Membrane Marker Enzyme Activities Research Articles

Oral administration of Nigella sativa oil attenuates arsenic-induced redox imbalance, DNA damage, metabolic distress, and histopathological alterations in rat intestine

BackgroundExposure to arsenic, a widespread environmental toxin, produces multiple organ toxicity, including gastrointestinal toxicity. Nigella sativa (NS) has long been revered for its numerous health benefits under normal and pathological states. In view of this, the present study attempts to evaluate the protective efficacy of orally administered Nigella sativa oil (NSO) against arsenic-induced cytotoxic and genotoxic alterations in rat intestine and elucidate the underlying mechanism of its action. MethodsRats were categorized into the control, NaAs, NSO, and NaAs+NSO groups. After pre-treatment of rats in the NaAs+NSO and NSO groups daily with NSO (2 ml/kg bwt, orally) for 14 days, NSO treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. Various biochemical parameters, such as enzymatic and non-enzymatic antioxidants, carbohydrate metabolic and brush border membrane marker enzyme activities were evaluated in the mucosal homogenates of all the groups. Intestinal brush border membrane vesicles (BBMV) were isolated, and the activities of membrane marker enzyme viz. ALP, GGTase, LAP, and sucrase were determined. Further, the effect on kinetic parameters viz KM (Michaelis-Menten constant) and Vmax of these enzymes was assessed. Integrity of enterocyte DNA was examined using the comet assay. Histopathology of the intestines was performed to evaluate the histoarchitectural alterations induced by chronic arsenic exposure and/or NSO supplementation. Arsenic accumulation in the intestine was studied by inductively coupled plasma-mass spectroscopy (ICP-MS). ResultsNaAs treatment caused substantial changes in the activities of brush border membrane (BBM), carbohydrate metabolism, and antioxidant defense enzymes in the intestinal mucosal homogenates. The isolated BBM vesicles (BBMV) also showed marked suppression in the marker enzyme activities. Severe DNA damage and mucosal arsenic accumulation were observed in rats treated with NaAs alone. In contrast, oral NSO supplementation significantly alleviated all the adverse alterations induced by NaAs treatment. Histopathological examination supported the biochemical findings. ConclusionNSO, by improving the antioxidant status and energy metabolism, could significantly alter the ability of the intestine to protect against free radical-mediated arsenic toxicity in intestine. Thus, NSO may have an excellent scope in managing gastrointestinal distress in arsenic intoxication.

Thymoquinone supplementation mitigates arsenic-induced cytotoxic and genotoxic alterations in rat liver

Arsenic, a widespread environmental toxin, produces multiple organ toxicity, including hepatotoxicity. Thymoquinone (TQ) is known to restore liver functions in several hepatic injury models. This study aims to assess the mitigative potential of TQ against sodium arsenate (NaAs)-induced cytotoxic and genotoxic alterations in the liver. Rats were randomly distributed to control, NaAs, TQ, and NaAs+TQ groups. NaAs+TQ and TQ group of rats were pre-treated with TQ (1.5 mg/kg bwt, orally) for 14 days, and the treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. The deleterious histological alterations in the liver of arsenic intoxicated animals were accompanied by an upsurge in the activities of serum ALT and AST, the diagnostic indicators of liver injury. NaAs caused pronounced alterations in the activities of membrane marker and carbohydrate metabolic enzymes and the enzymatic and non-enzymatic components of hepatic antioxidant defense. Significant hepatocyte DNA damage and hepatic arsenic accumulation were also observed in arsenic-exposed rats. TQ supplementation alleviated these adverse alterations and improved the overall hepatic metabolic and antioxidant status in NaAs-administered rats. Prevention of oxidative injury could be the key mechanism of TQ-elicited protective effects. TQ may have an excellent scope as a dietary supplement in the management of arsenic-induced hepatic pathophysiology.

