Identification, purification and characterization of a novel collagenolytic serine protease from fig (Ficus carica var. Brown Turkey) latex.

A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0-8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine.

Effect of atypical antipsychotics on antioxidant enzyme activities in human erythrocytes (in vitro study).

OBJECTIVE: This study was set out to examine the impact of atypical antipsychotic drugs: aripiprazole, clozapine, ziprasidone, olanzapine, quetiapine, sertindole and amisulpride on the activity of antioxidant defence enzymes in human erythrocytes in vitro. METHODS: Cu,Zn-superoxide dismutase (SOD1), catalase (CAT), selenium-dependent glutathione peroxidase and glutathione reductase activities were determined after drugs incubation with blood of 15 apparently healthy non-smoking male volunteers (ages 23-39) for 1 h at 37 °C. RESULTS: A statistically significant increase in SOD1 activity was found in samples incubated with aripiprazole (p < 0.01) and quetiapine (p < 0.05) compared with incubated control. SOD1 activity profile following native polyacrylamide gel electrophoresis indicates that aripiprazole and quetiapine protect enzyme activity from inhibition with hydrogen peroxide. Our results showed that sertindole decreases activity of CAT comparing with control non-treated erythrocytes. Moreover, in sertindole treated erythrocytes, negative correlation between SOD1 and glutathione peroxidase activities was found. Increased amount of hydrogen peroxide in such situation may leave erythrocytes and transform their role in circulation from anti-oxidative to pro-oxidative. CONCLUSIONS: Our results indicate that mechanism through sertindole could express its in vivo toxic effects and point toward possible (neuro)protective effects of aripiprazole and quetiapine.

Lipid status, anti-oxidant enzyme defence and haemoglobin content in the blood of long-term clozapine-treated schizophrenic patients.

OBJECTIVE: Despite clozapine's unique effectiveness in patients with schizophrenia, a number of adverse effects have been recognised including abnormalities in lipid and glucose metabolisms. A high clozapine level in red blood cells (RBCs) and disturbed anti-oxidant enzyme activities in blood from schizophrenic patients prompted us to investigate lipid status and anti-oxidant enzyme defence in the blood of chronic schizophrenic patients on long-term clozapine therapy. METHODS: Plasma lipids, RBC anti-oxidant enzyme activities and haemoglobin (Hb) content were measured using established procedures in a group of eighteen chronically-medicated (average 630 days of therapy) schizophrenic patients receiving clozapine (average dose of 295 mg/day) and data were compared with those from a group of eighteen well-matched normal controls. RESULTS: Significantly higher levels of plasma triglycerides (by 47%, p<0.01) and total cholesterol and phospholipids (by 8% and 11%, respectively p<0.05) in patients were found. CuZn-superoxide dismutase (SOD1) activity was markedly higher (by 35%, p<0.001) while selenium-dependent glutathione peroxidase (GSH-Px1) activity was markedly lower (by 41%, p<0.001) in patients. In addition, metHb and HbA1c levels in patients were significantly higher (by 58% and 25%, respectively p<0.001). SOD1 activity was negatively correlated (p<0.001) to GSH-Px1 activity in patients. CONCLUSIONS: The findings support the view that ongoing oxidative stress may be a mechanism by which clozapine induces some adverse effects that increase the risk of diabetes and metabolic syndrome. If valid, this would indicate that in parallel with long-term clozapine treatment, schizophrenic patients could be encouraged to make some lifestyle changes to limit the detrimental effects of the medication.

Dietary lipid intake influences the level of cholesterol bound to haemoglobin in human erythrocytes.

BACKGROUND: Blood cholesterol levels are affected by diet and in particular by the type of fat intake. We originally showed that a significant but variable amount of cholesterol is firmly bound to haemoglobin (Hb) yielding the Hb-lipid adduct (Hb-Ch) in erythrocytes isolated from normo-lipidemic males. AIM OF THE STUDY: To establish whether dietary lipids affect the level of Hb-Ch in human erythrocytes. METHODS: Seventy-four healthy free-living adults were separated according to their serum cholesterol levels into two groups: normo-cholesterolemic (LDL cholesterol <3.4 mmol/l and total cholesterol <5.2 mmol/l) (NC) and hyper-cholesterolemic (LDL cholesterol >or=3.4 mmol/l) (HC). Habitual dietary information was used to classify subjects in both study groups into sub-groups of low-fat (30% total energy as fat). The NC low-fat consumers were placed on a high-lipid (high-fat and high-cholesterol) diet whereas the HC subjects with high-fat intake were assigned to a low-lipid (low-fat and low-cholesterol) diet. Both types of dietary intervention were allowed to continue for 6 weeks. The main variable under scrutiny was the Hb-Ch concentration. RESULTS: In both study groups low-fat intake subjects had low levels of Hb-Ch (approx. 0.35 mmol/l RBC) compared with high-fat intake subjects (approx. 0.60 mmol/l RBC), and serum cholesterol was not correlated with Hb-Ch. The two dietary interventions produced substantial changes in the Hb-Ch level that paralleled variation in the serum cholesterol concentration. A high-lipid diet (35% fat, 15% saturated; 580 mg cholesterol) increased Hb-Ch (by approximately 47%, P<0.001) in subjects with low Hb-Ch at onset, whereas a low-lipid diet (28% fat, 9% saturated; 280 mg cholesterol) decreased Hb-Ch (by approximately 40%, P<0.001) in subjects with high Hb-Ch at onset. CONCLUSION: High consumption of dietary lipids, including saturated fat and cholesterol, has an important influence on the level of Hb-Ch in human erythrocytes.

