Enhanced benzene-induced DNA damage in PMA-stimulated cells in vitro and in LPS-treated animals

The present study investigated the interaction between inflammatory reactions and benzene in vitro and in vivo with respect to oxidative DNA damage. In the in vitro models the oxidative burst of cells was induced by the pretreatment with phorbol myristate acetate (PMA) and in the in vivo models of inflammation mice were pretreated with lipopolysaccharide (LPS). The oxidative DNA damage was indicated by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and strand breaks as assessed by alkaline single cell gel electrophoresis (SCGE, Comet assay). The results showed that combination of PMA and benzene enhanced the level of 8-oxodG in DNA from mouse bone marrow cells by 197%, from human lymphocytes by 188% and from human neutrophils by 205% (p < .05). Pretreatment of mice with LPS and benzene resulted in an enhanced Comet score formation in bone marrow cells by 98% and in lymphocytes by 39% in Comet score (p < .05) and in an enhanced 8-oxodG level in bone marrow cells by 290%. The effects of the combined treatment with PMA/LPS and benzene exceeded the sum of the effects induced by PMA/LPS or benzene alone. The production of nitrate/nitrite showed a two fold increase in the supernatant from incubation of benzene and PMA-pretreated neutrophils. The increase in the 8-oxodG level in the human neutrophil incubation system demonstrated a correlation with nitrate/nitrite production, indicating a possible relationship with the generation of reactive nitrogen species.     

Cytokines involved in the immunosuppressor period in experimental fasciolosis in rats

The aim of this study was to evaluate the kinetics of the cytokines interferon-gamma, interleukin-2, interleukin-10 and interleukin-4 produced by spleen mononuclear cells stimulated by Con A during an experimental infection in rats with Fasciola hepatica. The proliferative response to Con A of Spm cells from rats infected with F. hepatica was significantly decreased on day 7 post-infection (P<0.006) and simultaneously an increase of interferon-gamma, interleukin-10 and interleukin-4 production along with a decrease of interleukin-2 by spleen mononuclear cells were observed. Interleukin-4 and interleukin-10 were involved in ablating cellular proliferation in vitro, as the addition of neutralising antibodies to either cytokine reversed the proliferative block. The addition of exogenous recombinant interleukin-2 also restored the proliferative response by spleen mononuclear cells obtained 7 days after infection from infected rats. At the same time, we found an increase in interleukin-10 production by peritoneal cells (in close contact with the flukes) and decreased nitric oxide levels. In addition, histological studies on the liver on day 7 after infection showed the presence of parasite inside migratory tunnels in the parenchyma, and polymorphonuclear leukocytes, predominantly eosinophils, around the parasite. The transient suppression in proliferative response mediated by cytokines interleukin-4 and interleukin-10 in the spleen, and diminution of nitric oxide production in the peritoneum could be mechanisms to evade the protective immune response during the first stages of liver penetration by the parasite.     

Pyruvate oxidation in vivo and in vitro in Dictyostelium discoideum

The in vivo rate of the reaction catalyzed by the pyruvate dehydrogenase complex in Dictyostelium discoideum was measured by exposing cells to [1-14C]alanine and relating the rate of 14CO2 evolution to the specific radioactivity of the isolated [1-14C]pyruvate. The rate obtained, 0.33 mumol/min per ml packed cell volume, was very similar to that determined by independent calculations based upon the rate of utilization of endogenous protein as an energy source in this system.     

A tissue-specific increase in lipogenesis in rat brown adipose tissue in hypothyroidism.

1. Rats were made hypothyroid by feeding them with propylthiouracil together with a low-iodine diet for 4 weeks. 2. [U-14C]Glucose conversion into fatty acids was substantially enhanced in brown adipocytes isolated from hypothyroid rats. Incorporation of 3H2O into fatty acids in vivo was enhanced in hypothyroidism in interscapular brown fat, but not in epididymal white fat or in liver. Hypothyroidism increased the activities of fatty acid synthase and ATP citrate lyase in brown, but not in white, adipocytes. 3. Glycolytic flux in brown adipocytes, quantified by [3-3H]glucose detritiation, was increased by hypothyroidism. This change was accompanied by increased maximum activity of phosphofructokinase. In white adipocytes a large increase in phosphofructokinase maximum activity was observed in hypothyroidism, but this change was accompanied by only small increases in the rate of glucose detritiation by incubated cells. It is suggested that in the brown adipocyte the overall conversion of glucose into fatty acids is enhanced in thyroid deficiency, but that this change is muted in the white adipocyte, possibly because of limitation of glucose transport. 4. Fatty acid synthesis in brown adipocytes from hypothyroid animals was considerably less sensitive to inhibition by exogenous fatty acids than is the process in cells from euthyroid animals. Consequently, the effect of hypothyroidism to enhance lipogenesis is amplified in the presence of physiological concentrations of fatty acid.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

大肠杆菌棉子糖操纵子α—半乳糖苷酶表达的调节控制

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Laccase activity in lignin degradation by Coriolus versicolor in vivo and in vitro studies

Abstract Laccase activity in ligninolytic cultures of Coriolus versicolor was inhibited by the addition of antibodies. The degree and rate of lignin degradation was unaffected by this inhibition in comparison with cultures which had normal laccase activity. Although experiments in vitro showed that milled wood lignin was depolymerised by laccase in the presence of hydrogen peroxide, and that this reaction was inhibited by antibody, it is concluded that this degradative reaction was not the function of laccase in vivo.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

The role of PAR family receptors in activation of mast cells in the norm and in acute inflammation in rats

Dose-dependent release of beta-hexoaminidase induced with thrombin was shown to be mediated by the PAR-1. This was further confirmed by means of agonist, antagonist and PAR desensitization. Acceleration of the mast cell mediator secretion by the Xa factor and PAR-2 agonist, was revealed. An increase in the mast cell release induced by thrombin and TRAP-6 was shown in the acute peritonitis model.     

Changes in the serum protein composition in experimental hypercholesterolemia

Cholesterol accumulation, quantitative changes and composition of lipoproteins, total proteins of blood serum and protein fractions obtained by acid extraction are studied in hypercholesterinemia dynamics in rabbits. It is found that the initial period of cholesterinosis in blood serum is marked by an increased content of total proteins and proteins extracted by acid followed, however, by substantial lowering of the level of these compounds. The proteins obtained by acid extraction are characterized by more explicit changes. This permits assuming their important role in the pathogenesis of hypercholesterinemia. The obtained results make it possible to state that the investigated proteins possess the alkaline properties. The data available in literature on the ability of alkaline polypeptides to bind cholesterol permit assuming that the investigated proteins have the same properties and, hence, can participate in the molecular mechanisms of cholesterol transport as well as in the processes of synthesis and transformations of separate classes of lipoproteins.     

Ghrelin blunted vascular calcification in vivo and in vitro in rats

Ghrelin is a new peptide with regulatory actions in growth hormone secretion in the anterior pituitary gland and in energy metabolism. Currently, ghrelin has potently protective effects in cardiovascular diseases. We used an in vivo model of rat vascular calcification induced by vitamin D3 and nicotine and one of cultured rat vascular smooth muscular cells (VSMCs) calcification induced by beta-glycerophosphate to study the possible mechanism in the regulatory action of ghrelin in vascular calcification. Calcification increased total Ca2+ content and 45Ca2+ deposition in aortas and VSMCs and alkaline phosphatase (ALP) activation in plasma, aortas and VSMCs. However, calcified aortas and VSMCs showed a significant decrease in osteopontin (OPN) mRNA expression and a marked reduction of ghrelin levels in plasma and its mRNA expression in aortas. The aortic calcification was significantly attenuated by subcutaneous administration of ghrelin 30 and 300 nmol kg(-1) day(-1) for 4 weeks, and the latter dosage was more potent than the former. Ghrelin treatment at the two dosages reduced the total aorta Ca2+ content by 24.4% and 28.1%, aortic 45Ca2+ deposition by 18.4% and 24.9%, plasma ALP activity by 36.6% and 76.7%, and aortic ALP activity by 10.3% and 47.6% (all P < 0.01 or 0.05), respectively. Ghrelin at 10(-8)-10(-6) mol/L attenuated the calcification in cultured VSMCs, with decreased total Ca2+ content, 45Ca2+ deposition and ALP activity and increased OPN mRNA expression, in a concentration-dependent manner. In addition, endothelin levels in plasma and aortas and its mRNA expression in aortas significantly increased with calcification, but ghrelin treatment significantly decreased endothelin levels and mRNA expression, with the high dosage being more potent than the lower dosage. These results indicate that local ghrelin in vascular was down-regulated during vascular calcification, whereas administration of ghrelin effectively attenuated vascular and VSMCs calcification.     

