The 20S proteasome was purified from oocytes of the starfish Asterina pectinifera and its enzymatic properties were investigated. The chymotrypsin-like activities were potently inhibited by PSI as well as MG115, whereas the trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were not or only weakly inhibited by PSI and MG115. The inhibitory ability of MG115 toward germinal vesicle breakdown (GVBD) coincided with those toward the trypsin-like and PGPH activities, and PSI showed no inhibitory effect on GVBD. We have previously reported that the inhibition pattern toward GVBD of peptidyl-argininals, which potently inhibited the proteasomal trypsin-like activity rather than the chymotrypsin-like activity, correlated with the inhibition pattern toward the chymotrypsin-like activity of the proteasome. These results, together with the peptidyl-argininals scarcely inhibiting the PGPH activity at concentrations sufficient for the inhibition toward GVBD, indicate that both the chymotrypsin-like and trypsin-like activities, but not the PGPH activity, of the proteasome are responsible for degradation of the physiological substrate during starfish oocyte maturation. It was also suggested that the inhibition of a single catalytic site of the proteasome is not sufficient for prevention of the proteasomal function.
9-O-Acetyl neuraminic acid specific lectin (AchatininH) was isolated from the hemolymph of the land snail Achatina fulica by affinity chromatography on sheep submaxillary mucin (SSM) coupled cyanogen bromide activated Sepharose 4B. The molecular weight of the native protein was 2.42 kDa. UV-Vis absorption, fluorescence and circular dichroism spectroscopic studies on AchatininH revealed the importance of divalent metal ions (Ca2 +, Mg2+ and Mn2+) on lectin conformational change associated with activity of lectins. The binding of these cations changes lambdamax to shorter wavelength in the far UV region (blue shift) and longer wavelength in UV region (red shift), indicating substantial contribution of aromatic side chain in the far UV region on binding with metal ions. The results infer that divalent cations cause conformational changes in lectin which may be responsible for affinity with their carbohydrate moiety.
The effect of anticonvulsants on the burst firing of action potentials in snail central neuron elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac. Oscillation of membrane potential and burst firing of action potentials were elicited by d-amphetamine in a concentration-dependent manner. Voltage clamped studies revealed that d-amphetamine elicited a negative slope resistance (NSR) in steady-state I-V curve between - 40 and - 10 mV. The burst firing of action potentials was alleviated following extracellular application of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. The NSR elicited by d-amphetamine was blocked by phenytoin. However, the NSR was not altered if carbamazepine was added. These results suggest that of the four anticonvulsants tested, only phenytoin could alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron.
Estrogen binding activity was revealed in the cytosolic fraction of hepatic extracts from adult male and female eelpout (Zoarces viviparus). The binding moiety was characterized by a single class of high affinity binding sites (Kd=0.59±0.05 nM in males and 1.06±0.10 nM in females). The affinity was significantly higher in males. Binding sites were satiable and binding capacity was significantly elevated in vitellogenic females (2.92±0.28 pmol/g) compared to males (1.67±0.11 pmol/g). The binding was specific to known estrogens but not to other tested steroids. The binding moiety was able to bind to DNA–cellulose and was extractable by high salt concentrations. A time-course study of estrogen binding activity in liver cytosol and of vitellogenin (Vtg) in plasma, after intraperitoneal (i.p.) injections of 17β-estradiol (E2) in male eelpout, was carried out. It was shown that both are inducible by E2. Estrogen binding activity was significantly elevated 48 h and Vtg 72 h after E2 treatment. The binding moiety was hereafter designated as a cytosolic estrogen receptor (ER). The estrogenicity of 4-tert-octylphenol (OP) was evaluated by measuring ER and Vtg after i.p. treatment. OP-treatment increased both receptor levels and Vtg concentrations in male fish, indicating that OP acts as an estrogen in male eelpout.
