Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis.

A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.

Hydrogen sulfide inhibits calcium and phosphorus loss after fracture by negatively regulating glucocorticoid/glucocorticoid receptor α.

AIMS: Post-fracture calcium and phosphorus excretion is greater than influx, which might be caused by stress. Glucocorticoid is known to enhance calcium and phosphorous excretion, and hydrogen sulfide (H2S) has been shown to exert inhibitory effects on glucocorticoid. Therefore, this study explored whether H2S could inhibit calcium and phosphorus loss after fracture by regulating glucocorticoid and/or its receptor. MAIN METHODS: The following properties were analyzed in rats with femur fractures: serum and urinary calcium and phosphorus (by colorimetry); bone turnover markers alkaline phosphatase, serum type 1 collagen amino terminal peptide, type 1 procollagen carboxy terminal peptide, and anti-tartaric acid phosphatase (by ELISA); factors related to calcium-phosphorus metabolism including glucocorticoid, parathyroid hormone, calcitonin, fibroblast growth factor 23, and 1,25(OH)2D3 (by ELISA); and sulfhydration of glucocorticoid receptor α in the kidney (by immunoprecipitation linked biotin-switch assay), after supplementing with mifepristone, the H2S donor GYY4137 or H2S generating enzyme inhibitors aminooxyacetic acid and propargylglycine. KEY FINDINGS: Serum H2S decreased and glucocorticoid secretion increased in rats post-fracture. The glucocorticoid receptor inhibitor mifepristone partly blunted calcium and phosphorus loss. Furthermore, supplementation with GYY4137 reduced glucocorticoid secretion; inhibited glucocorticoid receptor α activity by sulfhydration; downregulated vitamin D 1α-hydroxylase expression; and upregulated 24-hydroxylase, calbindin-D28k, and sodium phosphate cotransporter 2a expression in the kidney; thereby inhibiting calcium and phosphorus loss induced by fracture. Moreover, inhibiting endogenous H2S generation showed opposite effects. SIGNIFICANCE: Our findings suggest that H2S antagonized calcium and phosphorus loss after fracture by reducing glucocorticoid secretion and inhibiting glucocorticoid receptor α activity by sulfhydration.

Hormetic Effects of Binaphthyl Phosphonothioates as Pro-oxidants and Antioxidants.

Organophosphorous compounds with such a wide variety in structure, application, and biochemical activities include pesticides, herbicides, nerve agents, medicines, reagents in organic chemistry, and additives for polymers. Binaphthyl phosphono-, phosphorothioates, and their derivatives, are useful chiral catalysts for various asymmetric reactions and are expected to act as heavy metal scavengers. In this study, we aimed to evaluate the neurotoxicity and biochemical properties of a new series of binaphthyl phosphonothioates called KK compounds using the mouse hippocampal HT22 cells. Despite negligible structural difference, the compounds exhibited differential general cytotoxic activity which was independent of acetylcholine esterase inhibition; on the other hand, all compounds tested prevented endogenous oxidative stress by suppressing generation of reactive oxygen species. Among them, KK397, KK387, KK410, and KK421 showed hormesis, i.e., biphasic dose responses to endogenous oxidative stress, characterized by beneficial effect at low dose and toxic effect at high dose. At cytotoxic concentrations, these compounds were potent radical generators and activated intracellular signaling molecules such as the p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, growth arrest- and DNA damage-inducible gene 153, X-box binding protein 1, and heme oxygenase 1, which are preferentially activated by cell stress-inducing signals, including oxidative and endoplasmic reticulum stress. These findings indicated that novel binaphthyl phosphonothioates can exhibit multiple biochemical properties, functioning as antioxidants and/or pro-oxidants, depending on the concentration, and chemical modification of binaphthyl organophosphorus compounds endowed them with unique characteristics and multiple beneficial functions.

Reactivation of VX-Inhibited Human Acetylcholinesterase by Deprotonated Pralidoxime. A Complementary Quantum Mechanical Study.

In the present work, we performed a complementary quantum mechanical (QM) study to describe the mechanism by which deprotonated pralidoxime (2-PAM) could reactivate human (Homo sapiens sapiens) acetylcholinesterase (HssAChE) inhibited by the nerve agent VX. Such a reaction is proposed to occur in subsequent addition-elimination steps, starting with a nucleophile bimolecular substitution (SN2) mechanism through the formation of a trigonal bipyramidal transition state (TS). A near attack conformation (NAC), obtained in a former study using molecular mechanics (MM) calculations, was taken as a starting point for this project, where we described the possible formation of the TS. Together, this combined QM/MM study on AChE reactivation shows the feasibility of the reactivation occurring via attack of the deprotonated form of 2-PAM against the Ser203-VX adduct of HssAChE.

Protective effect of GYY4137, a water­soluble hydrogen sulfide­releasing molecule, on intestinal ischemia­reperfusion.

The present study aimed to clarify the protective effects of p­methoxyphenyl morpholino­phosphinodithioic acid (GYY4137), a water­soluble hydrogen sulfide­releasing molecule, on a rat model of intestinal ischemia­reperfusion (IIR). A total of 40 healthy male Sprague Dawley (SD) rats were randomly divided into four groups (n=10/group): Group A, a sham­surgery group; Group B, the IIR group; group C, rats with IIR that were administered an abdominal injection of low­dose GYY4137 (40 mg/kg); and group D, rats with IIR that were administered high­dose GYY4137 (80 mg/kg). Intestinal histomorphology was observed using hematoxylin and eosin staining, and the concentrations of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. Apoptotic index (AI) was determined by terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling. Reverse transcription­quantitative PCR analysis was performed to assess the expression levels of intestinal caspase­3, Bax and Bcl­2. Notably, disordered arrangement of intestinal villi and mucosal necrosis were detected in group B, which was substantially improved by GYY4137 treatment (groups C and D). MDA content (nmol/mg) was 2.83±0.36, 9.23±0.78, 4.97±0.45 and 3.51±1.05 nmol/mg in groups A, B, C and D, respectively. In addition, SOD concentration (U/mg) was 135.37±3.34, 76.45±1.39, 95.13±1.64 and 115.13±2.54 in groups A, B, C and D, respectively. Furthermore, AI in group B (21.73±1.17%) was markedly higher than that in group A (4.53±0.28%) and in the GYY4137 intervention groups (9.53±0.96 and 6.53±0.76% in groups C and D, respectively). Compared with in group A, the mRNA expression levels of Bax and caspase­3 were markedly higher in group B (P<0.05), whereas the expression of Bcl­2 was significantly lower (P<0.05). Furthermore, compared with in group B, Bcl­2 expression was higher, and Bax and caspase­3 expression was lower in groups C and D (P<0.05). In conclusion, GYY4137 may alleviate IIR­induced damage in SD rats.

Hydrogen sulfide attenuates lung ischemia-reperfusion injury through SIRT3-dependent regulation of mitochondrial function in type 2 diabetic rats.

BACKGROUND: Lung ischemia-reperfusion injury is a complex pathophysiologic process associated with high morbidity and mortality. We have demonstrated elsewhere that diabetes mellitus aggravated ischemia-induced lung injury. Oxidative stress and mitochondrial dysfunction are drivers of diabetic lung ischemia-reperfusion injury; however, the pathways that mediate these events are unexplored. In this study using a high-fat diet-fed model of streptozotocin-induced type 2 diabetes in rats, we determined the effect of hydrogen sulfide on lung ischemia-reperfusion injury with a focus on Sirtuin3 signaling. METHODS: Rats with type 2 diabetes were exposed to GYY4137, a slow release donor of hydrogen sulfide with or without administration of the Sirtuin3 short hairpin ribonucleic acid plasmid, and then subjected to a surgical model of ischemia-reperfusion injury of the lung (n = 8). Lung function, oxidative stress, inflammation, cell apoptosis, and mitochondrial function were measured. RESULTS: Compared with nondiabetic rats, animals with type 2 diabetes at baseline exhibited significantly decreased Sirtuin3 signaling in lung tissue and increased oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction (P < .05 each). In addition, further impairment in Sirtuin3 signaling was found in diabetic rats subjected to this model of lung ischemia-reperfusion. Simultaneously, the indexes showed further aggravation. Treatment with hydrogen sulfide restored Sirtuin3 expression and decreased lung ischemia-reperfusion injury in animals with type 2 diabetes mellitus by improving lung functional recovery, decreasing oxidative damage, suppressing inflammation, ameliorating cell apoptosis, and preserving mitochondrial function (P < .05). Conversely, these protective effects were largely reversed in Sirtuin3 knockdown rats. CONCLUSION: Impaired lung Sirtuin3 signaling associated with type 2 diabetic conditions was further attenuated by an ischemia-reperfusion insult. Hydrogen sulfide ameliorated reperfusion-induced oxidative stress and mitochondrial dysfunction via activation of Sirtuin3 signaling, thereby decreasing lung ischemia-reperfusion damage in rats with a model of type II diabetes.

Experimental research into the potential therapeutic effect of GYY4137 on Ovariectomy-induced osteoporosis.

BACKGROUND: Evidence has shown that endogenous H2S plays an important role in the physiological and pathophysiological processes of many organs. The study aimed to explore whether exogenous H2S has a potential therapeutic effect on a rat ovariectomy-induced model of osteoporosis. METHODS: The OVX osteoporosis model was established in female Sprague-Dawley rats by full bilateral ovariectomy. The rats were randomly divided into four groups, with the two experimental groups receiving an intraperitoneal injection of GYY4137 or sodium alendronate. The level of H2S in the plasma was determined and common laboratory indicators to diagnose osteoporosis, such as alkaline phosphatase (ALP) activity and the levels of osteocalcin (OCN), calcitonin, parathyroid hormone and leptin were measured. The bone mineral density (BMD) of the 4th and 5th lumbar vertebrae was measured using dual-energy X-ray absorptiometry. The maximum stress of femoral fracture was obtained through a three-point bending test of the femur. RESULTS: The OVX osteoporosis model was successfully established. GYY4137 was injected to increase the level of H2S in the plasma in one group, designated OVX-GYY during the observation period (p < 0.05). At 12 weeks, the BMD value of the fourth lumbar vertebra in the OVX-GYY group had increased (p < 0.05). The BMD femur value in the OVX-vehicle group had decreased (p < 0.05). Bilateral ovariectomy leads to biochemical disorders related to bone metabolism and hormone levels in rat plasma (all p < 0.05). Ovariectomy also reduced blood calcium, blood phosphate and calcitonin, and increased parathyroid hormone and leptin. The opposite results were obtained for the groups with alendronate sodium or GYY4137 treatment (all p < 0.05). CONCLUSIONS: Through the slow release of H2S, GYY4137 did an excellent job of simulating endogenous neuroendocrine gaseous signaling molecules. Exogenous H2S had a regulatory effect on osteoporosis in ovariectomized rats, showing potential value for the treatment of human postmenopausal osteoporosis.

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

Hydrogen Sulfide and Sulfate Prebiotic Stimulates the Secretion of GLP-1 and Improves Glycemia in Male Mice.

Recently, the gastrointestinal microbiome, and its metabolites, has emerged as a potential regulator of host metabolism. However, to date little is known on the precise mechanisms of how this regulation occurs. Hydrogen sulfide (H2S) is abundantly produced in the colon by sulfate-reducing bacteria (SRB). H2S is a bioactive gas that plays regulatory roles in many systems, including metabolic hormone regulation. This gas metabolite is produced in close proximity to the glucagonlike peptide-1 (GLP-1)-secreting cells in the gut epithelium. GLP-1 is a peptide hormone that plays pivotal roles in both glucose homeostasis and appetite regulation. We hypothesized that H2S can directly regulate GLP-1 secretion. We demonstrated that H2S donors (NaHS and GYY4137) directly stimulate GLP-1 secretion in murine L-cells (GLUTag) and that this occurs through p38 mitogen-activated protein kinase without affecting cell viability. We then increased SRB in mice by supplementing the diet with a prebiotic chondroitin sulfate for 4 weeks. Mice treated with chondroitin sulfate had elevated Desulfovibrio piger levels in the feces and increased colonic and fecal H2S concentration. These animals also had enhanced GLP-1 and insulin secretion, improved oral glucose tolerance, and reduced food consumption. These results indicate that H2S plays a stimulatory role in GLP-1 secretion and that sulfate prebiotics can enhance GLP-1 release and its downstream metabolic actions.

H2S-induced S-sulfhydration of lactate dehydrogenase a (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells.

Cystathionine-ß-synthase (CBS) is upregulated and hydrogen sulfide (H2S) production is increased in colon cancer cells. The functional consequence of this response is stimulation of cellular bioenergetics and tumor growth and proliferation. Lactate dehydrogenase A (LDHA) is also upregulated in various colon cancer cells and has been previously implicated in tumor cell bioenergetics and proliferation. In the present study, we sought to determine the potential interaction between the H2S pathway and LDH activity in the control of bioenergetics and proliferation of colon cancer, using the colon cancer line HCT116. Low concentrations of GYY4137 (a slow-releasing H2S donor) enhanced mitochondrial function (oxygen consumption, ATP production, and spare respiratory capacity) and glycolysis in HCT116 cells. SiRNA-mediated transient silencing of LDHA attenuated the GYY4137-induced stimulation of mitochondrial respiration, but not of glycolysis. H2S induced the S-sulfhydration of Cys163 in recombinant LDHA, and stimulated LDHA activity. The H2S-induced stimulation of LDHA activity was absent in C163A LDHA. As shown in HCT116 cell whole extracts, in addition to LDHA activation, GYY4137 also stimulated LDHB activity, although to a smaller extent. Total cellular lactate and pyruvate measurements showed that in HCT116 cells LDHA catalyzes the conversion of pyruvate to lactate. Total cellular lactate levels were increased by GYY4137 in wild-type cells (but not in cells with LDHA silencing). LDHA silencing sensitized HCT116 cells to glucose oxidase (GOx)-induced oxidative stress; this was further exacerbated with GYY4137 treatment. Treatment with low concentrations of GYY4137 (0.3mM) or GOx (0.01U/ml) significantly increased the proliferation rate of HCT116 cells; the effect of GOx, but not the effect of GYY4137 was attenuated by LDHA silencing. The current report points to the involvement of LDHA in the stimulatory effect of H2S on mitochondrial respiration in colon cancer cells and characterizes some of the functional interactions between LDHA and H2S-stimulated bioenergetics under resting conditions, as well as during oxidative stress.

Protective effects of exogenous and endogenous hydrogen sulfide in mast cell-mediated pruritus and cutaneous acute inflammation in mice.

The recently described 'gasomediator' hydrogen sulfide (H2S) has been involved in pain mechanisms, but its effect on pruritus, a sensory modality that similarly to pain acts as a protective mechanism, is poorly known and controversial. The effects of the slow-releasing (GYY4137) and spontaneous H2S donors (Na2S and Lawesson's reagent, LR) were evaluated in histamine and compound 48/80 (C48/80)-dependent dorsal skin pruritus and inflammation in male BALB/c mice. Animals were intradermally (i.d.) injected with C48/80 (3µg/site) or histamine (1µmol/site) alone or co-injected with Na2S, LR or GYY4137 (within the 0.3-100nmol range). The involvement of endogenous H2S and KATP channel-dependent mechanism were also evaluated. Pruritus was assessed by the number of scratching bouts, whilst skin inflammation was evaluated by the extravascular accumulation of intravenously injected 125I-albumin (plasma extravasation) and myeloperoxidase (MPO) activity (neutrophil recruitment). Histamine or C48/80 significantly evoked itching behavior paralleled by plasma extravasation and increased MPO activity. Na2S and LR significantly ameliorated histamine or C48/80-induced pruritus and inflammation, although these effects were less pronounced or absent with GYY4137. Inhibition of endogenous H2S synthesis increased both Tyrode and C48/80-induced responses in the skin, whereas the blockade of KATP channels by glibenclamide did not. H2S-releasing donors significantly attenuate C48/80-induced mast cell degranulation either in vivo or in vitro. We provide first evidences that H2S donors confer protective effect against histamine-mediated acute pruritus and cutaneous inflammation. These effects can be mediated, at least in part, by stabilizing mast cells, known to contain multiple mediators and to be primary initiators of allergic processes, thus making of H2S donors a potential alternative/complementary therapy for treating inflammatory allergic skin diseases and related pruritus.

Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil.

Hydrogen sulfide (H2S) releasing agents: chemistry and biological applications.

Hydrogen sulfide (H2S) is a newly recognized signaling molecule with very potent cytoprotective actions. The fields of H2S physiology and pharmacology have been rapidly growing in recent years, but a number of fundamental issues must be addressed to advance our understanding of the biology and clinical potential of H2S in the future. Hydrogen sulfide releasing agents (also known as H2S donors) have been widely used in these fields. These compounds are not only useful research tools, but also potential therapeutic agents. It is therefore important to study the chemistry and pharmacology of exogenous H2S and to be aware of the limitations associated with the choice of donors used to generate H2S in vitro and in vivo. In this review we summarized the developments and limitations of currently available donors including H2S gas, sulfide salts, garlic-derived sulfur compounds, Lawesson's reagent/analogs, 1,2-dithiole-3-thiones, thiol-activated donors, photo-caged donors, and thioamino acids. Some biological applications of these donors were also discussed.

