Spleen Enzyme Research Articles

Triterpenes G-A and G-E from Galphimia glauca with anti-inflammatory and anti-arthritic activity in mice

Ethnopharmacological relevanceGalphimia glauca is a medicinal plant that treats inflammatory and anti-rheumatic problems. Its anti-inflammatory capacity has been reported pharmacologically, attributed to the triterpenes G-A and G-E. AimThe objective of the present work was to measure the anti-inflammatory and immunomodulatory effect of the methanolic extract (GgMeOH) of Galphimia glauca and the isolated galphimines G-A and G-E, first in an acute test of plantar edema with carrageenan, and later in the model of experimental-induced arthritis with CFA. The effect was measured by quantifying joint inflammation, the concentration of pro- (TNF-α, IL-6, IL-17) and anti-inflammatory (IL-10, and IL-4) cytokines, and the ADA enzyme in joints, kidneys, and spleen from mice with experimental arthritis. MethodThe extract and the active triterpenes were obtained according to established methods using different chromatographic techniques. Female ICR strain mice were subjected to intraplantar administration with carrageenan and treated with different doses of GgMeOH, G-A, and G-E; edema was monitored at different times. Subsequently, the concentration of TNF-a and IL-10 in the spleen and swollen paw was quantified. Meloxicam (MEL) was used as an anti-inflammatory control drug. The most effective doses of each treatment were analyzed using a complete Freunds adjuvant (CFA)-induced experimental arthritis model. Joint inflammation was followed throughout the experiment. Ultimately, the concentration of inflammation markers, oxidant stress, and ADA activity was quantified. In this experimental stage, methotrexate (MTX) was used as an antiarthritic drug. ResultsTreatments derived from G. glauca, GgMeOH (DE50 = 158 mg/kg), G-A (DE50 = 2 mg/kg), and G-E (DE50 = 1.5 mg/kg) caused an anti-inflammatory effect in the plantar edema test with carrageenan. In the CFA model, joint inflammation decreased with all natural treatments; GgMeOH and G-A inhibited the ADA enzyme in all organs analyzed (joints, serum, spleen, left and right kidneys), while G-E inhibited the enzyme in joints, serum, and left kidney. CFA caused an increase in the weight index of the organs, an effect that was counteracted by the administration of G. glauca treatments, which also modulate the response to the cytokines analyzed in the different organs (IL-4, IL-10, IL-17, IL-6, and TNF- α). ConclusionIt is shown, for the first time, that the GgMeOH extract and the triterpenes G-A and G-E of Galphimia glauca have an anti-arthritic effect (anti-inflammatory, immunomodulatory, antioxidant, and ADA inhibitor), using an experimental arthritis model with CFA. Therefore, knowledge of the plant as a possible therapeutic agent for this rheumatic condition is expanding.

Effects of Elephantopus scaber extract on growth, proximate composition, immunity, intestinal microbiota and resistance of the GIFT strain of Nile tilapia Oreochromis niloticus to Streptococcus agalactiae

This study aimed to investigate the effects of Elephantopus scaber extract on the GIFT (genetic improvement of farmed tilapia) strain of Nile tilapia Oreochromis niloticus. A total of 800 tilapia with an initial body weight of 1.34 ± 0.09 g each were randomly divided into five groups. The tilapia in the control group (E0 group) were fed on a basal diet only. Meanwhile, tilapia in the four experimental groups were fed on a basal diet supplemented with 1 g/kg (E1 group), 3 g/kg (E2 group), 5 g/kg (E3 group), and 7 g/kg (E4 group) of E. scaber extract for 10 weeks. Results showed that the survival rate was higher in the experimental groups than in the control group. Compared with the control group, some growth parameters (FW, WGR, SGR, VSI, and HSI) were significantly improved in the E1 group and E2 group. The crude lipid content in the dorsal muscle and liver was lower in the E1 group than in the control group. After E. scaber extract supplementation, activities of immunity-related enzymes (ACP, AKP, T-AOC, SOD, CAT, GSH-Px and LZM) in plasma, liver, spleen and head kidney, and expressions of immunity-related genes (IL-1β, IFN-γ, TNF-α, and CCL-3) in liver, spleen and head kidney showed various degrees of improvement, while MDA content and Hsp70 expression level were decreased. The survival rate of tilapia increased in all the supplementation groups after Streptococcus agalactiae treatment. E. scaber extract addition changed the species composition, abundance, and diversity of intestinal microbiota in tilapia. These results demonstrate that E. scaber extract supplementation in diet can improve the growth, immunity, and disease resistance of GIFT against S. agalactiae. E. scaber extract supplementation can also change intestinal microbiota and reduce crude lipid content in dorsal muscle and liver. The above indicators show that the optimal dose of E. scaber extract for GIFT is 1 g/kg.

