A 43 kD protein isolated from the herb Cajanus indicus L attenuates sodium fluoride-induced hepatic and renal disorders in vivo

The herb, Cajanus indicus L, is well known for its hepatoprotective action. A 43 kD protein has been isolated, purified and partially sequenced from the leaves of this herb. A number of in vivo and in vitro studies carried out in our laboratory suggest that this protein might be a major component responsible for the hepatoprotective action of the herb. Our successive studies have been designed to evaluate the potential efficacy of this protein in protecting the hepatic as well as renal tissues from the sodium fluoride (NaF) induced oxidative stress. The experimental groups of mice were exposed to NaF at a dose of 600 ppm through drinking water for one week. This exposure significantly altered the activities of the antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and the cellular metabolites such as reduced glutathione (GSH), oxidized glutathione (GSSG), total thiols, lipid peroxidation end products in liver and kidney compared to the normal mice. Intraperitoneal administration of the protein at a dose of 2 mg/kg body weight for seven days followed by NaF treatment (600 ppm for next seven days) normalized the activities of the hepato-renal antioxidant enzymes, the level of cellular metabolites and lipid peroxidation end products. Post treatment with the protein for four days showed that it could help recovering the damages after NaF administration. Time-course study suggests that the protein could stimulate the recovery of both the organs faster than natural process. Effects of a known antioxidant, vitamin E, and a non-relevant protein, bovine serum albumin (BSA) have been included in the study to validate the experimental data. Combining all, result suggests that NaF could induce severe oxidative stress both in the liver and kidney tissues in mice and the protein possessed the ability to attenuate that hepato-renal toxic effect of NaF probably via its antioxidant activity.     

Protective effect of a 43 kD protein from the leaves of the herb, Cajanus indicus L on chloroform induced hepatic-disorder

Cajanus indicus is a herb with medicinal properties and is traditionally used to treat various forms of liver disorders. Present study aimed to evaluate the effect of a 43 kD protein isolated from the leaves of this herb against chloroform induced hepatotoxicity. Male albino mice were intraperitoneally treated with 2 mg/kg body weight of the protein for 5 days followed by oral application of chloroform (0.75 ml/kg body weight) for 2 days. Different biochemical parameters related to physiology and pathophysiology of liver, such as, serum glutamate pyruvate transaminase and alkaline phosphatase were determined in the murine sera under various experimental conditions. Direct antioxidant role of the protein was also determined from its reaction with Diphenyl picryl hydroxyl radical, superoxide radical and hydrogen peroxide. To find out the mode of action of this protein against chloroform induced liver damage, levels of antioxidant enzymes catalase, superoxide dismutase and glutathione-S-transferase were measured from liver homogenates. Peroxidation of membrane lipids both in vivo and in vitro were also measured as malonaldialdehyde. Finally, histopathological analyses were done from liver sections of control, toxin treated and protein pre- and post-treated (along with the toxin) mice. Levels of serum glutamate pyruvate transaminase and alkaline phosphatase, which showed an elevation in chloroform induced hepatic damage, were brought down near to the normal levels with the protein pretreatment. On the contrary, the levels of antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferase that had gone down in mice orally fed with chloroform were significantly elevated in protein pretreated ones. Besides, chloroform induced lipid peroxidation was effectively reduced by protein treatment both in vivo and in vitro. In cell free system the protein effectively quenched diphenyl picryl hydroxyl radical and superoxide radical, though it could not catalyse the breakdown of hydrogen peroxide. Post treatment with the protein for 3 days after 2 days of chloroform administration showed similar results. Histopathological studies indicated that chloroform induced extensive tissue damage was less severe in the mice livers treated with the 43 kD protein prior and post to the toxin administration. Results from all these data suggest that the protein possesses both preventive and curative role against chloroform induced hepatotoxicity and probably acts by an anti-oxidative defense mechanism.     

