Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Termiticidal activity of diterpenes from the roots of Euphorbia kansui

Five ingenane compounds, 1-5, kansuinins A and B, isolated from Euphorbia kansui, and their derivatives 7 and 9 were tested for termiticidal activity against the Japanese termite, Reticulitermes speratus. At 72 hours after treatment, the ingenane compounds 1 to 5 caused 100% mortality in R. speratus at 50, 25 and 12.5 microg/disk, respectively, except for compound 1, which gave a mortality rate of (93.06 +/- 5.56)% at 12.5 microg/disk. At 36, 48 and 60 hours after treatment, compounds 1 to 5 showed more termiticidal activity than kansuinins A and B and their derivatives. The kansuinins showed no or only slight activity against termites in the filter paper bioassay under the conditions tested compared with a solvent control.     

"Inert" formulation ingredients with activity: toxicity of trisiloxane surfactant solutions to twospotted spider mites (Acari: Tetranychidae)

Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.     

Repellent and acaricidal activities of basil (Ocimum basilicum) essential oils and rock dust against Ixodes scapularis and Dermacentor variabilis ticks

Repellent and acaricidal activity of essential oils extracted from three varieties of basil (Ocimum basilicum L.) were evaluated on blacklegged ticks (Ixodes scapularis Say) and American dog ticks (Dermacentor variabilis Say) in laboratory conditions. Essential oils were extracted and characterized through gas chromatography-mass spectrometry, and tested at different concentrations for long-term repellent activity using horizontal bioassays. In addition, basil essential oils were combined with an inert material (i.e., granite rock dust) with known insecticidal and miticidal properties to assess acaricidal activities against adult ticks. Among the tested basil varieties, var. Jolina essential oil at 15% vol/vol concentration repelled 96% of tested ticks up to 2 h post-treatment. The EC₅₀ for I. scapularis nymphs was 4.65% vol/vol (95% confidence interval: 4.73–4.57). In acaricidal tests, the combination of essential oil from var. Aroma 2 at 10% wt/wt with rock dust resulted in 100% tick mortality after only 24 h post-exposure, with a LD₅₀ of 3.48% wt/wt (95% CI 4.05–2.91) for freshly prepared treatment tested on I. scapularis adults. The most common compounds detected in basil essential oils by GC–MS were linalool (52.2% in var. Nu Far, 48.2% in Aroma 2, 43.9% in Jolina), sabinene (6.71% in Nu Far, 8.99% in Aroma 2, 8.11% in Jolina), eugenol (11.2% in Jolina, 8.71% in Aroma 2), and estragole (18.2% in Nu Far). The use of essential oils alone and in combination with rock dust provides an innovative and environmentally friendly approach for managing ticks and inhibiting vector-borne disease transmission.

     

Biological effects of macrotetrolide antibiotics and nonactic acids

Published results as well as patent applications on biological effects of macrotetrolides and nonactic acids are reviewed. Their antimicrobial, antiprotozoan (coccidiostatic), antiparasitic (anthelminthic), insecticidal and acaricidal (miticidal) effects and also newly described immunosuppressive and plant growth stimulating activities are described. Both theoretical papers and practical applications including the effects of macrotetrolides on the environment are included; a particular target organism and precise dosage (e.g. LD₅₀) are reported, in agreement with the original papers. It appears that macrotetrolides and their homologs are very prospective bioactive compounds that find application in agriculture, forestry, human and veterinary medicine while their negative effects on the environment are restricted to a minimum (biological quality of soil and wateretc.).     

Antimicrobial and anti-dust mite efficacy of Cinnamomum camphora chvar. Borneol essential oil using pilot-plant neutral cellulase-assisted steam distillation

Cinnamomum camphora chvar. Borneol essential oil (BEO) was efficiently extracted by using pilot-plant neutral cellulase-assisted steam distillation (NCSD). Borneol, β-cadinene and α-caryophyllene were identified as major components. Bacillus subtilis was the most sensitive bacteria to BEO with the lowest minimal inhibition concentration (MIC) and minimal bactericial concentration (MBC) at 1·75 and 3·50 mg ml⁻¹, respectively. Antimicrobial activity of the BEO was also reasonably high against Salmonella typhimurium, Escherichia coli and Staphylococcus aureus, but not sensitive against two fungi, i.e. Aspergillus niger and Penicillium aurantiogriseum. Changes in permeability and integrity of cell membrane, damage of cell wall and further leakage out of metabolites and ions were determined as bactericidal mechanisms of BEO against the two gram-positive bacteria. The BEO showed a reasonably high repelling activity of dust mite, which achieved higher than 95% repelling dust mite activity after the treatment of BEO solution at 0·50 mg ml⁻¹. When the concentration of BEO was higher than 0·50 mg ml⁻¹, it was B-grade miticide with miticidal activity higher than 95%. Miticidal procedures were characterized as excitation, contraction, relaxation and lastly leading to the death of dust mite. It is speculated that the BEO would cause dehydration and death of dust mite as neuromuscular toxicity.     

The effects of beta acids from hops (Humulus lupulus) on mortality of Varroa destructor (Acari: Varroidae)

Hop (Humulus lupulus L.) beta acids (HBA) were tested for miticidal effects on varroa destructor Anderson and Trueman, a parasitic mite of the honey bee (Apis mellifera L.). When varroa were placed on bees that had topical applications of 1?% HBA, there was 100?% mite mortality. Bee mortality was unaffected. Cardboard strips saturated with HBA and placed in colonies resulted in mite drop that was significantly greater than in untreated hives. HBA was detected on about 60?% of the bees in colonies during the first 48?h after application. Mite drop in colonies lasted for about 7?days with the highest drop occurring in the first 2–3?days after treatment. There was a reduction in the percentages of bees with HBA and in the amounts on their bodies after 7?days. Bee and queen mortality in the colonies were not affected by HBA treatments. When cardboard strips saturated with HBA were put in packages of bees, more than 90?% of the mites were killed without an increase in bee mortality. HBA might have potential to control varroa when establishing colonies from packages or during broodless periods.     

Effects of anoxia and hypoxia on the two-spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

We investigated the potential use of anoxic (0% O₂) and hypoxic (lower O₂ concentration than in the atmosphere) conditions for controlling the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Adult T. urticae females were exposed to O₂ concentrations of 0, 0.5, 1, 2, or 21% (control) with a constant CO₂ concentration of 0.05% at 1 atm and 25 °C under continuous darkness for 24 h. The survival and fecundity at 8 days after treatment significantly decreased when the O₂ concentration was lower than 0.5% and 1%, respectively; the lethal concentration at 50% survival (LC₅₀) was 0.55%. The miticidal hypoxic condition (0.5% O₂) led to physiological disorders in host plants. The degree of physiological disorders differed among the plant species tested. Although tomato seedlings died after the hypoxia treatment, in kidney bean and cucumber seedlings the primary leaves remained and lateral buds developed instead of the apical buds that ceased. Hypoxia treatment could be useful as a physical measure for controlling spider mites depending on plant species or cultivars.     

Nitric oxide level, protein oxidation and antioxidant enzymes in rats infected by Trypanosoma evansi

The acaricidal (miticidal) activity of 90% ethanolic extracts of leaves and stem bark of Swietenia mahogani and Swietenia macrophylla were tested against Varroa destructor mite. Four concentrations were used over two different time intervals under laboratory and field conditions. In general, it was noticed that the acaricidal effect based on mortality and LC(50) of all tested extracts against the Varroa mite was concentration and time dependant. The acaricidal action against Varroa mites was relatively the least for the S. macrophylla stem bark extract at 500 ppm concentration after 48 h while it reached 100% and 95% in case of S. mahogani bark and S. macrophylla leaves, respectively. The% infestation with Varroa in colonies treated with the different extracts at various time intervals showed that the rate of infestation decreased to 0.0% after 12 days from the beginning of treatments with 500 ppm of S. mahogani leaves extract compared to 0.79% decrease after treatment with Mitac, a reference drug (60 mg/colony). The rate of infestation in case of treatments with S. mahogani bark, S. macrophylla leaves and S. macrophylla bark was decreased to 0.11%, 2.41% and 1.08%, respectively. The highest reduction was observed with S. mahogani leaves extract followed by S. mahogani bark. All the tested extracts showed less or no effect on honey bees at the different concentrations and at different bioassay times. This study suggested that the use of natural plant extracts or their products as ecofriendly biodegradable agents could be of high value for the control of Varroa mite.     

Isolation and Characterization of Burkholderia rinojensis sp. nov., a Non-Burkholderia cepacia Complex Soil Bacterium with Insecticidal and Miticidal Activities

Isolate A396, a bacterium isolated from a Japanese soil sample demonstrated strong insecticidal and miticidal activities in laboratory bioassays. The isolate was characterized through biochemical methods, fatty acid methyl ester (FAME) analysis, sequencing of 16S rRNA, multilocus sequence typing and analysis, and DNA-DNA hybridization. FAME analysis matched A396 to Burkholderia cenocepacia, but this result was not confirmed by 16S rRNA or DNA-DNA hybridization. 16S rRNA sequencing indicated closest matches with B. glumae and B. plantarii. DNA-DNA hybridization experiments with B. plantarii, B. glumae, B. multivorans, and B. cenocepacia confirmed the low genetic similarity (11.5 to 37.4%) with known members of the genus. PCR-based screening showed that A396 lacks markers associated with members of the B. cepacia complex. Bioassay results indicated two mechanisms of action: through ingestion and contact. The isolate effectively controlled beet armyworms (Spodoptera exigua; BAW) and two-spotted spider mites (Tetranychus urticae; TSSM). In diet overlay bioassays with BAW, 1% to 4% (vol/vol) dilution of the whole-cell broth caused 97% to 100% mortality 4 days postexposure, and leaf disc treatment bioassays attained 75% ± 22% mortality 3 days postexposure. Contact bioassays led to 50% larval mortality, as well as discoloration, stunting, and failure to molt. TSSM mortality reached 93% in treated leaf discs. Activity was maintained in cell-free supernatants and after heat treatment (60°C for 2 h), indicating that a secondary metabolite or excreted thermostable enzyme might be responsible for the activity. Based on these results, we describe the novel species Burkholderia rinojensis, a good candidate for the development of a biocontrol product against insect and mite pests.     

Studies on the Mechanisms of the Monofluoroacetanilides Poisoning to Mammals

Organophosphates, phosphorothioates, phosphorodithioates, phosphites, and phosphonates were found to inhibit the fluoroacetanilide amidohydrolase of chicken liver. Above all, the inhibition by triphenyl phosphate, tri-n-butyl phosphorothioate, triphenyl phosphite, Dipterex, and DDVP were extremely remarkable. Their concentrations for 50% inhibition were about 10^?8 m or 10^?7 m, and the inhibition of triphenyl phosphate was non-competitive to the substrate. These compounds were slightly or moderately toxic to mice even when administered intraperitoneally. The specificity of the inhibition was also discussed.

The effects of the fluoroacetanilide amidohydrolase (fluoroacetanilidase) inhibitors on the fluoroaceto-p-bromoanilide (FBA) poisoning to mammals and insects were studied. Triphenyl phosphate (TPP) and tri-n-butyl phosphorothioate (TBT) were mainly used as the inhibitors. TPP was effective for reducing the oral and cutaneous toxicities of FBA to mice and rats only when applied prior to the FBA application. However, in cases of the inhaled toxicity and the combined inhaled-cutaneous toxicity, TPP or TBT were effective even when they were applied simultaneously with FBA. When TPP and TBT were administered to mice by the combined inhaled-cutaneous application, the activity of fluoroacetanilidase in liver and kidney was rapidly decreased, but it recovered within 4 to 6 days after the application. The insecticidal and miticidal activities of FBA were not affected at all both in the previous and simultaneous applications of the inhibitors. However, it was found that the fluoroacetanilides hydrolyzing enzyme of Mealy plum aphid was not inhibited by TPP even in the concentration of 10^?4 m. The mechanisms of the fluoroacetanilides poisoning to mammals and insects were discussed.     

Effects of dispersal, predators (Acari: Phytoseiidae), weather, and ground cover treatments on populations of Tetranychus urticae (Acari: Tetranychidae) in apple orchards

In a 2-yr study of causes of mite outbreaks in apple (Malus spp.) orchards in Nova Scotia, we monitored immigration of Tetranychus urticae Koch from orchard ground cover into trees populated by the generalist phytoseiid predator Typhlodromus pyri Scheuten. In both years, T. urticae-days in the tree canopy increased with number of T. urticae caught in sticky bands on tree trunks. In 2000, T. urticae-days were negatively correlated with T. pyri-days. Lack of correlation in 2001 was attributed to higher rates of immigration, which would mask the effects of predation. Weather also affected mite dynamics. Rainfall in July and August was less in 2001 than in 2000. Heat units were sufficient for six generations of T. urticae in 2001 but only for five in 2000. Consequently, T. urticae-days in the tree canopy and immigration rates were significantly greater in 2001 than in 2000, despite three-fold greater use of miticides. We also tested the effects of herbicides on T. urticae immigration. Application of selective herbicides in laneways reduced coverage of reproductive hosts of T. urticae, but these changes did not reduce immigration. In 2001, application of a miticidal herbicide, glufosinate, in tree rows reduced captures of T. urticae on sticky bands in high immigration orchards but not in low immigration orchards. We conclude that generalist predators and modified herbicide use are insufficient remedies and that effective biological control of T. urticae in the ground cover by a specialist phytoseiid such as Amblyseius fallacis Garman is essential to prevent outbreaks.     