Gene expression and alterations of antioxidant enzymes in spleen of rats exposed to fluoride

BackgroundFluorosis is a slow and progressive process causing metabolic, functional and structural damages affecting many tissues particularly musculoskeletal, dental systems, kidney, liver and brain. It can be rapidly absorbed by passive diffusion through the stomach, small intestine, mouth and skin. Endemic fluorosis is connected to the high concentration of fluoride in drinking water. The present study aimed to evaluate the toxic effects of sodium fluoride (NaF) on splenic activity at the biochemical and molecular level. Materials and methodsWistar albino rats were randomly assigned to three groups. The control rats were given 1 ml deionized water orally for 40 days. Groups II and III were administered 300 and 600 mg NaF/kg b.w. /day for the same period. Animals were sacrificed under ether anaesthesia. The spleen tissue was excised and used for biochemical and real-time PCR analysis. The level of fluoride, malondialdehyde (MDA), reduced glutathione (GSH) and activities of different antioxidant enzymes such as cytosolic copper/zinc superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx) and catalase (CAT) were determined. The analysis of gene expression of Cu/Zn SOD, GPx and CAT in spleen was done using Real-time PCR. ResultsThe levels of fluoride and MDA were significantly (P < 0.0001) increased where as GSH content decreased significantly (P < 0.0001) in the spleen of fluoridated rats. The activities of antioxidant enzymes viz; Cu/Zn SOD, GPx and CAT declined significantly (P < 0.0001) compared to the control. Pearson’s bivariate correlation and simple linear regression analysis exhibited strong positive correlation between levels of splenic tissue fluoride and MDA(r = 0.985) while negative correlations existed in GSH (r = −0.907) as well as activities of Cu/Zn SOD (r = −0.982), GPx (r = −0.966), and CAT (r = −0.935). The gene expression of Cu/Zn SOD, GPx and CAT were significantly (P < 0.0001) reduced in fluorotic rats. ConclusionIt is concluded that fluoride intoxication leads to the development of oxidative stress and damaging the cellular metabolism resulting in the declined ability of free radical scavengers along with increased level of MDA and decreased expression level of antioxidant genes which helps to understand the possible mechanism of fluoride-induced toxicity at the molecular level.

Melatonin Alleviates Constant Light Induced Oxidative Stress Damages of Kidney and Spleen of Female Rat

Photoperiod refers to the relative duration of light and dark that an animal experiences over the course of a 24 h period. Light is the principal stimulus that causes profound physiological and behavioural changes in all living organism. By manipulating photoperiod, melatonin production changes and its secretion drives enduring changes in many physiological systems of our body. Taking into consideration that light can affect pineal function and melatonin can be used as reactive oxygen species scavenger, act as a potent antioxidant and immunomodulator in mammals, the present experiment was designed and conducted to explore the consequences of constant light exposure on oxidative stress and antioxidant status on kidney and spleen of rat. 16 young adult female rats were randomly divided into 3 groups. The 4 rats of first group were employed as Control. These animals were kept under a 12 h light (600 lux, 60 days)/12 h dark cycle. The 8 rats of second group were maintained under constant light condition (600 lux, 60 days). After 60 days of constant light exposure second group of rats were randomly divided into two. One of these group of rats were designated as constant light exposed rats (LL) and another group of rats exposed to constant light is treated with melatonin (LL+MEL) for 21 days. The 4 rats of third group were only supplemented melatonin (MEL). The results showed that constant light exposure (60 days, 600 lux) resulted in an increased lipid peroxidation rate in kidney and spleen tissue and could effectively disturb the immune cells function through excessive generation of free radicals and decreased antioxidant enzymes activity (superoxide dismutase, catalase, glutathione peroxidase) through physiological or functional pinealectomy. Exogenous melatonin administration (200 µg/100 g body weight/day) suppressed the constant light induced oxidative stress and increase the activity of antioxidant enzymes in kidney and spleen of rat. Hence, melatonin has a nephroprotective and immunoprotective effects following photoperiodic stress induced cellular damages in female rat model.

