terminal amide bond remains intact. In acetone solution, bidentate coordination of the tryptophanyl residue via the C(3) atom of indole and the amide oxygen atom produces complexes of spiro stereochemistry, which are characterized by (1)H, (13)C, and (195)Pt NMR spectroscopy, and also by UV-vis, IR, and mass spectroscopy. Upon addition of 1 molar equiv of water, these complexes undergo hydrolytic cleavage. This reaction is as much as 10(4)-10(5) times faster in the presence of platinum(II) complexes than in their absence. The hydrolysis is conveniently monitored by (1)H NMR spectroscopy. We report the kinetics and mechanism for this reaction between cis-[Pt(en)(sol)(2)](2+), in which the solvent ligand is water or acetone, and AcTrp-Ala. The platinum(II) ion as a Lewis acid activates the oxygen-bound amide group toward nucleophilic attack of solvent water. The reaction is unimolecular with respect to the metal-peptide complex. Because the tryptophanyl fragment AcTrp remains coordinated to platinum(II) after cleavage of the amide bond, the cleavage is not catalytic. Added ligand, such as DMSO and pyridine, displaces AcTrp from the platinum(II) complex and regenerates the promoter. This is the first report of cleavage of peptide bonds next to tryptophanyl residues by metal complexes and one of the very few reports of organometallic complexes involving metal ions and peptide ligands. Because these complexes form in nonaqueous solvents, a prospect for cleavage of membrane-bound and other hydrophobic proteins with new regioselectivity has emerged.

Comparison of electrostatic interactions and of protein-protein orientations in electron-transfer reactions of plastocyanin with the triplet state of zinc cytochrome c and with zinc cytochrome c cation radical.

Photoinduced reduction of cupriplastocyanin by the triplet state of zinc cytochrome c (the "forward" reaction) and the subsequent thermal oxidation of cuproplastocyanin by zinc cytochrome c cation radical (the "back" reaction) at ionic strengths from 40 mM to 3.00 M are studied by laser kinetic spectroscopy (so-called flash photolysis). Variation of the bimolecular rate constants over the entire range of ionic strength cannot be explained in terms of monopole-monopole interactions between the protein molecules, but it can be explained in terms of monopole-monopole, monopole-dipole, and dipole-dipole interactions. Analysis of the kinetic results in terms of these electrostatic interactions reveals the overall protein-protein orientation for electron transfer. In both the forward and back reactions the exposed heme edge in zinc cytochrome c apparently abuts the negatively-charged (acidic) patch on the plastocyanin surface, which is remote from the copper atom, and not the electroneutral (hydrophobic) patch, which is proximate to the copper atom. The acidic patch is large, and this analysis cannot rule out a relatively small difference in protein-protein orientations for the forward and back reactions. These two reactions are compared with the previously studied reduction of cupriplastocyanin by ferrocytochrome c. Although native cytochrome c and its zinc derivative have very similar structural and electrostatic properties, the reactive forms of the cytochrome c/plastocyanin and zinc cytochrome c/plastocyanin complexes may adopt somewhat different protein-protein orientations or may adopt similar orientations but differ in dynamic properties.