Toxicity of the entomopathogenic bacteria Photorhabdus and Xenorhabdus to the mushroom mite (Luciaphorus sp.; Acari: Pygmephoridae)

The mushroom mite, Luciaphorus sp. is a serious pest of tropical mushrooms. We determined the pathogenicity and toxicity of species and strains of the entomopathogenic bacteria, Photorhabdus and Xenorhabdus to the mite. As these bacteria are known to produce antifungal substances, we first determined the effect of 21 species and strains of the bacteria on the mycelial growth of the mushroom, Lentinus squarrosulus. We then determined the toxicity of the eight species and strains of bacteria that did not show any effect on mushroom growth against both the female and male mites. All eight species and strains of the bacteria were toxic to the female mite resulting in significant mite mortality within 24-48 h. Cell-free supernatants from all the eight bacterial species and strains were also toxic to the female mite inflicting significant mortality within 24-48 h. The supernatants of two strains, GPS12 and GPS11, of Photorhabdus luminescens ssp. laumondii were significantly more toxic than the other species and strains to the female mite, resulting in 90-95% mite mortality within 48 h. Both the concentration and age of the bacteria had significant effect on the toxicity of the supernatants to the female mite. None of the bacteria showed toxicity to the male mite which has undeveloped mouthparts. These results indicate that P. luminescens ssp. laumondii and its byproducts are directly toxic to the female mite, suggesting the potential of developing a novel biological approach for the control of this mushroom pest. This is the first report on the miticidal activity of the entomopathogenic bacteria, Photorhabdus and Xenorhabdus.     

Inhibitory effect of synthetic C-C biflavones on various phospholipase A(2)s activity

Several prototypes of C-C biflavones (a-f) were synthesized and evaluated their inhibitory activity against phospholipase A(2)s (PLA(2)s) activity. The synthetic C-C biflavones (a-f) showed rather different inhibitory activity against various PLA(2)s. Most synthetic C-C biflavonoids exhibited potent and broad inhibitory activity against various sPLA(2)s and cPLA(2) tested regardless of their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C-C biflavonoid (d) only showed inhibitory activity against sPLA(2) X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA(2) IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C-C biflavones which possess more selective inhibitory activity against isozymes of PLA(2).     

Response of the endophytic diazotroph Gluconacetobacter diazotrophicus on solid media to changes in atmospheric partial O(2) pressure

Gluconacetobacter diazotrophicus is an N(2)-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O(2) pressures (pO(2)) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO(2) (5 to 60 kPa). Nitrogenase activity was measured by H(2) evolution in N(2)-O(2) and in Ar-O(2), and respiration rate was measured by CO(2) evolution in N(2)-O(2). To validate the use of H(2) production as an assay for nitrogenase activity, a non-N(2)-fixing (Nif(-)) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup(+)) activity (0.016 +/- 0.009 micromol of H(2) 10(10) cells(-1) h(-1)) when incubated in an atmosphere enriched in H(2). However, Hup(+) activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO(2) tested. However, when the assay atmospheric pO(2) was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO(2) for nitrogenase activity was 0 to 20 kPa above the pO(2) at which the bacteria had been grown. As atmospheric pO(2) was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO(2) was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO(2) from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O(2), 80% of nitrogenase activity was recovered within 10 min, indicating a "switch-off/switch-on" O(2) protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N(2) at a wide range of atmospheric pO(2) and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO(2).     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

The effect of Cu2+, Zn2+ cations and biogenic amines on the nuclear poly(ADP-ribose) polymerase activity in the rat brain