Hypoglycemic and antioxidative activities of ethanol seed extract of Hunteria umbellate (Hallier F.) on streptozotocin-induced diabetic rats

BackgroundDiabetes, a global cause of mortality in developing countries is a chronic disorder affecting the metabolism of macromolecules and has been attributed to the defective production and action of insulin characterized by persistent hyperglycemic properties. This global disorder harms organs of the body such as the liver, kidney and spleen. Medicinal plants such as Hunteria umbellate have been shown to possess hypoglycemic, antioxidative and anti-diabetic properties owing to the high concentration of active phytochemical constituents like flavonoids and alkaloids.The present study seeks to evaluate the hypoglycemic activities of ethanolic seed extract of Hunteria umbellate on streptozotocin-induced diabetes rats.MethodsThirty (30) female experimental rats were randomly divided into five groups with six rats per group and were administered streptozotocin (STZ) and Hunteria umbellate as follows. Group 1 served as control and was given only distilled water, group 2 rats were administered 60 mg/kg STZ; Group 3 was administered 60 mg/kg STZ and 100 mg/kg metformin; group 4 rats were administered 60 mg/kg STZ and 800 mg/kg Hunteria umbellate, group 5 rats 60 mg/kg STZ and 400 mg/kg Hunteria umbellate. The fasting blood glucose level of each rat was measured before sacrifice. Rats were then sacrificed 24 h after the last dose of treatment.ResultsThe results showed that Hunteria umbellate significantly reversed STZ-induced increase in fasting blood glucose and increase in body and organs weight of rats. Hunteria umbellate significantly reversed STZ-induced decrease in antioxidant enzyme in liver, kidney and spleen of rats. Hunteria umbellate significantly reversed STZ-induced increase in oxidative stress markers in liver, kidney and spleen of rats.ConclusionCollectively, our results provide convincing information that inhibition of oxidative stress and regulation of blood glucose level are major mechanisms through which Hunteria umbellate protects against streptozotocin-induced diabketes rats.

Effects of deltamethrin subacute exposure in snakehead fish, Channa argus: Biochemicals, antioxidants and immune responses

To evaluate the effects on biochemicals, antioxidants, immune responses and disease resistance of the snakehead fish, following exposure to deltamethrin at 0.061, 0.121, 0.242, 0.485 and 0.970 μg/L. After 28 d, the biochemical, the levels of antioxidant enzymes and immune enzymes in liver, spleen, kidney and intestine were negatively related to the concentrations of deltamethrin exposure. Likewise, the survival rates of the fish after 7 d challenge with Aeromonas veronii were negatively related. The levels of IL-1β, IL-8, TNF-α, Hsp70 and malondialdehyde in liver, spleen, kidney and intestine were positively connected to the concentrations of deltamethrin exposure. Results demonstrated that environmentally relevant concentrations (0.121, 0.242, 0.485 and 0.970 μg/L) inhibited the biochemicals, antioxidants and immune responses and disease resistance of snakehead fish.

