Nazmi Özer

Mitochondrial complex I and IV activities in leukocytes from patients with parkin mutations

The parkin protein functions as a RING‐type ubiquitin protein ligase. Considering the possibility that impaired ubiquitin‐proteosomal system activity may impair antioxidant defenses and enhance oxidative stress, we have investigated the activity of mitochondrial respiratory enzymes in patients with parkin gene mutations. A significant decrease in the leukocyte complex I activity was found both in patients with parkin mutations (62.5%) and idiopathic PD (64.5%) compared with age‐matched controls (P < 0.001). Complex IV activity was also decreased significantly in idiopathic PD patients (60%), but no difference was detected between controls and patients with parkin mutations.

Kinetics and equilibria of S-nitrosothiol-thiol exchange between glutathione, cysteine, penicillamines and serum albumin.

The kinetics and equilibria of S‐nitrosothiol‐thiol (SNO—SH) exchange reactions were determined using differential optical absorption. At pH 7.4 and 37°C, k 2 values ranged from 0.9 M−1 · s−1 for the reaction between S‐nitroso‐glutathione (GSNO) and N‐acetyl‐penicillamine, and up to 279 M−1 · s−1 for the exchange between S‐nitroso‐penicillamine (penSNO) and GSH. SNO—SH exchange involving GSH/GSNO and cysteine/cySNO was relatively rapid, k 2 approx. 80 M−1 · s−1 with an equilibrium constant slightly in favour of GSNO. GSNO was strongly favoured in equilibrium with penSNO, k eq 0.0039. In the case of SNO—SH exchange between S‐nitroso human serum albumin (albSNO) and GSH or cysteine k 2 values were 3.2 and 9.1 M−1 · s−1, respectively. The results show that the initial rate of SNO—SH exchange between physiological albSNO (7 μM) and venous plasma levels of GSH and cysteine is very slow, < 1%/min. On the other hand, if a nitrosothiol such as cySNO were to enter a cell, it would be rapidly converted to GSNO (43%/s).

The role of free oxygen radicals in the aetiopathogenesis of rosacea

Summary A possible link between superoxide dismutase activity and malondialdehyde level with the clinical manifestations of rosacea was investigated. We found differences in superoxide dismutase activities between mild rosacea (stages I and II) and severe involvement (stage III) groups, as well as between disease and control groups that were statistically significant (P < 0.05). In the mild involvement group (stages I and II), the superoxide dismutase activity was higher than in the control group (P < 0.05), while the malondialdehyde levels did not differ from the control. In the severe involvement group (stage III), the superoxide dismutase activity was lower than in the control group (P < 0.05), and this was coupled to a raised level of malondialdehyde (P < 0.05). These findings clearly show that in the mild involvement phase of rosacea patients, superoxide dismutase activity was stimulated to protect the skin against reactive oxygen species so that the malondialdehyde levels were maintained. In contrast, in more severe disease, due to a decrease in the capacity of the antioxidant defence system, the malondialdehyde levels were increased. These findings support the ‘antioxidant system defect hypothesis’ in rosacea patients.

Poly(hydroxybutyrate-co-hydroxyvalerate) nanocapsules as enzyme carriers for cancer therapy: an in vitro study

In the present paper, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocapsules were prepared by a double emulsion-solvent evaporation procedure (w/o/w) for the encapsulation of model enzymes (L-asparaginase, catalase, glucose oxidase) and bovine serum albumin. To increase the encapsulation efficiency and activity of the encapsulated enzyme, numerous modifications were made in the compositions of the phases of double emulsion. For the preparation of low molecular weight PHBV, the polymer was treated with sodium borohydride. A 14-fold decrease in molecular weight (from 297 000 to 21 000) was observed upon 4 h of incubation. Although the amount of encapsulated protein was not increased, the enzyme activity increased upon use of low molecular weight PHBV, indicating that these nanocapsules have a higher permeability to solutes (reactants and products). The adjustment of the second water phase to the isoelectric point of the proteins significantly increased the encapsulation yields of catalase, L-asparaginase and BSA. Likewise, polyethylene glycol coupling significantly increased the entrapment efficiency as well as the activity of catalase and L-asparaginase. A combination of the various optimum preparation conditions further increased the encapsulated catalase activity (about sixfold) in comparison to the initial basic conditions (with no modification and no isoelectric point adjustment).

In vivo half life of nanoencapsulated L-asparaginase

In the present study, antileukemic enzyme L-asparaginase (ASNase) was encapsulated into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules in order to decrease the immunogenicity and toxicity of the enzyme and to increase its in vivo half life in mice. Nanocapsules were prepared by water-in-oil-in-water approach and each phase was changed systematically. By changing the pH of the w2 phase to the isolelectric point of L-ASNase, the encapsulation efficiency was increased from 23.7% to 28.0%. Also, modification of ASNase with PEG2 increased the encapsulation efficiency from 23.7% to 27.9% and protected the enzyme against denaturation. Combination of the various optima enabled a substantial increase in the activity (0.074–0.429 U/mg nanocapsule). The enzyme activity in the blood due to unmodified PHBV nanocapsules dropped to 38% of its initial value 4 h after injection. When the same sample was tested for the enzyme content in the circulation by using the radio-labeled enzyme a much lower enzyme (30% of initial) could be detected after a shorter time (3 h). The PHBV nanocapsules with heparin conjugated on their surface had a longer presence in the circulation than unmodified PHBV nanocapsules. After 6 h, around 50% of the enzyme was still present in the blood. Radioactivity measurements using the same sample showed a sharp decrease in enzyme amount in the circulation in the early stages. However, radioactivity was still detectable at the eighth hour. No adverse effects and symptoms of anaphylaxis were observed upon injection of encapsulated ASNase-PHBV nanocapsules to mice i.v. through the tail vein.