Sub-fractions from Carica Papaya Seed Extracts Can Prevent Potassium Bromate- induced Changes in Activities of Renal Brush Border Membrane Enzymes and Some Enzymes of Carbohydrate Metabolism in the Kidney of Rats

Aim: To investigate the effect of the chromatographic fractions of Carica papaya seed on KBrO3 –induced reduction in the activities of renal brush border membrane (BBM) marker enzymes and the changes in activities of some enzymes of carbohydrate metabolism in the kidney of rats.
 Study Design: twenty male Wistar rats were divided into four groups, five rats per group; normal control, KBrO3 control, papaya fraction control and KBrO3 group administered with 126mg/kg body weight of the most active fraction of partially purified methanol extract of C. papaya for 48 hours.
 Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria.
 Methodology: The activities of renal BBM marker enzymes: γ-glutamyl transferase, alkaline phosphatase, maltase and leucine aminopeptidase were assayed in homogenates of renal cortex and medulla, and in brush border membrane vesicle (BBMV) isolated from cortex using standard methods. Furthermore, activities of the following enzymes representing different pathways of carbohydrate metabolism were determined in renal homogenates: hexokinase (HK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose 6-phosphatase (G6P), fructose 1,6-bisphosphatase (FBP), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME).
 Results: KBrO3 administration significantly (P<0.05) decreases the activities of all the BBM marker enzymes in renal homogenates and BBMV. It also decreases the activities of MDH, G6P, FBP and G6PD, and significantly increases (P<0.05) that of HK, LDH and ME in renal homogenates however co-administration of most active fraction of C. papaya seed prevented all the KBrO3 -induced changes in these biochemical parameters.
 Conclusion: Chromatographic fractions of C. papaya seed extract possesses potent phytochemicals that could prevent KBrO3 –induced reduction in activities of renal BBM marker enzymes and the changes in enzymes of carbohydrate metabolism studied and therefore could be analyzed further to isolate the bioactive compounds.

Thymoquinone supplementation ameliorates cisplatin-induced hepatic pathophysiology.

Hepatotoxicity is a major dose-limiting side effect of CP chemotherapy besides nephrotoxicity and gastrointestinal dysfunction. TQ, a principal Nigella sativa seed oil constituent, has been shown to improve hepatic functions in various in vivo models of acute hepatic injury. In view of this, the present study aimed to evaluate the effect of TQ against CP-induced hepatotoxicity. Rats were divided into four experimental groups; control, CP, CP+TQ and TQ. Animals in CP+TQ and TQ groups were administered TQ (1.5 mg/kg bwt, orally), with or without a single hepatotoxic dose of CP (6 mg/kg bwt, i.p.) respectively, for 14 days before and four days following the CP treatment. CP induced an upsurge in serum ALT and AST activities, indicating liver injury, as also confirmed by the histopathological findings. CP caused significant alterations in the activities of membrane marker enzymes, carbohydrate metabolic enzymes, and the enzymatic and nonenzymatic components of the antioxidant defense system. TQ supplementation ameliorated all these adverse biochemical and histological changes in CP-treated rats. Thus, TQ may have excellent scope for clinical applications in combating CP-induced hepatic pathophysiology.

Oral Nigella sativa oil administration alleviates arsenic-induced redox imbalance, DNA damage, and metabolic and histological alterations in rat liver.

Arsenic, an omnipresent environmental contaminant, is regarded as a potent hepatotoxin. Nigella sativa oil (NSO) consumption has been shown to improve hepatic functions in various in vivo models of acute hepatic injury. The present study evaluates the protective efficacy of NSO against sodium arsenate (As)-induced deleterious alterations in the liver. Male Wistar rats were divided into four groups, namely, control, As, NSO, and AsNSO. After pre-treating rats in AsNSO and NSO groups with NSO (2 mL/kg bwt, orally) for 14 days, NSO treatment was further extended for 30 days, with and without As treatment (5 mg/kg bwt, orally), respectively. As induced an upsurge in serum ALT and AST activities indicating liver injury, as also confirmed by the histopathological findings. As caused significant alterations in the activities of membrane marker enzymes and carbohydrate metabolic enzymes, and in the vital components of antioxidant defense system. Marked DNA damage and hepatic arsenic accumulation were also observed in As-treated rats. Oral NSO administration ameliorated these deleterious alterations and improved overall hepatic antioxidant and metabolic status in As-treated rats. Prevention of oxidative damage could be the underlying mechanism of NSO-mediated protective effects. The results suggest that NSO could be a useful dietary supplement in the management of arsenic hepatotoxicity.