Cardioprotective effect of alpha-mangostin, a xanthone derivative from mangosteen on tissue defense system against isoproterenol-induced myocardial infarction in rats

Increased oxidative stress and antioxidant deficit have been suggested to play a major role in isoproterenol-induced myocardial infarction. The present study was designed to evaluate the effect of alpha-mangostin on the antioxidant defense system and lipid peroxidation against isoproterenol-induced myocardial infarction in rats. Induction of rats with ISO (150 mg/kg body weight, ip) for 2 days resulted in a marked elevation in lipid peroxidation, serum marker enzymes (LDH, CPK, GOT, and GPT) and a significant decrease in the activities of endogenous antioxidants (SOD, CAT, GPx, GST, and GSH). Pre-treatment with alpha-mangostin (200 mg/kg of body weight per day) orally for 6 days prior to the ISO administration and 2 days along with ISO administration significantly attenuated these changes when compared to the individual treatment groups. These findings indicate the protective effect of alpha-mangostin on lipid peroxidation and antioxidant tissue defense system during ISO-induced myocardial infarction in rats.     

The effect of menadione-induced oxidative stress on the in vivo reactive oxygen species and antioxidant response system of Phanerochaete chrysosporium

The antioxidant response system of Phanerochaete chrysosporium against menadione-induced oxidative stress was investigated in this study. The superoxide anion radical levels in tested menadione-supplemented conditions generally decreased over the incubation period. The level of hydrogen peroxide and the activities of NAD(P)H oxidase, superoxide dismutase (SOD) and catalase (CAT) were higher than those in the controls at all incubation times. The highest NADH and NADPH oxidase activities were determined to be 4.9- and 5.0-fold higher than those in the control, respectively in cells exposed to 0.75 mM menadione. The SOD and CAT activities increased with increasing menadione, and their highest activities were 5.4- and 5.1-fold higher than those in the control, respectively. In 0.1–0.5 mM menadione exposed cells, the lipid peroxidation levels did not change significantly when compared to each other, except 8th hour of incubation (p > 0.01). Our result shows that although menadione induces the formation of reactive oxygen species, the antioxidant response system of P. Chrysosporium is able to negate menadione-induced oxidative stress up to relatively high menadione concentrations, as 0.75 mM. These results are important to determine the effects of menadione, as a medicine, on the antioxidant response system of eukaryotic models and the resulting level of damage.     

Prophylactic role of D-Saccharic acid-1,4-lactone in tertiary butyl hydroperoxide induced cytotoxicity and cell death of murine hepatocytes via mitochondria-dependent pathways

D-Saccharic acid 1,4-lactone (DSL) is a derivative of D-glucaric acid. It is a beta-glucuronidase inhibitor and possesses anticarcinogenic, detoxifying, and antioxidant properties. In the present study, the protective effects of DSL were investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity and cell death in vitro using murine hepatocytes. Exposure of TBHP caused a reduction in cell viability, enhanced the membrane leakage, and disturbed the intracellular antioxidant machineries in murine hepatocytes. Investigating the signaling mechanism of TBHP-induced cellular pathophysiology and protective action of DSL, we found that TBHP exposure disrupted mitochondrial membrane potential, facilitated cytochrome c release in the cytosol, and led to apoptotic cell death via mitochondria-dependent pathways. DSL counteracted these changes and maintained normalcy in hepatocytes. Combining, results suggest that DSL possesses the ability to ameliorate TBHP-induced oxidative insult, cytotoxicity, and apoptotic cell death probably due to its antioxidant activity and functioning via mitochondria-dependent pathways.     

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.     

Marker enzyme assessment in the liver of cyprinus carpio (L.) exposed to 2,4-D and azinphosmethyl

The potential utility of antioxidant enzymes and lipid peroxidation as indicators of exposure to 2,4-D and azinphosmethyl together with the toxic effects of these compounds in freshwater fish Cyprinus carpio were evaluated. Biochemical parameters were recorded spectrophotometrically in fish liver, which were exposed to a single dose of 2,4-D and azinphosmehtyl (1/3 LC(50)), and their mixture at 1:1 ratio for 24, 48, 72, and 96 h. The most sensitive parameter was glutathione S-transferase (GST) activity, which significantly increased with experimental exposures. Glucose 6-phosphate dehydrogenase activity did not change after 24 and 48 h while there was an elevation after 72 h in all exposure groups. The activity decreased only when these were applied in combination at 96 h. Superoxide dismutase activity increased after azinphosmethyl exposure for 48 and 96 h. 2,4-D decreased the activity after 24 h while the activity remained at the same level with control after 48 h. An elevation was found between 72 and 96 h. Mixture treatment did not changed the activity. Glutathione reductase and catalase enzyme activities, and malondialdehyde levels remained constant in all the treatment groups compared with controls. These results suggest that induction of GST activity may be used as biomarker for the assessment of water pollution in C. carpio.     