Efflux of cholesterol and phospholipids derived from the haemoglobin-lipid adduct in human red blood cells into plasma.

OBJECTIVE: The interior of red blood cells (RBCs) contains a variable amount of cholesterol and phospholipids bound to haemoglobin (Hb). This current study was devised to determine if this pool of lipids (termed Hb-Ch) was available for exchange with plasma lipoproteins. DESIGN AND METHODS: We studied the in vitro efflux of lipids from human RBCs into fasting plasma in men with either low (control group) or high Hb-Ch (study group). RESULTS: When plasma was incubated with a two-fold excess of autologous RBCs the plasma cholesterol level increased due to a decrease in the level of cholesterol from the RBC membrane (in the control group) and due to a decrease in the level of cholesterol both from the RBC membrane and the Hb-Ch fraction (in the study group). The loss of Hb-Ch-derived phospholipids during lipid efflux was roughly equal to that of Hb-Ch-derived cholesterol. The loss of RBC cholesterol into plasma high-density lipoproteins (HDL) was more pronounced in our study group and correlated with the loss of cholesterol from Hb-Ch. CONCLUSION: The Hb-Ch adduct significantly contributes to the lipid efflux from RBCs into plasma. The majority of cholesterol released from Hb-Ch appears in the plasma HDL fraction suggesting that Hb-Ch may play a role in reverse cholesterol transport in vivo.

Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide.

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (*NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed *NO disproportionation (dismutation) into nitrosonium (NO+) and nitroxyl (NO-) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to *NO, MnSOD-derived NO- species initiate the formation of peroxynitrite (ONOO-) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO- decomposition and ONOO(-)-dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO- is accompanied by the formation of substantial amounts of H2O2. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of *NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of *NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of *NO.

Could cholesterol bound to haemoglobin be a missing link for the occasional inverse relationship between superoxide dismutase and glutathione peroxidase activities?

The concept of an anti-oxidant defence system as a means to prevent oxidative cell damage implies balanced activities of anti-oxidant defence enzymes. As well as positive correlations between anti-oxidant enzyme activities in human erythrocytes, it has been observed that sometimes when glutathione peroxidase activity is increased, CuZn-superoxide dismutase activity is decreased. In our current study we have examined the plasma lipid profile and the anti-oxidant defence enzymes in erythrocytes from humans, pigs, and bulls. We found that a negative correlation existed between CuZn-superoxide dismutase and glutathione peroxidase activities in human erythrocytes when the concentrations of both plasma triglycerides and total cholesterol were high. This correlation was also found in pig erythrocytes, but not in bull erythrocytes. We propose that cholesterol could affect membrane lipid peroxidation and superoxide generation in erythrocytes via the recently found fraction of cholesterol bound to haemoglobin, termed haemoglobin-cholesterol.

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients.

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.

Iron catalyzed conversion of NO into nitrosonium (NO+) and nitroxyl (HNO/NO-) species.

The conversion of NO into its congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, has important consequences in NO metabolism. Dinitrosyl iron complex (DNIC) combined with thiol ligands was shown to catalyze the conversion of NO into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) both in vitro and in vivo. The formation mechanism of DNIC was proposed to involve the intermediate release of nitroxyl. Since the detection of hydroxylamine (as the product of a rapid reaction of HNO/NO- with thiols) is taken as the evidence for nitroxyl generation, we examined the formation of hydroxylamine, RSNO, and nitrite (the product of a rapid reaction of NO+ with water) in neutral solutions containing iron ions and thiols exposed to NO under anaerobic conditions. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione (GSH). The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions, and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. Results described here contribute to the understanding of the role of iron ions in catalyzing the conversion of NO into HNO/NO- and point to the role of uric acid not previously described.

Cholesterol bound to hemoglobin in normal human erythrocytes: a new form of cholesterol in circulation?

OBJECTIVE: To study lipid fraction that is occasionally observed in red blood cell (RBC) hemolysate (supernatants from which membranes were separated). STUDY DESIGN: Plasma lipid profiles, cholesterol (Ch) and phospholipids (PL) in intact RBCs, RBC membranes and hemolysates were examined in young healthy male population in winter and summer. RESULTS: The RBC Ch and PL content was significantly higher than in membranes, both in winter and summer. The "excess" of cholesterol (associated with phospholipid) was bound to hemoglobin yielding Hb-lipid adduct (Hb-Ch), the pools in the RBC membrane remaining virtually unaltered. Levels of hemoglobin-lipid complex (Hb-Ch), which were significantly higher in winter than in summer (30% and 19% of the total Hb, respectively), positively correlated with plasma HDL cholesterol levels. CONCLUSION: To our knowledge, this is the first demonstration of cholesterol binding to Hb. The results suggest influence of plasma lipoprotein metabolism on the formation of Hb-Ch.