Effect of supplementing moringa (Moringa oleifera) leaf meal and pomegranate (Punica granatum) peel meal on performance, carcass attributes, immune and antioxidant responses in broiler chickens

An experiment was conducted to study the benefits of dietary supplementation of moringa (Moringa oleifera) leaf meal (MLM) and pomegranate (Punica granatum) peel meal (PPM) on the performance, carcass attributes, immune and antioxidant responses in commercial broiler chickens during 1–42 days of age. In total, 450 Cobb-400 broiler male chicks raised in wire-floor stainless-steel battery brooder pens in an open-side poultry house were divided into five experimental groups with 18 replicates of five chicks each and fed a standard control diet (CD) and the test diets supplemented with MLM (500 and 1000 mg/kg) or PPM (250 and 500 mg/kg), each at two different concentrations. Bodyweight gain, feed conversion efficiency and carcass attributes were not affected (P &amp;gt; 0.05). The cell-mediated immune response to phytohaemagglutinin phosphate was not influenced (P &amp;gt; 0.05), but the antibody titre against Newcastle disease (ND) vaccine was significantly (P &amp;lt; 0.05) improved by supplementation of MLM or PPM in broiler diet at 42 days of age. However, at 21 days of age, the ND titre was similar in broilers fed CD and 500 mg/kg PPM fed group. Activities of glutathione reductase and super oxide dismutase in liver were not influenced (P &amp;gt; 0.05), while lipid peroxidation (LP) reduced significantly (P &amp;lt; 0.05) with the herbal additives. The LP in the PPM group was significantly (P &amp;lt; 0.05) lower than that in groups fed MLM. The activity of glutathione peroxidase (GSHPx) was significantly (P &amp;lt; 0.05) improved in the PPM group. Activities of antioxidant enzymes (super oxide dismutase, glutathione reductase, GSHPx) in spleen were not influenced (P &amp;gt; 0.05). The degree of LP in the 500 mg/kg PPM group was significantly (P &amp;lt; 0.05) lower than that in groups fed the CD. It is concluded that supplementation of MLM (500 and 1000 mg/kg) or PPM (250 and 500 mg/kg) in diets improved humoral immune response (against ND vaccine) at 42 days of age and reduced LP in liver in commercial broilers, without showing any negative effects on performance and carcass attributes. Supplementation of PPM (250 and 500 mg/kg) significantly improved the activity of GSHPx in the liver of broiler chickens.

Diet companied with cold stimulation changed the activity of cytochrome P450 and phase II drug metabolism enzymes in spleen of rats

Diet companied with cold stimulation changed the activity of cytochrome P450 and phase II drug metabolism enzymes in spleen of rats

Naringenin Ameliorates Doxorubicin Toxicity and Hypoxic Condition in Dalton's Lymphoma Ascites Tumor Mouse Model: Evidence from Electron Paramagnetic Resonance Imaging.

Doxorubicin (DOX) is a well-known cytotoxic agent used extensively as a chemotherapeutic drug to eradicate a wide variety of human cancers. Reactive oxygen species (ROS)-mediated oxidative stress during DOX treatment can induce cardiac, renal, and hepatic toxicities, which can constrain its use as a potential cytotoxic agent. The present work investigates the antioxidant potential of naringenin (NAR) against DOXinduced toxicities of a Dalton's lymphoma ascites (DLA) tumor-bearing mouse model. Mice were randomized into four groups: a negative control, positive control, DOX (2.5 mg/kg) treated, and DOX (2.5 mg/kg) + NAR (50 mg/kg/d) treated. DOX administration significantly altered the levels of functional markers in blood and antioxidant enzymes in kidney, heart, lung, liver, spleen, and tumor tissues. These changes in antioxidant enzymes and successive lipid peroxidation were prevented by NAR supplementation, resulting in decreases in the risk of toxicity due to DOX therapy. Histopathology results and electron paramagnetic resonance imaging (EPRI) of the tumor microenvironment confirmed this evidence. Using EPRI, pharmacokinetics of the nitroxide, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3-CP) was monitored intratumorally before and after chemotherapy. EPRI of the DOX + NAR-treated mouse model showed reduced tumor size with significant modification of the hypoxic condition inside the tumor microenvironment. Consequently, these findings suggest that NAR treatment significantly reduces DOX-induced toxicity and the hypoxic condition in a DLA tumor-bearing mouse model.

