Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology最新文献

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865±334 nmol/mg microsomal protein per min versus 895±27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159±370 nmol/mg microsomal protein per min. V_max values and K_m values for the terpene treated possum, control, possum and koala were 1932–2225 nmol/mg microsomal protein per min and 0.80–0.81 mM; 1406–1484 nmol/mg microsomal protein per min and 0.87–0.92 mM and 5895–6403 nmol/mg microsomal protein per min and 0.067–0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (K_i 15 μM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.

This study examines the effects of Fenoxycarb® on larval growth, and lipid class and fatty acid composition in first crabs of the mud crab Rhithropanopeus harrisii reared through total larval development in nominal water concentrations from 1 to 100 μg/l. In first crabs of R. harrisii, dry weight (μg) decreased significantly (P<0.05) from 228.8±38.2 μg (n=9) in the controls to 131.8±10.1 μg (n=4) in animals exposed throughout larval development to 100 μg/l. A significant (P<0.05) reduction was found between total lipid content in the controls and first crabs reared at concentrations greater than 50 μg/l. In relative terms (% dry weight), different lipid classes predominated in the controls and the various fenoxycarb exposure concentrations. There were no significant (P>0.05) differences among the treatment groups in phospholipid level, while the triglyceride content was significantly lower in crabs exposed to 10 and 100 μg/l. No significant differences in the percent of free fatty acids were found in crabs exposed to 1–10 μg/l and the controls. Free sterols in crabs exposed to concentrations higher than 10 μg/l were below the detection limit. Control animal fatty acid profiles were dominated by palmitic, stearic, and oleic acid, accounting for 48% of total fatty acids (TFA). The fatty acid composition of crabs exposed to 100 μg/l significantly (P<0.05) differed from the controls. The results suggest that fenoxycarb has substantial effects on growth, lipid class and fatty acid composition in developing larvae of R. harrisii at water concentrations greater than 10 μg/l.

Whether free choline levels are changeable in vivo in response to different types of autonomic agonists was examined in several mouse organs. Upon one subcutaneous injection of isoproterenol, phenylephrine and pilocarpine, choline levels in whole organ decreased, increased and decreased, respectively, in various organs within 30 min and returned to initial levels in a day. In the three major salivary glands, a delayed choline elevation also appeared on day 2 after one isoproterenol injection and subsided by day 6. Only in the three salivary glands more choline was accumulated after 10 once-a-day injections of isoproterenol than after one isoproterenol injection. Neither phenylephrine nor pilocarpine induced comparable choline accumulation in any organs examined. Isoproterenol injection repeated at a 2-day interval augmented the subsequent, delayed choline elevation. Examination with dobutamine and the adenylyl cyclase activator 6-(3-dimethylaminopropionyl)forskolin suggested that isoproterenol-induced immediate choline lowering was downstream of cAMP synthesis and linked to cAMP more tightly than the choline accumulation, though both choline changes occurred via β₁-adrenergic receptors. Choline levels in the salivary glands also changed depending on the form of diet given and particularly in the parotid gland in parallel with gland weights. These results provide the first evidence for the autonomic control of intracellular choline levels; intracellular choline levels might be an integral part of the autonomic signalling pathway.

We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3′-triiodothyronine (T3) levels to an intraperitoneal injection (5–500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3′-4,4′-tetrachlorobiphenyl) or 126 (3,3′,4,4′,5-pentachlorobiphenyl). T4 and 3,3′5′-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent K_m values of 0.6–4 nM) fell in the same rage as those of low-K_m deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25°C but uniformly low over the natural range of 0–9°C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P<0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.

In leech P neurones caffeine activates unselective ion channels in the plasma membrane and induces intracellular Ca²⁺ release (Schoppe, J., Hochstrate, P., Schlue, W.-R., 1997. Caffeine mediates cation influx and intracellular Ca²⁺ release in leech P neurones. Cell Calcium 22, 385–397). These effects are prominent only upon the first caffeine exposure, while subsequent applications are largely ineffective; i.e. both plasma membrane channels and intracellular Ca²⁺ release mechanism desensitize irreversibly. In order to examine whether this desensitization is paralleled by irreversible changes in the electrophysiological parameters of the cells, we investigated the action of caffeine on changes in membrane potential and the cytosolic free Ca²⁺ concentration, which were induced by varying the ionic composition of the extracellular fluid or by application of 5-hydroxytryptamine. Neither the resting values nor any of the experimentally induced shifts in membrane potential or cytosolic Ca²⁺ concentration were affected by caffeine, which suggests strongly that activation and/or desensitization of the caffeine-sensitive ion channels and Ca²⁺ stores have no long-lasting effect on the relevant electrochemical gradients, membrane conductances, or transport mechanisms.

Interaction of bilirubin with different types of erythrocyte membrane vesicles such as unsealed, heterogeneous, sealed and inside-out membrane vesicles prepared from human and goat erythrocytes was studied. Out of various types of membrane vesicles, in both species, unsealed membrane vesicles bound quantitatively higher amounts of bilirubin followed by heterogeneous and sealed membrane vesicles whereas inside-out membrane vesicles bound the lowest amount of bilirubin. These differences in the amount of bound bilirubin to different membrane vesicles were correlated well with the percentage accessibility of sialic acid to neuraminidase in these membranes suggesting that bilirubin bound preferentially to the outer layer of erythrocyte membranes than the inner layer. Further, membrane vesicles prepared from human erythrocytes bound higher amounts of bilirubin than those prepared from goat erythrocytes. This can be ascribed to different phospholipid composition of these membranes.

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.

The aim of the present study was to determine whether short-term administration of hexachlorobenzene (HCB) (1 g/kg body wt., suspended in water, 5 days/week), could cause and maintain marked porphyria in the absence of the exogenous drug, and whether porphyria parameters can be useful as biomarkers of HCB persistence in rats. Hepatic uroporphyrinogen decarboxylase activity, its inhibitor formation, porphyrin content and composition were studied in Wistar rats treated with the fungicide for 1, 2, 3, or 4 weeks and then withdrawn for a 20-week period. The time course of urinary porphyrin excretion was studied for 7 weeks either by continuous treatment for the entire period, or a 1-week HCB administration. The degree of porphyria achieved by rats after 20 weeks of suspended HCB administration was severe, independent of the length of the treatment, and even higher than that observed in animals analysed immediately at the end of each treatment. Rats treated with HCB for 1 week showed a modest decrease in uroporphyrinogen decarboxylase and low inhibitor formation, and exhibited a greater enzyme inhibition, inhibitor formation, hepatic porphyrin accumulation, and an altered pattern of porphyrin composition in the absence of the exogenous drug. Independent of the treatment, urinary porphyrins rose after a delay of 5 weeks. Substantial amounts of HCB were still found in fat of rats treated with HCB for 1 week, after a withdrawal period of 20 weeks. These results suggest that the high persistence of HCB in tissues acts as a continuous source of the xenobiotic, and stimulus for heme biosynthesis derangement. The alterations induced by HCB within 1 week of treatment could be regarded as an initial trigger for irreversible damage on heme metabolism. Thus, abnormalities in heme biosynthesis can be considered effective markers of HCB persistence in rats or of irreversible HCB-induced damage. Taking into account the delayed and enhanced metabolic effects of HCB, it is advisable that porphyria parameters should be evaluated not only immediately after exposure, but also some time afterwards, especially in susceptible and occupationally-exposed populations.

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCl extraction, 70°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°C, but unstable at neutral pH at >60°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.

9-O-Acetyl neuraminic acid specific lectin (Achatinin_H) 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 Achatinin_H 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 λ_max 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.