The in-vitro and in-vivo characterization of PLGA:L-PLA microspheres containing dexamethasone sodium phosphate.

Dexamethasone sodium phosphate (DSP) is a widely used corticosteroid in the treatment of brain oedema associated with brain tumours. DSP has many side effects that limit its usage at an effective concentration. The objective of this study was to minimize these side effects by encapsulating DSP using biodegradable synthetic polymers, to extend the release time from microspheres and to evaluate the effectiveness in the treatment of brain oedema. Microspheres containing 5% DSP were formulated by the solvent evaporation method by using a 1:1 mixture of two synthetic polymers, poly(lactic-co-glycolic acid) and L-polylactic acid (PLGA and L-PLA). The surface morphologies and particle size distribution of the microspheres were investigated. The in-vitro release studies were performed in pH 7.4 phosphate buffer solution. For determining the effectiveness of microspheres in the treatment of brain oedema, Sprague-Dawley rats weighing 200-250g were used as an animal model. Brain oedema was generated by the cold lesion method, and the effectiveness of the microspheres in treatment of oedema was investigated by the wet-dry weight method, lipid peroxidation ratios and histological evaluations. The average particle size of the microspheres was 13.04 +/- 2.05 microm, and the in-vitro release time of the microspheres was 8 h for 100/release. The degree of oedema was significantly different from the control group for the wet-dry weight method and lipid peroxidation ratio (p < 0.05). Similarly, histological evaluation of the tissues shoved that degree of oedema was significantly decreased with respect to the control group. All these results showed that implantation of microspheres was significantly more effective with respect to the systemic administration of DSP.Dexamethasone sodium phosphate (DSP) is a widely used corticosteroid in the treatment of brain oedema associated with brain tumours. DSP has many side effects that limit its usage at an effective concentration. The objective of this study was to minimize these side effects by encapsulating DSP using biodegradable synthetic polymers, to extend the release time from microspheres and to evaluate the effectiveness in the treatment of brain oedema. Microspheres containing 5% DSP were formulated by the solvent evaporation method by using a 1:1 mixture of two synthetic polymers, poly(lactic-co-glycolic acid) and L-polylactic acid (PLGA and L-PLA). The surface morphologies and particle size distribution of the microspheres were investigated. The in-vitro

Determination of Pseudocholinesterase Activity in the Gingival Crevicular Fluid, Saliva, and Serum from Patients with Juvenile Periodontitis and Rapidly Progressive Periodontitis

By use of a spectrophotometric method, pseudocholinesterase (PCE) activities were determined in gingival crevicular fluid (GCF), saliva, and serum from patients with juvenile periodontitis (JP) and rapidly progressive periodontitis (RPP) and from controls. The PCE activity in the GCF samples was 181 ± 48 U/L in the JP group, 588 ± 135 U/L in the RPP group, and 88.5 ± 29.1 U/L in the control group. Saliva PCE activity levels were 9.1 ± 1.7 U/L in the JP group, 21.8 ± 4.5 UlL in the RPP group, and 12.7 ± 0.8 UlL in the control group. GCF contained a higher PCE activity than saliva but a lower one than that of serum. The RPP group had a significantly higher PCE activity in both the GCF and saliva samples. No significant differences could be found regarding serum enzyme levels. Also, no significant correlations were present between biochemical values and the severity of periodontal disease. GCF may be an important source for the PCE content of saliva. It is suggested that the increased PCE activity seen in RPP patients might be caused by either the direct production of esterases by bacteria or the induction of esterases during periodontal destruction.

A new enzyme-coupled spectrophotometric method for the determination of arginase activity

Abstract A spectrophotometric method for the determination of arginase (EC 3.5.3.1) is presented. Arginase is coupled to urease and glutamate dehydrogenase and the decrease in absorbance at 340 nm due to the oxidation of NADPH is followed. The method is rapid, is sensitive, is economical and permits continuous monitoring. The initial velocities were directly proportional to the enzyme concentrations between 0.06 and 0.30 units per 0.5 ml. The Lineweaver-Burk plot yielded positive allosteric behavior for the tetrameric enzyme (16). The K ′ and the Hill coefficient, n , calculated from Hill plot were found to be 4.7 m m and 1.26 ( r = 1.00), respectively. These values are in good agreement with the literature (12,16,17).

Simple, high-yield purification of xanthine oxidase from bovine milk.

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

A SIMPLE AND SENSITIVE METHOD FOR THE ACTIVITY STAINING OF XANTHINE OXIDASE

Abstract Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 μU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 μU takes about 1 min whereas staining of 0.5 μU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources.