The relationship between serum thiol levels and thiol/disulfide homeostasis in women with tubal ectopic pregnancy.

OBJECTIVE: The aim of this study was to investigate the thiol/disulfide homeostasis in tubal ectopic pregnancies in terms of early diagnosis of the disease. DESIGN: A prospective case-control study was carried out between June 2017-February 2018 in the Gynaecology Department of Umraniye Medical and Research Hospital. MATERIALS AND METHODS: A total of 42 women with ectopic pregnancy were compared with 44 healthy women who have intrauterine first trimester pregnancies. The thiol/disulfide homeostasis is evaluated with the spectrophotometric measurement method that was recently developed by Erel&Neselioglu. RESULTS: Disulfide/native thiol and disulfide/total thiol ratios were increased (p = 0.018 and p = 0.023 respectively), while native thiol/total thiol ratios and native thiol levels were decreased in tubal ectopic pregnancy group according to control group (p = 0.023). Between control and tubal ectopic pregnancy groups no differences were measured in disulfide levels (p = 0.350). The area under curve for native thiol and total thiol were 0.937 and 0.927, respectively. The optimum cut off value for native thiol was 379.95 µmol/l with a sensitivity of 90% and specificity of 81%. The optimum cut off value for total thiol was 432.5 µmol/l had 92% sensitivity and 79% specificity. LIMITATIONS: In the study, whether intrauterine pregnancies resulted in miscarriage or delivery can be examined. CONCLUSION: Increased disulfide/native thiol levels, disulfide/total-thiol ratio and decreased native/total thiol ratio were found to be significantly associated with the presence of tubal ectopic pregnancy which can be useful for the early diagnosis of the disease.

Redox compartmentalization and cellular stress.

Mammalian cells are highly organized to optimize function. For instance, oxidative energy-producing processes in mitochondria are sequestered away from plasma membrane redox signalling complexes and also from nuclear DNA, which is subject to oxidant-induced mutation. Proteins are unique among macromolecules in having reversible oxidizable elements, 'sulphur switches', which support dynamic regulation of structure and function. Accumulating evidence shows that redox signalling and control systems are maintained under kinetically limited steady states, which are highly displaced from redox equilibrium and distinct among organelles. Mitochondria are most reducing and susceptible to oxidation under stressed conditions, while nuclei are also reducing but relatively resistant to oxidation. Within compartments, the glutathione and thioredoxin systems serve parallel and non-redundant functions to maintain the dynamic redox balance of subsets of protein cysteines, which function in redox signalling and control. This organization allows cells to be poised to respond to cell stress but also creates sites of vulnerability. Importantly, disruption of redox organization is a common basis for disease. Research tools are becoming available to elucidate details of subcellular redox organization, and this development highlights an opportunity for a new generation of targeted antioxidants to enhance and restore redox signalling and control in disease prevention.

[Peculiarities of diagnosis and treatment of patients with hyposialosis].

33 patients with complaints of hyposalivation (21 in a 33 with syaloadenopatya and 12 in a 33 with syalosis out of the acute stage of disease) were examined. Complex examination of the saliva glands, analysis of the haemokynes in the various mediums of organism and thyoles compounds in the blood plasma was carried out. Tendency to increase in contents of haemokynes in the mixed saliva and in the tissue samples (from low in the syaloadenopatya to expressed in the syalosis) was revealed. Reduction of thyosulfide index in the blood plasma of these patients was determined. Use of antioxidant remedy in complex treatment of syaloadenopatya and syalosis was substanciated in this paper.

Purification of NADPH-free glutathione disulfide reductase from human erythrocytes.

Human erythrocyte glutathione disulfide reductase was purified using serially connected 2',5'-ADP-Sepharose 4B affinity and anion-exchange columns. About 11,000-fold purification was achieved with 90% yield. The specific activity of the final preparation was 140 units per milligram of protein. The purified enzyme gave a single band on both native and SDS-PAGE with a subunit mass of 58 kDa. Its pH optimum was 7.20. The Michaelis constants determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [K(m)(NADPH) = 18 microM, at 30-200 microM NADPH; K(m)(GSSG) = 72 microM, at 40-1000 microM glutathione disulfide, both at saturating concentrations of the second substrate]. The affinity eluent NADPH and its oxidized form NADP+ were successfully removed from the enzyme on the ion-exchange column. The purification method developed is very useful when the enzyme source material is scarce (e.g., in preparations from human tissues) and may find further application in the purification of other NAD(P)H-dependent enzymes which might be inactivated by their affinity eluent(s).