Atrazine (1,000 ppm), endosulfan (1 ppm) or butylated hydroxyanisole (BHA) (1,000 ppm) added to a semi-synthetic diet of Orthosia gothica for 2 days in the last instar did not change the soft tissue cytosolic glutathione-S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and cumene hydroperoxide (CU). However, all three pesticides changed the GST subunit composition compared with the control as observed by reverse phase high performance liquid chromatography of the isozymes purified by glutathione-Sepharose affinity chromatography. The changes seem to have occurred mainly in the GST class 2 subunit. There is no obvious explanation for the changes, which may be a result of interactions between xenobiotic and GST in the cytosol as well as changes in the level of regulation of synthesis. However, the observation added to our knowledge of the processes involved when pesticides are degraded by GSTs in vivo.
Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [3H]17beta-estradiol (E2) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B(SP)) of [3H]E2 was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B(SP) in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B(SP) in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k(+1)) for cytosol and nuclear extract preparations were 1.10 +/- 0.02 x 10(8) M(-1) min(-1) and 1.27 +/- 0.04 x 10(8) M(-1) min(-1), respectively. Equilibrium bound B(SP) dissociated from cytosol preparations with a half life (t1-2) of 42 min and a dissociation rate constant (k(-1)) of 1.01 +/- 0.03 min(-1). B(SP) dissociated from nuclear extract preparations with a t1-2 = 45 min and k(-1)= 0.92 +/- 0.01 min(-1) x B(SP) was saturable in both extract preparations with a calculated equilibrium dissociation constant (Kd) of 1.46 +/- 0.1 nM (cytosol) and 2.37 +/- 0.2 nM (nuclear), and a maximum number of binding sites (B(MAX)) of 50.85 +/- 3.2 fmol mg(-1) protein and 61.74 +/- 2.65 fmol mg(-1) protein, respectively. In both preparations, B(SP) was differentially displaced by structurally similar compounds with a rank order of potency of E2 > estrone > estriol > 17alpha-ethynyl estradiol > testosterone >> progesterone = tamoxifen >> cortisol > dexamethasone >> > beta-sitosterol. These properties of specifically bound [3H]E2 suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.
In order to study temporal and spatial variation of environmental fluoride levels, we analyzed the mandibular bone fluoride content of 157 roe deer (age range: 1-11 years) from two industrialized regions (Ruhr area: n = 76, sampling period 1955-1998; area W of Cologne: n = 81, sampling period 1983 1998) in the federal state of North Rhine-Westphalia, Germany. Bone fluoride values (dry weight basis) ranged between 150 mg F/kg (2 year-old specimen taken in 1997) and 5724 mg F/kg (10 year-old specimen taken in 1957). In both study areas, a pronounced decline in mandibular bone fluoride concentrations occurred over the respective sampling periods. In consequence, bone fluoride content of animals (both study areas pooled) taken during the period 1990-1998 was significantly (P < 0.00001) lower than that of roe deer from the period 1955 1989, while the two animal groups did not significantly differ in age. These findings are regarded as indicative of a considerable reduction of fluoride deposition into the animals' habitats, due to effective emission control measures. Bone fluoride values for the period 1990-1998 in the roe deer from the Ruhr area significantly (P < 0.005) exceeded those of the individuals from the study area W of Cologne, while the difference in age between the two groups was not significant. In both study areas, a significant (P < 0.00001) positive correlation between age and mandibular bone fluoride content (Ruhr area: rs = 0.601; area W of Cologne: rs = 0.725) was found for animals taken during this period. The present study underscores the suitability of analyzing skeletal fluoride concentrations in wild roe deer in order to monitor the magnitude of environmental contamination by fluoride and thereby to assess the efficiency of measures taken to reduce fluoride emissions from industrial sources.
Reactive oxygen species are formed in physiological and pathological conditions in mammalian tissues. Because of their high reactivity, they may interact with biomolecules, inducing oxidative injury. Increases in lipid peroxidation can result in oxidative damage to cellular membranes. Protection against oxidative damage is provided by enzymatic and non-enzymatic antioxidant defenses. Antioxidant enzyme activities and lipid peroxidation, as an index of oxidative stress injury, were evaluated in different seasons over one year in the heart and liver of rats, maintained on a 12 h light and dark cycle. Glutathione peroxidase and catalase activities, in both tissues, were maximal in the summer season. Lipid peroxidation in the heart was maximal in the spring as compared to the other seasons and it did not vary in the liver during the year. These findings suggest that any study of antioxidants or oxidative stress must take into account such seasonal variations for a more precise analysis of changes due to any pathological condition.
Ovarian samples from fear sea bass, Dicentrarchus labrax, were collected for the in vitro incubations during the spawning period. Follicles with fully developed vitellogenic oocytes showing central germinal vesicle (stage I follicles) and follicles with oocytes showing initial germinal vesicle migration (stage II follicles) were treated with either (1) 20 microg sea bass hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), (2) 20 microg hypophysis alone, (3) 50 ng 17-P alone and (4) media alone. Structure-activity experiments used stage II follicles treated with several dosages (0.1, 1.0 and 10.0 ng/ml) of either 17-P, 17,20beta-P, or 17,20beta,21-P. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P) were measured in the incubation media by high performance liquid chromatography. Our results show that the synthesis of free and conjugated 17,20beta-P is constant (0.1-0.2 ng/ml) in all incubates. In contrast, the synthesis of free and conjugated 17,20beta,21-P is higher in incubates containing stage II follicles (up to 5 ng/ml) than in those having stage I follicles (up to 3 ng/ml; P<0.01). Structure-activity data reveal that 17-P is not effective at inducing in vitro germinal vesicle breakdown whereas both 17,20beta-P and 17,20beta,21-P are equally potent and highly effective. These results demonstrate that 17,20beta-P and 17,20beta,21-P are synthesized in vitro by follicles of sea bass and that sulfation is the main route for the metabolism of the C(21)-steroids in riper follicles. The highest levels of 17,20beta,21-P, found in incubates containing stage II follicles, points at 17,20beta,21-P, rather than 17,20beta-P, as the most probable MIS in sea bass, nonetheless, this hypothesis requires further confirmation.
Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68–85 kD and two high molecular mass bands in the range of 190–221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.
Treatment of experimental animals subjected to 90 days physical training programme plus repeated doses of salbutamol, a β-adrenergic agonist, administered under two different regimes: therapeutic (16 μg/kg body weight, twice a day) and doping (3 mg/kg body weight, twice a day), caused a marked increase in size of skeletal (soleus, gastrocnemius and plantaris) leg muscles. Adrenergic involvement of salbutamol-linked hypertrophy was demonstrated by co-administration of the non-specific β-adrenergic antagonist d,l-propranolol (10 mg/kg body weight twice a day). The salbutamol-induced muscle hypertrophy was associated with an early increase in creatine phosphokinase (CK) and its myocardial isozyme (CKmb), without significant changes in lactate dehydrogenase (LDH), alanine aminotransferase (AAT) and aspartate aminotransferase (DAT). The induction of muscle-injury biomarkers was completely abolished by co-administration of propranolol, thus suggesting the adrenergic involvement of these alterations.
Sea bass (Dicentrarchus labrax) were injected intraperitoneally once (single dose) or three times (fractionated dose) with phenol or OH-phenols (hydroquinone, resorcinol, and pyrocatechol). On the basis of the lethal doses, OH-phenols were more toxic than phenol, and pyrocatechol was the most powerful compound. Hematological, metabolic and antioxidant blood parameters were measured 3 days after the end of the treatment. Metabolic variations as specific effects on erythrocytes were revealed and differences between single and fractionated doses were observed. OH-phenols-treated fish showed disorders in the metabolic toxicity indicators as hypoglycemia, low blood urea nitrogen level (BUN) and decrease of alkaline phosphatase activity (ALP). In addition, quantitative structure-activity relationships were developed using the n-octanol:water partition coefficient (log K(ow)). Positive correlations were found with ALP, plasma glucose and hemoglobin.