In vitro and in vivo studies on the antioxidant activity of fish peptide isolated from the croaker (Otolithes ruber) muscle protein hydrolysate

Peptide from croaker (Otolithes ruber) muscle protein hydrolysate was purified, characterized and evaluated for its in vitro and in vivo antioxidant activity. Results showed that purified peptide contained the amino acid sequence as Lys-Thr-Phe-Cys-Gly-Arg-His (861.6Da), which were expected to contribute to its antioxidant activities. This peptide efficiently quenched 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals (84.5±1.2 and 62.4±2.9%), and successfully inhibits the lipid peroxidation and DNA damage and proven to be a potent antioxidant at different in vitro systems. It also improved the endogenous cellular antioxidant enzymes in Wistar rat by increasing the activities of catalase (CAT), glutathione-S-transferase (GST) and superoxide dismutase (SOD) after supplementation of the peptide (283.6±7.25, 4.3±0.78 and 28.42±1.97) compared to the negative control (196.4±5.65, 1.3±0.45 and 15.1±0.35). Therefore, croaker muscle peptide can increase an endurance capacity and facilitate recovery from oxidative stress.     

Crystal structure of reduced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer and a non-disulfide-bonded intermediate in the catalytic cycle

Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.     

Antihyperglycemic effect of a new thiazolidinedione analogue and its role in ameliorating oxidative stress in alloxan-induced diabetic rats

Thiazolidinediones (TZDs) are a new class of antidiabetic drugs, having an insulin sensitizing effect in patients with type 2 diabetes. The contribution of oxidative stress from the standpoint of lipid and protein damage, alteration in endogenous antioxidant enzymes and effects of newly synthesized compounds, 5-[4-2-(6,7-Dimethyl-1,2,3,4-tetrahydro-2-oxo-4-quinoxalinyl)ethoxy]phenyl]methylene]thiazolid- ine-2,4-dione, (C(1)) in normal/alloxan-induced diabetic rats form the focus area of this study. Its effect was compared to two well-known TZDs, namely pioglitazone and rosiglitazone. It has been concluded from results that after thirty days of administration of C(1), Pg and Rg in alloxan-induced diabetic animal groups, the blood glucose level decreased, more remarkably in C(1) treated group. Also oxidative damage has been studied by estimating hepatic superoxide dismutase (SOD) activity, which was found to be increased (p<0.001 vs. control). An inverse change in SOD values between hepatic and pancreatic/kidney tissues were observed. Treatment with the test compounds lowered the activity of SOD in liver while increased its activity in kidney and pancreas. Similar normalizing effect of C(1) on liver, pancreatic and renal catalase (CAT)/ glutathione peroxidase (GPx) activities were pronounced in diabetic rats (p<0.001 vs. diabetic rats). Decreased reduced glutathione (GSH) content, found in diabetic animals, was significantly elevated to normal levels by C(1) treatment. The treatment with C(1) also decreased the levels of nitric oxide and increased the activities of glutathione-s-transferase and glutathione reductase, as compared to diabetic animals. Evidence of oxidative damage to lipids and proteins was shown through the quantification of protein carbonyl (in tissues) and malondialdehyde levels (both serum and tissues). It was observed that the protein/lipid damage in diabetic rats was improved by treatment with C(1). Total antioxidant activity (TAA) was found to be enhanced in C(1) treated rats (p>0.05 vs. group3, p<0.001 vs. group2, p<0.001 vs. group 4). These results suggest that the newly synthesized TZD derivative (C(1)) has a potential to act as antihyperglycemic and antioxidant agent. In addition, for all parameters checked, it has better efficacy than rosiglitazone and is as effective as pioglitazone.     

Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation.     

The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop’s region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections.     

Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant

 We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for IFN-γ- and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases. Received: 27 July 2000 / Accepted: 13 March 2001     

Increase in antioxidant enzyme activity,stress tolerance and biocontrol efficacy of Pichia kudriavzevii with the transition from a yeast-like to biofilm morphology

Morphological change, such as from yeast-like to biofilm, has been recently considered to be involved in the mode of action of some antagonistic yeasts used as postharvest biocontrol agents. In the present study, the biocontrol yeast, Pichia kudriavzevii, reversibly shifted from a yeast-like morphology on yeast peptone dextrose (YPD) medium with 2% agar to a biofilm morphology on YPD with 0.3% agar. The tolerance of P. kudriavzevii to heat and oxidative stresses, as well as the biocontrol efficacy against postharvest diseases on pear fruit, increased significantly from the yeast-like form to the biofilm form. The activity of antioxidant enzymes, including catalase and superoxidase dismutase, in the biofilm form was also significantly higher. The elevated activity of antioxidant enzymes was associated with less protein and lipid oxidation in the biofilm form, compared to the yeast-like form, under heat and oxidative stresses. These results suggest that activation of antioxidant system with the morphology shift contributes to the enhancement of abiotic stress tolerance and biocontrol performance of P. kudriavzevii. These findings provide new information on the biology of yeast antagonists that is essential for their potential application and development.     

Caffeic acid derivatives isolated from the aerial parts of Galinsoga parviflora and their effect on inhibiting oxidative burst in human neutrophils

Four new caffeoyl -glucaric and -altraric acid derivatives along with eleven known compounds were isolated from aerial parts of Galinsonga parviflora. Their structures were elucidated by high-resolution spectroscopic studies. The four new compounds were determined as being 2,3,4,5-tetracaffeoylglucaric acid (1), 2,4,5-tricaffeoylglucaric acid (2), 2,3,4- or 3,4,5-tricaffeoylaltraric acid (3) and 2,3(4,5)-dicaffeoylaltraric acid (4). A reliable criterion for the determination of the linkage position of caffeic acids moieties in glucaric acid derivatives has been proposed, on the basis of detailed analysis of the respective J-couplings, including substitution and solvent influence on the observed values. All hexaric acids derivatives appeared as inhibitors of reactive oxygen species production by stimulated neutrophils.     

Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers

The response of the glutathione S-transferase (GST, EC 2.5.1.18) and related proteins of Mytilus edulis to environmental pollution load was assessed. Mussels were reciprocally transplanted between an industrial estuary (Douglas), a rural estuary (Youghal) and a m arine site (Bantry). In addition, m ussels were sam pled along a pollution gradient in an estuary receiving leather tannery effluent (Colligan). These latter m ussels were previously shown to be subject to oxidative stress resulting from the discharges. GST specific activity of cytosolic extracts from the gill and digestive gland tissues was determ ined for all anim als. Specific activity was shown to vary significantly in anim als from different sites, with highest specific activity always observed in sam ples (local and transplanted) taken from the industrial site. By com parison, the m ussels exposed to tannery discharges displayed no significant alteration in GST specific activity. Total intracellular glutathione (GSH) was also determ ined for sam ples taken from the Douglas and Youghal estuaries but no correlation with pollution load was observed. Using FPLC analysis, we observed no specific effect on the relative levels of the GST and the individual GST related proteins in gill or digestive gland sam ples from local or reciprocally transplanted anim als from Douglas or Youghal. The increase in GST specific activity observed in samples from the industrial estuary are indicative of a possible, specific inductive agent at this site. The results from the tannery site, by com parison, indicate that general oxidative stress does not result in elevated G ST specific activity in M. edulis.     

The origins of asymmetry in the folding transition states of protein L and protein G

Topology has been shown to be an important determinant of many features of protein folding; however, the delineation of sequence effects on folding remains obscure. Furthermore, differentiation between the two influences proves difficult due to their intimate relationship. To investigate the effect of sequence in the absence of significant topological differences, we examined the folding mechanisms of segment B1 peptostreptococcal protein L and segment B1 of streptococcal protein G. These proteins share the same highly symmetrical topology. Despite this symmetry, neither protein folds through a symmetrical transition state. We analyzed the origins of this difference using theoretical models. We found that the strength of the interactions present in the N-terminal hairpin of protein L causes this hairpin to form ahead of the C-terminal hairpin. The difference in chain entropy associated with the formation of the hairpins of protein G proves sufficient to beget initiation of folding at the shorter C-terminal hairpin. Our findings suggest that the mechanism of folding may be understood by examination of the free energy associated with the formation of partially folded microstates.