Oxidation of Tris to One-Carbon Compounds in a Radical-Producing Model System,in Microsomes,in Hepatocytes and in Rats

The buffer substance tris(hydroxymethyl)aminomethane (Tris) is converted to formaldehyde in an hydroxyl radical producing model system and in rat liver microsomes, and to CO₂ in rat hepatocytes and in the intact rat. In microsomes, formaldehyde formation from Tris is inhibited by catalase, by the antioxidant propylgallate and by the iron chelator deferoxamine, formaldehyde formation is stimulated by the addition of Fe (II) EDTA. In hepatocytes, the formation of [¹⁴C] CO₂ from [¹⁴C] Tris is inhibited by propylgallate and by the iron chelator o-phenanthroline and is stimulated by the presence of a xanthine oxidase system plus Fe (II) EDTA in the medium. In the intact rat, the administration of [¹⁴C] Tris results in the exhalation of [¹⁴C] CO². The results indicate that an oxidant formed via a Fenton-type reaction, possibly the hydroxyl radical, may be involved in the formation of one-carbon compounds from Tris.     

Directional cues in tactile stimuli involved in agonistic encounters in cockroaches

ABSTRACT. The ability of male cockroaches, Nauphoeta cinerea and Periplaneta americana , to respond directionally to tactile agonistic acts was tested using stimulation by artificially manipulated appendages. Responses by Nauphoeta included turning towards the stimulus at preferred angles of c. 40°, 90° and 180°, apparently relying on internally-stored directional sensory information. This turning responsiveness depended in part on the social status of the receiving individual, since subordinate individuals often retreated or failed to respond. Periplaneta males reacted to tactile stimuli by quick movements away from the stimulus or by kicking towards it. The leg used in kicking was that nearest to the part of the body which was stimulated.     

Changes in blood cortisol, glucose and lactate in carp retained in anglers'keepnets

Capture of carp Cyprinus carpio from holding tanks by dipnet, or from semi-natural conditions by rod and line, elicits a physiological stress response characterized by elevation of plasma cortisol levels. The transfer of carp to keepnets subsequent to capture does not increase or reduce the magnitude or duration of this response and in both cases plasma cortisol levels have returned to pre-stress levels within 24 h of the initial disturbance. The postcapture plasma cortisol elevation is accompanied by disturbances in plasma glucose and lactate levels but these are less consistent in severity and duration than the cortisol response. These data suggest that the retention of fish in keepnets following capture, does not represent a source of stress additional to that imposed by capture and has no effect on the rate of recovery of the fish from the initial capture stress.     

临汾市山区水保封禁治理对野生药用植物的恢复的研究

通过对临汾市山区水保封禁治理区域内药用植物的恢复研究,得出:连翘、翅果油树作为该区最主要的两种药用植物,经三年的封禁,其密度恢复分别增加183.8%、8.0%,产量增加9.8%、460.2%;其它药材增加密度为5.6%、产量255.0%,而对其它药用植物的研究可知,耐干旱的、阳性的药用植物恢复好于耐阴,阴性的药用植物,草本药用植物优于灌木、乔木,土石山区的药用植物恢复优于黄土区.封禁治理应是一个长期的植被恢复过程.     

Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.