In order to elucidate the regulatory mechanism of blood glucose concentrations specific to chickens, carbohydrate metabolism in the liver, muscle and kidney and metabolite concentrations in the blood were investigated in chickens with acute and persistent hypoglycemia. Acute and persistent hypoglycemia were experimentally induced by a single injection of insulin (8 U/kg BW) or by continuous infusion of insulin (22.5 U/kg BW/day) for 4 days. Non-esterified fatty acid (NEFA) concentration in plasma and D-3-hydroxybutyrate (3HB) concentrations in liver and muscle increased in the acute hypoglycemia. Plasma NEFA concentration and 3HB concentration in the blood and liver were not changed at day 3 of persistent hypoglycemia, while 3HB concentration in the muscle was decreased. Phosphofructokinase (PFK) activity in the liver tended to increase but PFK and pyruvate kinase (PK) activities were unchanged in acute hypoglycemia. In persistent hypoglycemia, increase of hepatic PFK activity at day 1 in which it was reversed at day 3, and a small increase of muscle PK activity were observed, while PK and phosphoenolpyruvate carboxykinase (PEPCK) activities in the liver and kidney were not significantly changed. These results show that in the persistent hypoglycemic chickens, hepatic glycolysis transiently increases, which is followed by a small decrease, while glycolysis in muscles and gluconeogenesis in the liver and kidney are not significantly changed.
A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculetum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCl extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (M-r) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K-i of 1.4 nM and is not a glycoprotein. Tryptic peptides (M-r 26, 13 kDa) and most chymotryptic peptides (M-r 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower M-r cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor. (C) 2000 Elsevier Science Inc. All rights reserved.
Planaria represents the most primitive example of centralization and cephalization of nervous system. Previous reports indicate that planaria shows specific behavioral patterns, analogous to mammalian stereotypes, in response to drugs acting on acetylcholine or dopamine transmission. Here we further characterized these responses, and investigated the interactions between cholinergic and dopaminergic systems by means of behavioral methods. Exposure to cholinergic agonists physostigmine or nicotine produced hypokinesia with 'bridge-like' and 'walnut' positions, respectively. Blockade of muscarinic receptors by atropine produced 'screw-like' hyperkinesia. Exposure to dopamine agonists (nomifensine, apomorphine) produced marked hyperkinesia with 'screw-like' movements. Finally, exposure to dopamine antagonists produced immobility or 'bridge-like' position. Pre-exposure to physostigmine blocked the behavioral effects of nomifensine and reduced and markedly delayed the behavioral effects of apomorphine. Pre-exposure to apomorphine slightly reduced and delayed the behavioral changes by physostigmine. Finally, planaria exposed to atropine after either SCH23388 or sulpiride showed 'C-like' or 'screw-like' hyperkinesia, respectively. Thus, reduction of cholinergic transmission seems to play a pivotal role in determining hyperkinesia in planaria. Under these conditions, different patterns of hyperkinetic activities occur, according to the subpopulation of dopamine receptors stimulated by drugs. These findings suggest that interactions between cholinergic and dopaminergic systems occur very early in animal phylogeny.
The anti-cancer taxoids, Taxol (paclitaxel) and Taxotere (docetaxel), are the most promising anti-mitotic agents developed for cancer treatment in the past decade. The effectiveness of this new class of compounds lies in their unique mechanism of action on the cytoskeleton. Both taxol and taxotere bind to microtubules and shift the normal equilibrium between monomeric and polymerized tubulin to favor the polymerized form by strongly promoting tubulin assembly and inhibiting microtubule depolymerization. Although very similar in structure, these two compounds have recently demonstrated different in vitro, in vivo, and clinical activities; however, no study to date has effectively compared specific cytoskeletal alterations induced by taxol and taxotere in cultured cells. Using specific staining techniques for both F-actin and alpha-tubulin, this study provides the first detailed immunohistochemical comparison of the effects of equimolar concentrations of taxol and taxotere on both the microfilament and microtubule networks in a cultured cell line. Using human MCF7 breast adenocarcinoma cells, new observations of taxotere/taxol alterations of the cytoskeleton include: an increased abundance of parallel microtubule 'bundles' in taxotere treated cells and a definitive reorganization of the microfilament network which results in novel ring-like formations of F-actin condensed exclusively in the perinuclear zone. Reorganization of the actin cytoskeleton induced by a taxoid disruption of the microtubule equilibrium is indicative of the interdependence between microtubules and microfilaments in this transformed cell line and suggests that the indirect role of the taxoids on the microfilament network may have been overlooked in their mechanism of action as chemotherapeutic agents.
Superfusion with 200 microM 2-amino-4-phosphonobutyrate (APB) of dark and chromatically adapted frog eyecups caused marked potentiation of the ERG OFF-response (d-wave). Blockade of the glycinergic synapses by strychnine did not change this effect at all. Blockade of the GABAergic synapses by picrotoxin slightly diminished the effect of APB in chromatically-adapted eyes with isolated cones' activity, and did not change it in dark-adapted eyes. The results indicate that the action of APB on ERG OFF-response does not depend significantly on GABAergic and glycinergic neurotransmission in frog retina.
The presence and function of the P-glycoprotein mediated multixenobiotic resistance (MXR) mechanism was demonstrated in numerous aquatic organisms. The aim of this study was to investigate whether in aquatic organisms exists the inherent, species-specific basal level of MXR activity. Here the results of the direct comparison of the basal (noninduced) level of MXR activity measured in several marine (Mytilus galloprovincialis, Monodonta turbinata, Patella lusitanica) and freshwater (Dreissena polymorpha, Viviparus viviparus, Anodonta cygnea) molluscs species are presented. The primary criterion for the assessment and quantification of the basal level of MXR activity was the ratio (R) between the accumulation or efflux of the fluorescent model MXR substrates (rhodamine B or rhodamine 123) in or from the gills, measured with and in the absence of model MXR inhibitors verapamil or cyclosporin A. Significantly different levels of MXR activity were found in the species investigated. These levels generally show a relatively good correlation with the level of pollution present in their natural habitats. Considering these results a conclusion was reached that in aquatic organisms indeed exist the different inherent, species-specific levels of MXR activity. The identified levels might be, at least partly, responsible either for the resistance to, or for the sensitivity of a particular species to organic pollution.
The antibacterial properties and cDNA sequences of two types of antibacterial peptides from the haemolymph of immunized common cutworm, Spodoptera litura larvae, were determined. Since the primary structures of peptides deduced from cDNA sequences showed significant homologies to cecropins A and B, they were named as Spodoptera cecropins A and B. Spodoptera cecropins were broadly effective against Gram-positive and negative bacteria. They also retained antibacterial activities in all conditions tested (at pH 5.6-8.0 and in the presence of 50-150 mM NaCl) that was adapted to confirm the antibacterial properties of Spodoptera cecropins. These results indicate that the change of pH and the increase of salt concentration in the media do not influence the activities of Spodoptera cecropins. For the reverse transcription (RT)-polymerase chain reaction (PCR) to obtain the complete primary sequence, the primer designed according to the conserved region of the cecropin leader sequences was used, which was determined by the comparison of the cDNA sequences of known cecropins. The results from RT-PCR presented that the partial cDNAs of Spodoptera cecropins A and B encode 57 and 58 amino acids, including the sequences of mature peptides, respectively. In addition, Northern blot analysis with (32)P-labeled PCR product coding for Spodoptera cecropin A revealed that Spodoptera cecropin genes are expressed in immunized fat body, but not in normal fat body.