Dose-Dependent Chemopreventive Effect of Methanol Extract of Carica papaya Seed on Potassium Bromate- Induced Nephrotoxicity in Rats

Aim: To investigate the effect of Carica papaya seed extract on KBrO3 - induced nephrotoxicity in rats. Renal toxicity was induced by a single oral dose of 100 mg/kg body weight of KBrO3.
 Study Design: Thirty (30) male albino rats were divided into six groups, five rats per group; normal control, KBrO3 control, papaya control and KBrO3 group administered with methanol seed extract of 200 mg/kg, 400 mg/kg and 600 mg/kg body weight for 48 hours.
 Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria, from April 2018 to August 2018.
 Methodology: Serum urea, creatinine, uric acid and electrolytes were determined using kits from randox laboratories. Furthermore, activities of renal brush border membrane marker enzymes namely γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), maltase (Mal) and leucine aminopeptidase (LAP) and some parameters of oxidative stress including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and malondialdehyde (MDA) were determined in homogenates prepared from renal cortex and medulla of the kidney of rats using colorimetric methods.
 Results: Administration of KBrO3 significantly (P<0.05) increases the serum levels of urea, creatinine, uric acid and all electrolytes studied in a dose-dependent fashion from 200mg/kg to 400mg/kg and 600mg/kg in that order. Furthermore, the activities of GGT, ALP, Mal and LAP decrease in renal homogenates with KBrO3 administration. Also the activities of CAT, SOD, GPx and level of GSH decreases while the level of MDA significantly (P<0.05) increases however concurrent administration of Carica papaya seed extract prevented all the KBrO3- induced changes in the biochemical parameters studied .
 Conclusion: It was suggested that methanol seed extract of Carica papaya possess nephroprotective effect against KBrO3 –induced renal toxicity and oxidative stress, and the most effective dose was 600 mg/kg body weight.

Dillenia indica fruit prevents cisplatin-induced kidney injury in experimental rats through modulation of oxidative stress, marker enzyme, and biochemical changes

BackgroundDillenia indica fruit is known for its numerous health benefits in folk medicine including its use to cure kidney diseases. The present study was designed to investigate the protective effect of D. indica fruit extracts on cisplatin-induced nephrotoxicity.MethodsA nephrotoxic dose of cisplatin (3 mg/kg b.w./day, i.p) was administered every fifth day to the animals receiving vehicle or fruit extracts (methanol, ethyl acetate, and petroleum ether) daily for 25 days. Rats were sacrificed on the 25th day, and the effect of extracts was assessed by determining the alterations in various serum and urine parameters, membrane-bound enzyme, and antioxidant defense system in kidney tissue.ResultsIncrease in serum urea, uric acid, creatinine, blood urea nitrogen, phospholipid, and cholesterol and a decrease in urine urea, uric acid, creatinine, and creatinine clearance rate were reported in the cisplatin control group. Cisplatin alters electrolyte balance, brush border membrane marker enzyme (i.e., alkaline phosphatase, γ-glutamyl transferase, leucine aminopeptidase) activity, and redox balance significantly. Methanol and ethyl acetate extracts of D. indica fruit produced beneficial effect and ameliorated serum and urine parameter to normal. Extract administration increases Na+/K+-ATPase activity and different enzymatic and non-enzymatic antioxidants positively, whereas lipid peroxidation reduced significantly. Extracts exhibited a potent in vitro antioxidant activity.ConclusionTaking into account these results, it can be assumed that D. indica fruit could be the future key candidate which may maximize the clinical use of cisplatin in the treatment of different cancer without nephrotoxicity.