The complex effects of the slow-releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells.

The role of hydrogen sulfide (H2 S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund's adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1-0.5 mM) decreased LPS-induced production of nitrite (NO2 (-) ), PGE2 , TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-ß-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1ß, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

Survey for resistance to four insecticides in Myzus persicae clones from peach trees and weeds in south-central Washington.

The insecticides esfenvalerate, endosulfan, imidacloprid, and methamidophos were screened against 74 clonal populations of the green peach aphid, Myzus persicae (Sulzer), collected as fundatrices from peach (Prunus spp.) and as apterous virginoparae from various weeds near potato (Solanum spp.) fields in the Yakima Valley and Columbia Basin regions of Washington state. Response to diagnostic concentration of four insecticide products demonstrated a bimodal pattern of survival to endosulfan (Phaser), suggesting two phenotype classes responding to this insecticide. This pattern was not observed with the other products. Moderately significant correlations in mortality of aphid clones treated with diagnostic concentrations of endosulfan versus clones treated with methamidophos and similarly with the correlation of mortalities for imidacloprid-treated versus methamidophos-treated clones suggested modest levels of cross tolerance. No significant correlations were observed with the remaining four possible comparisons. Concentration-response bioassays were conducted on 16 clones with the four insecticides. The greatest difference between resistant and susceptible clones (expressed as the ratio of lethal concentrations producing 50% mortality; RR) was only seven-fold observed in the endosulfan-treated clones. The greatest RR was five-fold for imidacloprid, four-fold for esfenvalerate, and three-fold for methamidophos-treated clones. Only the endosulfan response is likely to be ofbiological significance and reflects the same cyclodiene resistance discovered in this region over a decade ago.

Effect of methamidophos and urea application on microbial communities in soils as determined by microbial biomass and community level physiological profiles.

In this study, we evaluated the effect of the application by two agrochemicals, methamidophos (O,S-dimethyl phosphoroamidothioate) and urea, on microbial diversity in soil, using the combined approaches of soil microbial biomass analysis and community level physiological profiles (CLPPs). The results showed that both a low and a high level of methamidophos application (CS2 and CS3) and urea application (CS4) significantly decreased microbial biomass C (Cmic) by 41-83% compared with the control (CS1). The soil organic C (Corg) values of CS3 and CS4 were significantly higher and lower by 24% and 14%, respectively, than that of CS1. Similarly to Cmic, the values of Cmic/Corg of the three applied soils which decreased were lower by 31-84% than that of CS1. In contrast, the respiration activity of the three applied soils were significantly higher than the control. Agrochemical application also significantly increased the soil total of N and P (Ntol and Ptol) and decreased the Corg/Ntol and Corg/Ptol values. The CLPPs results showed that the AWCD (average well color development) of the three applied soils were significantly higher than that of CS1 during the incubation period. Substrate richness, Shannon and Simpson indices of microbial communities under chemical stresses, increased significantly. In addition, the CFU (colony-forming unit) numbers of methamidophos metabolized bacteria in CS2 and CS3 also increased significantly by 86.1% and 188.9% compared with that of CS1. The combined results suggest that agrochemicals reduce microbial biomass and enhance functional diversities of soil microbial communities; meanwhile, some species of bacteria may be enriched in soils under methamidophos stress.

Interactions between nematophagous fungi and consequences for their potential as biological agents for the control of potato cyst nematodes.

The efficacies of three nematophagous fungi, Paecilomyces lilacinus, Plectosphaerella cucumerina and Pochonia chlamydosporia, for controlling potato cyst nematodes (PCN) as part of an Integrated Pest Management (IPM) regime were studied. The compatibility of the nematophagous fungi with commonly used chemical pesticides and their ability to compete with the soil fungi Rhizoctonia solani, Chaetomium globosum, Fusarium oxysporum, Penicillium bilaii and Trichoderma harzianum were tested in vitro. Paecilomyces lilacinus was the most successful competitor when the ability to grow and inhibit growth of an opposing colony at both 10 and 20 degrees C was considered. P. lilacinus also showed potential for control of the soil-borne fungal pathogen R. solani, releasing a diffusable substance in vitro which inhibited its growth and caused morphological abnormalities in its hyphae. Pochonia chlamydosporia was least susceptible to growth inhibition by other fungi at 20 degrees in vitro, but the isolate tested did not grow at 10 degrees. Plectosphaerella cucumerina was a poor saprophytic competitor. Radial growth of Paecilomyces lilacinus and Plectosphaerella cucumerina was slowed, but not prevented, when grown on potato dextrose agar incorporating the fungicides fenpiclonil and tolclofos-methyl, and was not inhibited by the addition of pencycuron or the nematicide oxamyl. Radial growth of Pochonia chlamydosporia was partially inhibited by all the chemical pesticides tested. The efficacy of Paecilomyces lilacinus as a control agent for R. solani was further investigated in situ. Treatment with P. lilacinus significantly reduced the symptoms of Rhizoctonia disease on potato stems in a pot trial. The effectiveness of P. lilacinus and P. cucumerina against PCN was also tested in situ. Three application methods were compared; incorporating the fungi into alginate pellets, Terra-Green inoculated with the fungi and applying conidia directly to the tubers. Both formulations containing P. lilacinus and formulation mixtures alone, particularly alginate pellets, significantly reduced multiplication of PCN in soil. We conclude that P. lilacinus showed the greatest potential for use in combination with selected fungicides and nematicides as part of an IPM programme for the control of PCN, but further work is required to confirm whether it is effective against PCN in soil.

Insecticide use and organophosphate resistance in the coffee leaf miner Leucoptera coffeella (Lepidoptera: Lyonetiidae).

Increasing rates of insecticide use against the coffee leaf minerLeucoptera coffeella(Guérin-Méneville) and field reports on insecticide resistance led to an investigation of the possible occurrence of resistance of this species to some of the oldest insecticides used against it in Brazil: chlorpyrifos, disulfoton, ethion and methyl parathion. Insect populations were collected from ten sites in the state of Minas Gerais, Brazil and these populations were subjected to discriminating concentrations established from insecticide LC99s estimated for a susceptible standard population. Eight of the field-collected populations showed resistance to disulfoton, five showed resistance to ethion, four showed resistance to methyl parathion, and one showed resistance to chlorpyrifos. The frequency of resistant individuals in each population ranged from 10 to 93% for disulfoton, 53 to 75% for ethion, 23 to 76% for methyl parathion, and the frequency of resistant individuals in the chlorpyrifos resistant population was 35%. A higher frequency of individuals resistant to chlorpyrifos, disulfoton and ethion was associated with greater use of insecticides, especially other organophosphates. This finding suggests that cross-selection, mainly between organophosphates, played a major role in the evolution of insecticide resistance in Brazilian populations of L. coffeella. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide and triphenyl phosphate) suggested that cytochrome P450-dependent monooxygenases may play a major role in resistance with minor involvement of esterases and glutathione S-transferases.

Acute effects of acephate and methamidophos and interleukin-1 on corticotropin-releasing factor (CRF) synthesis in and release from the hypothalamus in vitro.

Acute effects of Ace, Meth and IL-1 on AChE activity, ACh and CRF mRNA levels in, and CRF-release from the hypothalamus were studied in vitro. The hypothalamus samples were dissected from the rat brain and were incubated in vitro with IL-1, Ace or Meth in the presence or absence of Dex, Atrop, PTL, PROP and GABA. Ace and Meth, but not IL-1, inhibited AChE activity, while all three compounds; (1) increased ACh and CRF mRNA levels in and CRF release from; (2) activated the CRE promoter region of CRF-gene in: and (3) increased cFos binding to the AP-1 region of the CRF-gene in the hypothalamus. Dex suppressed the effects of IL-1, possibly by inducing the nGRE regulatory sites of the CRF-gene. Dex, however, did not modulate the effects of Ace and Meth on the hypothalamus, which may be attributed to the failure of Dex to modulate the CRF-gene's nGRE regulatory sites. Atrop caused 80-90% inhibition of the effects of IL-1, but caused only 50-65% inhibition of the effects of Ace or Meth on CRF mRNA levels in and CRF release from the hypothalamus. PTL did not affect, while PROP slightly attenuated the effects of IL-1 and the insecticides on the hypothalamus. GABA attenuated the effects of the insecticides but not the effects of IL-1 on the hypothalamus. This suggests that the IL-1-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through a cholinergic pathway, while the insecticide-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through the cholinergic and GABAergic pathways. The insecticides, but not IL-1, disrupt feedback regulation of CRF synthesis in and release from the hypothalamus.