Effects of desipramine on the antioxidant status in rat tissues at carrageenan‐induced paw inflammation

The pathogenesis of many diseases and different pathological conditions, including inflammation, is associated with excess production of reactive oxygen species (ROS). The present study aimed to investigate the effects of the antidepressant desipramine (DES) on carrageenan (CG)-induced inflammation, as well as on the endogenous levels of cell enzyme and non-enzyme antioxidants in rat liver and spleen, 4 and 24 h after CG injection. The intra-plantar CG injection into the right hind paw resulted in a time-dependent increase in the paw volume; the maximum of CG-induced edema peak was in 2-4 h. A single DES dose of 20 mg · kg(-1) , administered 30 min before CG, had no effect on paw edema, whereas the higher drug dose used (50 mg · kg(-1) ) suppressed the edematous response to CG. The latter drug dose protected CG-induced decrease of glutathione (non-enzyme antioxidant) in the liver; it did not affect CG-unchanged activities of superoxide dismutase, glutathione peroxidase (enzyme antioxidants) and glucose-6-phosphate dehydrogenase (enzyme, important for the activity of glutathione-conjugated antioxidant enzymes) in both liver and spleen. The drug showed an efficient antioxidant capacity in ROS-generating chemical systems; it was higher than that of fluoxetine (another type of antidepressant). The present results suggest that the good antioxidant activity of DES might contribute to its beneficial effects in liver injuries.

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies

Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.

Quinoid radio-toxin (QRT) induced metabolic changes in mice: An ex vivo and in vivo EPR investigation

Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from γ-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2 mg QRT. A dose of 2 mg QRT was fatal to mice within 45–60 min of treatment. Nitrosyl hemoglobin complexes α-(Fe 2+–NO)α-(Fe 2+)β-(Fe 2+) 2 were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO 2 in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with γ-irradiation.

Determination of toxicity of subacute treatment of some plant growth regulators on rats

The effects of some plant growth regulators (PGRs), 2,3,5-triiodobenzoic acid (TIBA), Naphthaleneacetic acid (NAA) and 2,4-Dichlorofenoxyacetic acid (2,4-D), at sublethal concentrations on antioxidant defense system [glutathione peroxidases (GPx), reduced glutathione (GSH), glutathione reductase (GR), glutathione-S-transferase (GST) and catalase (CAT)], immune potential enzymes [adenosine deaminase (ADA) and myeloperoxidase (MPO)], and lipid peroxidation content [Malondialdehyde, (MDA)] were investigated in lung and speen tissues of rats. Sprague-Dawley albino rats were exposed to 0, 50, or 100 ppm (parts per million) TIBA, NAA, or 2,4-D in drinking water ad libitum for 25 days continuously. According to the results, MDA concentration significantly increased in the tissues treated with 100 ppm dosage of NAA or 2,4-D without any change in the tissues of rats treated with both dosage of TIBA. The GSH depletion in the spleen tissue of rats treated with both the dosage of NAA and 2,4-D were found to be significant. Also, GSH level in the spleen was significantly reduced with 100 ppm of 2,4-D and NAA. The activity of antioxidant enzymes were also seriously affected by PGRs; GPx significantly decreased in the lung of rats treated with both dosages of the PGRs, whereas GPx activity in the spleen were significantly increased with 100 ppm dosage of 2,4-D and NAA. On the other hand, CAT activity significantly decreased in the lung of rats treated with both dosages of NAA, 100 ppm of 2,4-D and 50 ppm of TIBA, and also in the spleen treated with 50 ppm NAA and 2,4-D. The ancillary enzyme GR activity significantly decreased in the spleen with both doses of the PGRs, also in the lung treated with both dosages of 2,4-D, 50 ppm of NAA and 100 ppm of TIBA. The drug metabolizing enzyme GST activity significantly reduced in the lung of rats treated with both dosages of the PGRs and also in the spleen treated with 100 ppm dosage of 2,4-D and TIBA and 50 ppm of NAA. Meanwhile, immune potential enzyme MPO activity significantly increased in the spleen of rats treated with both doses of NAA and TIBA whereas ADA activity significantly decreased in the spleen of rats treated with 100 ppm dose of NAA and TIBA. The observations presented led us to conclude that the administrations of subacute NAA, 2,4-D, and TIBA promote MDA content, inhibit the antioxidative defense system and activate or inhibit immune potential enzymes in the rat's spleen and lung tissues. These data suggest that PGRs produced substantial organ toxicity in the lung and spleen during the period of a 25-day subacute exposure.

Effect of Steroid on Mitochondrial Oxidative Stress Enzymes, Intestinal Microflora, and Bacterial Translocation in Rats Subjected to Temporary Liver Inflow Occlusion

Protective effects of steroids against ischemia-reperfusion (I/R) injury are well known, but there is little information about the influence of temporary inflow occlusion on intestinal barrier function or bacterial translocation. The aim of this experimental study was to investigate the effects on liver, kidney, spleen, ileal mitochondrial stress enzymes, and bacterial translocation of methylprednisolone (MP) in rats undergoing temporary liver inflow occlussion. Twenty-seven pathogen-free Wistar albino rats were randomized into three groups: group A: I/R ( n = 10); group B: I/R + MP ( n = 10); and group C: sham ( n = 7). Rats in groups A and B were subjected to 20 minutes of portal vein and hepatic artery occlusion with 3 mg/kg MP injected into group B animals intraperitoneally during the occlusion. Twenty-two hours later, all rats were sacrificed to measure mitochondrial oxidative stress enzymes in liver, kidney, spleen, and ileum. We evaluated intestinal bacterial counts, intestinal mucosal histopathology, bacterial translocation to mesenteric lymph nodes (MLN), liver, spleen, and kidney. Decreased levels of malondialdehyde and increased levels of glutathione were observed in all examined tissues of group B compared to those of group A rats. Statistically significant increases in the intestinal counts of Klebsiella spp and Proteus spp and of bacterial translocation to liver, kidney, spleen, and MLN were measured in group B with respect to group A.

Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: Generation of new tools to study peroxisome proliferation

Aquatic organisms living in coastal/estuarine areas show peroxisome proliferation after exposure to different environmentally relevant pollutants. In order to generate new tools to assess peroxisome proliferation in aquatic animals, peroxisomal enzymes were cloned using degenerate primers in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus. Fragments of catalase (CAT), thiolase (THIO), polyamine oxidase (POX) and xanthine oxidoreductase (XOR) were cloned and their expression pattern studied in different tissues by semi-quantitative RT-PCR. In mussels, CAT, THIO, POX and XOR were expressed in digestive gland, mantle and gills while in mullets CAT, THIO and POX were expressed in liver, spleen, brain, heart, muscle and gills. XOR was mainly expressed in liver and heart. Mature mullets showed the highest expression of peroxisomal enzymes in liver, spleen and brain, while in juveniles expression was mainly found in muscle tissues, liver and gills. Laboratory experiments of exposure to organic pollutants are being performed to study the usefulness of these tools to study peroxisome proliferation in pollution biomonitoring programmes.

Protective role of sanumgerman against γ-irradiation–induced oxidative stress in Ehrlich carcinoma-bearing mice

This study was conducted to evaluate the potency of organogermanium sanumgerman (germanium lactate citrate) as a protective agent against γ-irradiation–induced toxicity, which may be extended to affect normal organs in mice bearing solid Ehrlich tumor. Another aim was to evaluate the antitumor effect of this agent, alone or in combination with irradiation, by measuring the change in tumor volume. Female Swiss albino mice were injected intraperitoneally with sanumgerman 1 day before tumor inoculation, then once every other day for a total of seven injections during 13 days. The last injection was given 20 minutes before exposure to a single dose of whole body γ-irradiation at a dose level of 6.5 Gy. Lipid peroxidation (LPx), glutathione (GSH) levels, and activity of the antioxidant scavenger enzymes glutathione reductase, glutathione peroxidase, glucose 6-phosphate dehydrogenase, superoxide dismutase, and catalase were estimated in blood, liver, and spleen at days 1 and 7 postirradiation. Ehrlich carcinoma–bearing mice, whether exposed to irradiation or not, showed marked elevation in LPx level accompanied by a decline in GSH content and in the activity of related antioxidant enzymes that was more apparent at day 7 postirradiation. Pretreatment with sanumgerman potentially reversed the investigated parameters in nonirradiated, tumor-bearing mice. Additionally, it prevented LPx, and ameliorated to a great extent GSH content and the activity of antioxidant scavenger enzymes in blood, liver, and spleen of mice exposed to γ-irradiation. Moreover, sanumgerman significantly suppressed the growth of tumor and efficiently produced a synergistic effect with irradiation in reducing tumor volume. Therefore, this agent may be a good candidate for use with irradiation to enhance tumor cell killing without affecting normal organs of the tumor host.

Effect of iron lactate overloading on adenine nucleotide levels and adenosine 3'-monophosphate forming enzyme in rat liver and spleen.

To elucidate the pathophysiological significance of adenosine 3'-monophosphate (3'-AMP) forming enzyme in rats, the effect of iron lactate overloading on the enzyme activities and adenine nucleotide levels in the liver and spleen was examined. Sprague-Dawley rats were fed a diet supplemented with 0%, 0.625% or 5.0% of iron lactate for 4 weeks. Iron deposition was found in periportal hepatocytes, Kupffer cells and macrophages of red pulp of the spleen. No significant changes in hematological parameters were detected. Although serum alkaline phosphatase and inorganic phosphorus levels elevated slightly in the 5.0% group, activities of alanine aminotransferase and aspartate aminotransferase, and levels of serum urea nitrogen and creatinine were not changed significantly. The ATP levels in the liver and spleen of iron fed groups were significantly decreased, but adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) levels were within control levels. On the other hand, the levels of ATP, ADP and AMP in the erythrocytes without mitochondria were not suppressed by the iron lactate overloading. Free activity of 3'-AMP forming enzyme, one of ribonucleases (RNase), was not changed in the liver of iron-overloaded rat, and total amount of 3'-AMP and adenosine formed after the treatment of the crude enzyme(s) with p-chloromercuribenzensulfonic acid, a SH blocker of RNase inhibitors, was decreased dose-dependently. On the contrary, free activity of 3'-AMP forming enzyme was enhanced dose-dependently in the spleen of iron-overloaded rat but the total activity was not changed. However, the free and total 3'-AMP forming enzyme activities in the liver and spleen of iron-overloaded rats became equal at the dosage of 5.0% of iron lactate. The results obtained suggested that iron loading might induce significant decrease in hepatic and splenic ATP levels via malfunction of their mitochondria and might lead dissociation of RNase-RNase inhibitor complex to activate 3'-AMP forming enzyme in both tissues.

Design, synthesis, and biological evaluation of novel nucleoside and nucleotide analogues as agents against DNA viruses and/or retroviruses.