Oral administration of Nigella sativa oil and thymoquinone attenuates long term cisplatin treatment induced toxicity and oxidative damage in rat kidney

Cisplatin (CP) is an effective anti-cancer drug which causes remarkable toxicity to the kidney, particularly to proximal tubules, by generating reactive oxygen species. Nigella sativa (NS), commonly known as “black cumin” reduces the progression of various kidney disorders. Thymoquinone (TQ), the major bioactive constituent of NS seeds, has been credited for various pharmacological effects of NS. Since, a typical clinical CP dosing regimen involves CP administration in multiple cycles over a long time duration, hence the present study aimed to evaluate the renoprotective efficacy of NS oil and TQ against multiple dose CP treatment induced deleterious biochemical and histological alterations in rat kidney. Adult male Wistar rats were divided into six groups viz. control, CP, CPNSO, CPTQ, NSO and TQ. Animals in CPNSO and CPTQ groups were pre-administered NSO (2ml/kg bwt, orally) and TQ (1.5mg/kg bwt, orally) respectively for 14 days and were then treated with CP (3mg/kg bwt, i.p), every fourth day for 20 days while still receiving NSO/TQ. NSO and TQ administration, prior to and along with CP treatment, attenuated CP induced renal functional impairment as evident by significantly restored serum creatinine and blood urea nitrogen levels. CP treatment alone led to significant decline in the specific activities of brush border membrane (BBM) marker enzymes viz. ALP (−46.64%), GGTase (−50.24%) and LAP (−42.15%), while NSO or TQ administration to CP treated rats significantly prevented the decline in the activities of these enzymes in isolated BBM vesicles (BBMVs) as well as in the homogenates of renal cortex and medulla. Furthermore, both NSO and TQ administration also mitigated the CP induced perturbations in renal metabolic and redox status. Histological studies supported these biochemical results showing significant attenuation of CP induced kidney damage in CPNSO and CPTQ cotreated groups. Thus, NSO and TQ have excellent scope for use as functional food or combinatorial nutraceuticals in CP chemotherapy to ameliorate the accompanying nephropathy in long term cancer chemotherapy.

Optical Manipulation Reveals Strong Attracting Forces at Membrane Contact Sites between Endoplasmic Reticulum and Chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies

Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua, Manduca sexta, and Helicoverpa armigera. Higher membrane protein recovery was always obtained from lyophilized midguts compared to frozen midguts, and similar membrane marker enzyme activities were found in BBMV from either treatment. Comparable equilibrium dissociation constants and binding site concentrations, calculated from binding experiments with labeled 125I-Cry1Ab toxin, were found using BBMV from either method. In the light of these results, lyophilization is a good preserving method of lepidopteran midguts to study binding of B. thuringiensis Cry toxins.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

The Malonyl-CoA-sensitive Form of Carnitine Palmitoyltransferase Is Not Localized Exclusively in the Outer Membrane of Rat Liver Mitochondria

The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15-19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane "ghosts," contained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.

Specific Non-Genomic, Membrane-Localized Binding Sites for Progesterone in the Bovine Corpus Luteum1

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

Role of glutathione on renal mitochondrial status in hyperoxaluria.