The thiol-proteindisulfide-oxidoreductase--a new marker enzyme of monocytes from peripheral blood.

Monocytes were enumerated by three different methods, cytochemical staining for alpha-naphthyl-acetate esterase-activity, immunofluorescence test using the monoclonal antibody BL-M/G and immunochemical staining for the enzyme thiol-proteindisulfide-oxidoreductase (TPO, EC 1.8.4.2) using a polyclonal rabbit anti-rat TPO immunoglobulin. For the comparison of these methods mononuclear cells from peripheral blood of healthy volunteers, patients with different diseases and adherent cell populations were tested. Statistical analysis showed no differences between the markers within these groups (H-test) and a significant correlation between the numbers of monocytes. TPO was found to be also valid for enumeration of monocytes obtained by adherence methods.

Insulin degradation: radioimmunoassay for glutathione-insulin transhydrogenase and its application.

A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [125I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunoassay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human gamma-globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620 +/- 80 versus 1070 +/- 30 fmol/l, p less than 0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue and lowest in the muscle.

Characterization of an insulin degrading enzyme from cultured human lymphocytes.

An insulin degrading enzyme from cultured human lymphocytes, IM-9 cells, has been purified and characterized. The biochemical, enzymatic and immunological characteristics of this enzyme were all found to be similar to the characteristics of insulin degrading enzymes previously isolated from rat and pig skeletal muscle. Furthermore, this insulin degrading enzyme was found to have no effect on the structure of the insulin receptor nor to be linked to the insulin receptor either on the plasma membrane of cells or when they are shed into the media. The present studies suggest that the IM-9 lymphocytes, which have been extensively used to study the human insulin receptor, may also be a good system for studying human insulin degrading enzymes.

[Isolation of monocytes by adherence to gelatin-layered surfaces]. / Gewinnung von Monozyten durch Adhärenz an Gelatine-beschichteten Flächen.

The method for purification of monocytes using adherence to gelatin coated glass surfaces described by Chien et al. was optimized by drastic shortening the incubation time and modifying the culture media. After one adherence step we obtained monocytes with a purity of 73-78% and a recovery of 53%. Thiol-protein-disulfide-oxidoreductase (TPO), a new enzyme marker of monocytes, was found to be also valid for monocytes obtained by adherence methods. Comparing the number of TPO-containing monocytes with other markers (alpha-naphthylacetate esterase, peroxidase, phagocytosis of latex particles, acridine orange fluorescence, antigens detected by the monoclonal BL-M/G antibody) almost identical values were found.

Insulin-degrading activity in mononuclear and polymorphonuclear circulating leukocytes of nondiabetic and diabetic subjects.

Insulin-degrading activity in mononuclear (MN) and polymorphonuclear (PMN) fractions of circulating leukocytes obtained from 7 nondiabetic and 13 insulin-dependent diabetic subjects was studied. Insulin-degrading activity in both MN and PMN fractions was activated by reduced glutathione and was inhibited completely by N-ethylmaleimide. Both fractions had Michaelis-Menten constant (Km) (insulin) values within the range of values reported for purified glutathione-insulin transhydrogenase (GIT). In double immunodiffusion tests with antibody to human liver GIT, the MN fraction showed immunoprecipitin bands continuous with those of purified liver enzyme, but the PMN fraction showed little or no reaction with the antibody. These data indicate that both leukocyte fractions contain thiol-dependent insulin-degrading activity; however, only in the MN fraction was the degrading activity immunologically similar to that of liver GIT. Kinetic studies showed that the insulin-degrading activity of MN and PMN cells from diabetic patients had a 3.6- and 14.5-fold, respectively, higher maximal capacity (Vmax) than the insulin-degrading activity of these cells from nondiabetic subjects, without any change in the half-saturation constant for the substrate (Km for insulin). These results demonstrate that diabetes and/or insulin therapy result in increased leukocyte glutathione-dependent insulin-degrading activity.