A novel strategy was developed for the synthesis of N(7)-purine acyclic nucleosides 9 and 14. The key step involved the reaction between [2-(p-methoxyphenyloxy)ethoxy]methyl chloride and N(9)-tritylated nucleobases 6 or 11 followed by concomitant self-detritylation. N(7)-Guanine acyclic nucleoside 9 exhibited antiviral activity, but was phosphorylated by both HSV and Vero cell thymidine kinases. Thus, it showed more potent cellular toxicity than acyclovir (2). N(7)-Adenine acyclic nucleoside 14 was found to be an excellent antiviral agent as well as a good inhibitor of calf mucosal adenosine deaminase. This inhibitory property allows for a greater expression of antiviral activity of antiviral agents, such as N(9)-adenine acyclic nucleoside 1 and ara-A (3). Compound 14 was phosphorylated neither by herpes simplex virus (HSV) thymidine kinase nor by Vero cell thymidine kinase, yet it enhanced the rate constant for the monophosphorylation of acyclovir (2) by HSV thymidine kinase. Consequently, the combination of acyclovir (2) and 14 exhibited greater antiviral activity than acyclovir alone. 7-[2-(Phosphonomethoxy)ethyl]adenine (20) was also synthesized. The key step involved the reaction of 9-(2-cyanoethyl)adenine (15) with methyl iodoacetate in the presence of lithium 2,2,6,6-tetramethylpiperidine in THF. Unlike 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 4), the N(7)-isomer 20 was not phosphorylated effectively by 5-phosphoribosyl 1-pyrophosphate synthetase (PRPP synthetase). Thus, it did not exhibit pronounced antiviral activity. Dinucleotide 5'-monophosphate 24 and its butenolide ester 25 were also synthesized. Compound 24 showed substrate activity toward PRPP synthetase and exhibited notable activity against DNA viruses. The antiviral activity of the ester derivative 25 was found to be higher than that of the parent molecule 24. Dinucleotide 5'-monophosphate 24 is susceptible to degradation by snake venom and spleen phosphodiesterases. However, its respective butenolide ester derivative 25 was completely resistant to snake venom and spleen enzymes. Butenolide ester derivatives 28 and 29 were also synthesized and exhibited notable anti-DNA virus and anti-retrovirus activity in vitro. Compounds 2, 4, 9, 14, 20, 24, 25, and 28 were also evaluated for their inhibitory effect on HSV-1-induced mortality in NMRI mice. N(7)-adenine acyclic nucleoside 14 [LD(50) (intraperitoneal, ip) 950 mg/kg], nucleotide-containing butenolide 25 [LD(50) (ip) 675 mg/kg], and butenolide 28 [LD(50) (ip) 710 mg/kg] were found to be potent anti-HSV-1 agents in vivo. In addition, butenolide 28 efficiently decreased tumor formation induced by Moloney murine sarcoma virus (MSV) in NMRI mice while significantly increasing the survival time of MSV-infected mice.

Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P2 12 12 1 with unit cell dimensions a = 64.1 Å, b = 108.9 Å, c = 119.3 Å and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 °C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 Å resolution. The ternary “dead-end” complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuK α radiation, and the three-dimensional structure refined at 2.4 Å resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central β-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved β- and γ-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1· · ·Glu204 O ϵ1· · ·guanine NH 2· · ·Glu204 O ϵ2. The exocyclic O6 of the base is bridged via a water molecule to Asn246 N δ, which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.

XENOBIOTIC-METABOLIZING ENZYMES IN CARP (CYPRINUS CARPIO) LIVER, SPLEEN, AND HEAD KIDNEY FOLLOWING EXPERIMENTAL LISTERIA MONOCYTOGENES INFECTION

Infection of carp with Listeria monocytogenes 4b resulted in decreased liver, spleen, and head kidney enzyme activities, involved in the metabolism of xenobiotics. After infection, cytochrome P-450 levels and ethoxyresorufin O-deethylase (EROD) activity were decreased while conjugation enzymes remained unaffected. The maximum decrease for phase I enzymes occurred on d 3. This loss of monooxygenase levels and activity could not be directly correlated with an increase in the number of organisms, as consistently high bacterial counts were observed in all three organs during infection. The effect of L. monocytogenes infection was also measured in carp exposed to 3-methylcholanthrene (MCA). Cytochrome P-450 levels and EROD activity were significantly reduced, especially on d 3. A significant decreased activity of conjugation enzymes such as glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) was also observed for all days studied. Listeria infection inhibited MCA-induced increases in xenobiotic-metabolizing enzyme activities. These results indicate that infection may have deleterious effects on basal cytochrome P-450 monooxygenase levels. Futhermore, MCA treatment aggravates the insult to xenobiotic biotransformation enzymes by L. monocytogenes infection, by impairing a number of detoxification enzymes. These findings could result in significant changes in the susceptibility of fish to pollutants.