Role of glutathione on kidney mitochondrial integrity and function during stone forming process in hyperoxaluric state was investigated in male albino rats of Wistar strain. Hyperoxaluria was induced by feeding ethylene glycol (EG) in drinking water. Glutathione was depleted by administering buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis. Glutathione monoester (GME) was administered for supplementing glutathione. BSO treatment alone or along with EG, depleted mitochondrial GSH by 40% and 51% respectively. Concomitantly, there was remarkable elevation in lipid peroxidation and oxidation of protein thiols. Mitochondrial oxalate binding was enhanced by 74% and 129% in BSO and BSO + EG treatment. Comparatively, EG treatment produced only a 33% increase in mitochondrial oxalate binding. Significant alteration in calcium homeostasis was seen following BSO and BSO + EG treatment. This may be due to altered mitochondrial integrity and function as evidenced from decreased activities of mitochondrial inner membrane marker enzymes, succinate dehydrogenase and cytochrome-c-oxidase and respiratory control ratio and enhanced NADH oxidation by mitochondria in these two groups. NADH oxidation (r = -0.74) and oxalate deposition in the kidney (r = -0.70) correlated negatively with mitochondrial glutathione depletion. GME supplementation restored normal level of GSH and maintained mitochondrial integrity and function, as a result of which oxalate deposition was prevented despite hyperoxaluria. These results suggest that mitochondrial dysfunction resulting from GSH depletion could be a contributing factor in the development of calcium oxalate stones.

Reactive oxygen species-mediated tissue injury in experimental ascending pyelonephritis

Pyelonephritis is the most common urinary tract infection in females, but the pathogenetic mechanisms are not well understood. Reactive oxygen species (ROS) have been implicated as cause of injury in several renal diseases. In this study, we have demonstrated the role of ROS in pathogenesis of pyelonephritis in Balb/c mice. A clear correlation between extent of ROS generation and subsequent lipid peroxidation and DNA damage in kidneys was observed during the course of infection, from 2 to 14 days. Activities of brush border membrane marker enzymes were also significantly altered. Administration of antioxidants, superoxide dismutase, catalase and dimethylsulfoxide significantly reversed the histopathological changes, reduced the extent of lipid peroxidation in renal brush border membrane, and also reversed the altered enzyme activities to near normal situation. These results clearly suggest that interaction of ROS with various cellular organelles in kidneys has a significant deleterious effect, and this could be the underlying mechanism for renal dysfunction in pyelonephritis.

A survey of gtp-binding proteins and other potential key regulators of exocytotic secretion in eosinophils : Apparent absence of rab3 and vesicle fusion protein homologues

We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (G alpha i3, G alpha o, G alpha q11, G alpha s and G beta subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (G alpha i1 and G alpha z) could not be detected. Separation of subcellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while G beta subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.

Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier

Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that gamma-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5'-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Na+/K(+)-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K(+)-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was already investigated.

Separation of K+-and Cl−-selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots were separated by isopycnic sucrose density gradient centrifugation. The ion channels present in gradient fractions were assayed by reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers (PLB) and the distributions of ion channel activities were compared with membrane markerenzyme activities. A number of ion channel activities were observed and could be distinguished on the combined bases of their conductance, selectivity, kinetics and pharmacology. A voltage-dependent maxi (498 pS) cation-channel, a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K + channels, and a 148-pS Cl − channel (all unitary conductances determined in asymmetrical cis: trans 325: 100 mM KCl) colocalized with the plasma membrane marker-enzyme, vanadatesensitive ATPase. A weakly K + -selective (108 pS) channel, a 1249-pS cation-channel and a 98-pS K + channel colocalized with the tonoplast markerenzyme, nitrate-sensitive ATPase. A 706-pS K + channel colocalized with the expected distribution of intact plastids and a 38-pS Cl − channel colocalized with either plastid or ER membranes. The membrane location of several other channels including a hypervoltage-sensitive maxi (497 pS) cation-channel, a 270-pS K + channel, an 8-pS K + channel and a 4-pS K + channel was equivocal, but they were tentatively assigned to the Golgi. Thus, the plasma membrane and tonoplast origin of ion channels previously characterized following the incorporation of plasma membrane prepared by aqueous-polymer two-phase partitioning or tonoplast derived from isolated vacuoles into PLB was confirmed and the ion channel complement of previously unassayed membranes was defined. This demonstrates the usefulness of PLB in identifying and characterizing ion channels from plant cell membranes, in particular, those of membranes which are inaccessible to patch-clamp electrodes