Study of the effects of Cu2+, Zn2+ cations and polyamines, spermine and spermidine, on the nuclear poly(ADP-ribose)polymerase activity of rat brain was carried out. It was shown that low concentrations of Cu2+ stimulate the activity of purified poly(ADP-ribose)polymerase. The poly(ADP-ribose)polymerase activity was increased 1.4-fold at 5 microM Cu2+. A further increase of Cu2+ concentration inhibited the enzymatic activity; at 50 microM Cu2+ the polymerase activity appeared to be fully inhibited. It was shown that Zn2+ inhibited only the poly(ADP-ribose)polymerase activity. Zn2+ at a concentration of 125 microM fully inhibited the enzymatic activity. Spermine and spermidine stimulated the poly(ADP-ribose)polymerase activity of brain nuclei of newborn and old rats.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein

We sought to determine the roles of PKCalpha and G(i)alpha in regulating cPLA(2) activity in bovine pulmonary artery endothelial cell membrane under peroxynitrite (ONOO(-)) stimulation. Treatment of bovine pulmonary artery endothelial cells with ONOO(-) markedly stimulates the cell membrane associated protease activity, protein kinase C (PKC) activity, phospholipase A(2) (PLA(2)) activity, and arachidonic acid (AA) release from the cells. ONOO(-) significantly increases (Ca(2+))(i) in the cells, and pretreatment with the intracellular Ca(2+) chelator BAPTA-AM prevents the increase in (Ca(2+))(i), protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF(3)) (a cPLA(2) inhibitor) prevents ONOO(-)-stimulated cPLA(2) activity and AA release without producing a significant alteration of the protease activity. Pretreatment with vitamin E and aprotinin prevents ONOO(-)-induced increase in the protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment with the PKC inhibitor calphostin C prevents ONOO(-)-caused increase in PKC activity and cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot study of the cell membrane isolated from the ONOO(-)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. An immunoblot study with anti-nitrotyrosine antibody revealed that ONOO(-) induces nitration of tyrosine residues in PKCalpha. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. An immunoblot study of the endothelial cell membrane with polyclonal cPLA(2) antibody revealed that treatment of the cells with ONOO(-) markedly increases the cPLA(2) immunoreactive protein profile in the membrane. Pretreatment of the endothelial cells with Go6976, a PKCalpha inhibitor, prevents the increase in PKC activity and cPLA(2) activity in the cell membrane under ONOO(-)-triggered condition. It, therefore, appears from the present study that treatment of the cells with ONOO(-) causes an increase in the protease activity, and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot assay with polyclonal G(i)alpha antibody elicited an immunoreactive band having a molecular mass of 41 kDa. Pretreatment of the cells with pertussis toxin markedly inhibits ONOO(-)-induced increase in cPLA(2) activity and AA release without significantly altering (Ca(2+))(i), protease activity, and PKC activity in the cell membrane. Treatment of the cells with ONOO(-) causes phosphorylation of G(i)alpha in the cell membrane, and pretreatment with Go6976 prevents its phosphorylation. We suggest the existence of a pertusssis toxin sensitive G protein-mediated mechanism for activation of cPLA(2) by ONOO(-) in bovine pulmonary artery endothelial cell membrane, which is regulated by PKCalpha-dependent phosphorylation and sensitive to aprotinin for its inhibition.     

The effects of modulation of gamma-glutamyl transpeptidase activity in HepG2 cells on thiol homeostasis and caspase-3-activity

The present studies aimed to elucidate how the modulation of gamma-glutamyl transpeptidase (gammaGT) activity in human hepatoma (HepG2) cell line influences H(2)O(2) production, caspase 3 activity, protein S-thiolation by glutathione (GSH), cysteinyl-glycine (Cys-Gly) and cysteine (Cys), and the level of other redox forms of these thiols. The experiments showed that 1-h stimulation of gammaGT elevated H(2)O(2) production, leading to prooxidant conditions. After 24-h stimulation, H(2)O(2) concentration was at the control level, while Cys-Gly-, Cys- and GSH-dependent S-thiolation was markedly increased, which was accompanied by a drop in caspase-3 activity. The inhibition of gammaGT activity by acivicin led to H(2)O(2) decrease after 1-h incubation which still persisted after 24 h. The inhibition of gammaGT activity in HepG2 cells was also connected with the lowering of S-thiolation with Cys and Cys-Gly and with increasing of caspase-3 activity. The results of our studies indicate that the modulation of gammaGT activity can be used to change cellular redox status, and can affect Cys- and Cys-Gly-dependent S-thiolation and caspase-3 activity. We suggest that the role of high gammaGT activity in HepG2 cells can be connected with production of reactive oxygen species and with S-thiolation with Cys and Cys-Gly that can influence activity of caspase 3.