Synthesis of tentacle-type magnetic beads as immobilized metal-chelate affinity support for cytochrome c adsorption

Magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads with an average diameter of 100-140 microm were produced by suspension polymerization in the presence of magnetite particles (i.e. Fe3O4). Specific surface area and average pore size of the magnetic beads was found to be 50 m2/g and 819 nm, respectively. Ester groups in the mPHEMA structure were converted to imine groups by reacting with poly(ethyleneimine) (PEI) in the presence of NaH. Amino (-NH2) content of PEI-attached mPHEMA beads was determined as 102 mg PEI/g. Then, Cu2+ ions were chelated on the magnetic beads in the range of 20-793 micromol Cu2+/g. Cytochrome c (cyt c) adsorption was performed on the metal chelating beads from aqueous solutions containing different amounts of cyt c at different pHs, Cu2+ loadings and temperatures. Cyt c adsorption on the mPHEMA/PEI beads was 4.6 mg/g. Cu2+ chelation increased the cyt c adsorption significantly (40.1 mg/g). Adsorption capacity increased with Cu2+ loading and then reached a saturation value. Cyt c adsorption decreased with increasing temperature. Cyt c molecules could be reversibly adsorbed and eluted ten times with the magnetic adsorbents without noticeable loss in their cyt c adsorption capacity. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. In the last part of this article, cyt c adsorption experiments were performed in a magnetically stabilized fluidized bed (MSFB) system at optimum conditions determined from the batch experiments. The adsorption capacity decreased significantly from 46.8 to 15.4 mg/g polymer with the increase of the flow-rate from 0.5 to 4.0 ml/min. The resulting magnetic chelator beads possessed excellent long-term storage stability.     

L-Cysteine influx and efflux: a possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied L-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of L-cysteine and then measured the intracellular free -SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free -SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free -SH depletion on L-cysteine transport is also investigated. We also determined the rate of L-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our L-cysteine influx studies demonstrated that erythrocytes can respond to increases in L-cysteine concentration in the extracellular media and influx L-cysteine in a concentration-dependent manner. Free -SH concentrations in erythrocytes treated with 1 mM L-cysteine reached to 1.64 +/- 0.06 mM in 1 h whereas this concentration reached to 4.30 +/- 0.01 mM in 10 mM L-cysteine treated erythrocytes. The L-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher L-cysteine concentrations displayed a higher efflux process. Outside concentration of free -SH in 1 mM L-cysteine pretreated erythrocytes reached to 0.200 +/- 0.005 mM in 1 h whereas this concentration reached to 1.014 +/- 0.002 with 10 mM L-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of L-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the L-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the L-cysteine efflux.     

The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.     

Impact of adrenal versus ovarian androgen ratio on signs and symptoms of polycystic ovarian syndrome

The aim of this study was to examine the effect of adrenal versus ovarian androgen (dehydroepiandrosterone sulfate/total testosterone [DHEAS/TT]) on clinical presentation and related metabolic disturbances in Turkish women with polycystic ovarian syndrome (PCOS). Two hundred eighty PCOS cases were taken into the study. For all cases, the DHEAS/TT ratio was calculated. The median value of this ratio was 4.40. Patients with an androgen ratio lower than 4.40 were included in Group 1 and cases with a ratio higher than 4.40 were Group 2. The two groups were compared in terms of hormonal, biochemical and clinical parameters. Body mass index and waist circumference were lower, the Ferriman-Gallwey score was higher and the cycle length was shorter in Group 2. High DHEAS level was associated with better lipid profiles and lower levels of inflammatory markers, meaning good metabolic control in these women, in spite of increased hirsutism rates. In patients with PCOS, both androgens are usually high in proportion to each other. Therefore, it would be more meaningful to use the DHEAS/TT ratio for an assessment of the metabolic and phenotypic effects of PCOS.     

Time dependent influence of etonogestrel on the caspase-3 immunoreactivity and apoptotic indexes of rat uterus and ovaries

Apoptosis is necessary for the balance between cell proliferation and loss. Thirty-six Wistar-Albino rats were subjected to investigate apoptotic effect of widely used implantable progestins on ovarian and uterine tissues. Rats were divided into 6 groups. In the first five groups, we applied etonogestrel (IMP) subcutaneous implants (n = 30). The rats in groups were sacrificed sequentially every 10 days after application. The rats in the last group (n = 6) were accepted as controls. Apoptotic index (AI) values and Caspase-3 immunoreactivities of ovaries and uterus were recorded. In IMP groups, AI and Hscore values in stroma and glandular epithelium of uterus, granulosa and teca-lutein cells of the ovary increased with the longer progesterone exposure. Increase in AI and Hscore values were more prominent after 30 days of exposure for teca-lutein cells of ovary. Progestins increased apoptosis in ovaries and uterus by the longer exposure. Apoptosis increased in ovaries by chronic progesterone exposure. The apoptotic effect of progestin on endometrium is clear but long-term systemic application may lead to alterations in ovarian physiology. We evaluated time dependent apoptotic effect of etonogestrel on reproductive physiology and discussed progestins effect from another point of view in this study.     

Chromone containing sulfonamides as potent carbonic anhydrase inhibitors

A series of sulfonamide derivatives incorporating substituted 3-formylchromone moieties were investigated for the inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, and VI. All these compounds, together with the clinically used sulfonamide acetazolamide, were investigated as inhibitors of the physiologically relevant isozymes I, II (cytosolic), and VI (secreted isoform). These sulfonamides showed effective inhibition against all these isoforms with K(I)'s in the range of 0.228 to 118 μM. Such molecules can be used as leads for discovery of novel effective CA inhibitors against other isoforms with medicinal chemistry applications.     

α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO(2) to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K(I)-s of these compounds ranged between 32.7-423 μM against hCA I, 2.13-32.4 μM against hCA II, 13.7-234 μM against hCA IV and 76-278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols including natural products

(2-Bromo-3,4-dimethoxyphenyl) (3,4-dimethoxyphenyl)methanone (10) and its derivatives with Br, one dibromide and isomeric three tribromides, were synthesized. Demethylation of these compounds afforded a series of new bromophenols. Inhibition of human cytosolic carbonic anhydrase II (hCA II) isozyme by these new bromophenols and naturally occurring 3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)benzene-1,2-diol (3), and 5,5'-methylenebis(3,4,6-tribromo-benzene-1,2-diol) (4) was investigated. The synthesized compounds showed carbonic anhydrase inhibitory capacities with IC(50) values in the range of 0.7-372 μM against hCA II. Some bromophenols investigated here showed effective hCA II inhibitory activity and might be used as leads for generating novel carbonic anhydrase inhibitors which are valuable drug candidates for the treatment of glaucoma, epilepsy, gastric and duodenal ulcers, neurological disorders, or osteoporosis.     

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9mM Cd(NO(3))(2).4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination.