Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.     

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75–1400 ng/ml for PMZ and 2.5–1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within ±11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett–Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.     

Peritoneal vascular injuries induced by gaseous oxygen]

When introduced as a gas into the abdominal cavity of the rat, oxygen above the initial concentration of 10 % provokes vasodilatation, perivascular oedematous infiltrations and fluid formation. These lesions are entirely reversible. Antihistaminics (promethazine and mepyramine 2 mg/100 g completely inhibit vascular lesions.     

Selective inhibition of brain Na,K-ATPase by drugs

The effect of drugs from the class of cardiac (methyldigoxin, verapamil, propranolol), antiepileptic (carbamazepine), sedative (diazepam) and antihistaminic (promethazine) drugs on Na,K-ATPase activity of plasma membranes was studied in rat brain synaptosomes. Methyldigoxin in a concentration of 0.1 mmol/l inhibits enzyme activity by 80 %. Verapamil, propranolol and promethazine in concentrations of 20, 20 and 2 mmol/l respectively, entirely inhibit the ATPase activity. Carbamazepine and diazepam in concentrations of 0.02-60 mmol/l have no effect on the activity of this enzyme. According to the drug concentrations that inhibit 50 % of enzyme activity (IC(50)), the potency can be listed in the following order: methyldigoxin promethazine verapamil ? propranolol. From the inhibition of commercially available purified Na,K-ATPase isolated from porcine cerebral cortex in the presence of chosen drugs, as well as from kinetic studies on synaptosomal plasma membranes, it may be concluded that the drugs inhibit enzyme activity, partly by acting directly on the enzyme proteins. Propranolol, verapamil and promethazine inhibitions acted in an uncompetitive manner. The results suggest that these three drugs may contribute to neurological dysfunctions and indicate the necessity to take into consideration the side effects of the investigated drugs during the treatment of various pathological conditions.     

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.     

The inhibitory effects in vitro of phenothiazines and other drugs on lipid-peroxidation systems in rat liver microsomes, and their relationship to the liver necrosis produced by carbon tetrachloride

1. The effects of several phenothiazine derivatives on lipid-peroxidation systems in rat liver microsomes were studied and the results are considered in relation to the hepatotoxic action of carbon tetrachloride. 2. The lipid-peroxidation system coupled to NADPH(2) oxidation and stimulated by an ADP-Fe(2+) mixture is strongly inhibited in vitro by promethazine (50% inhibition at 29mum). Chlorpromazine and Stelazine also inhibit the peroxidation system but are less effective than promethazine. 3. The effects of promethazine on three other systems involving oxygen uptake (sulphite oxidation, orcinol oxidation and mitochondrial succinate oxidation) were also studied. Promethazine does not inhibit these systems to the same extent as it does the NADPH(2)-ADP-Fe(2+) lipid-peroxidation system. 4. Promethazine also produces an inhibition of the NADPH(2)-ADP-Fe(2+) system in liver microsomes after administration in vivo. It is concluded that the inhibition involves the interaction of the drug (or a metabolite of it) with the microsomal electron-transport chain. 5. Several other compounds known to protect the rat against liver necrosis after the administration of carbon tetrachloride were tested for inhibitory action on the NADPH(2)-ADP-Fe(2+) system. No clear correlation was observed between effectiveness in vivo as a protective agent and inhibitory effects on the NADPH(2)-ADP-Fe(2+) system in vitro. 6. Promethazine was found to inhibit the stimulation of lipid peroxidation produced in rat liver microsomes by low concentrations of carbon tetrachloride. This effect occurs at a concentration similar to that observed in vivo after administration of a normal clinical dose.     

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.     

Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells.

The factors that control oncornavirus formation were analyzed in Friend leukemia cells that undergo hematopoiesis when treated with dimethyl sulfoxide. Suspension cultures of Ostertag FSD-1 cell line were found to enter a G or resting state at the end of their proliferative phase and to simultaneously cease producing helper and dependent components of Friend virus. Whereas the decline in virus production is at least 100-fold, rates of cellular RNA and protein synthesis are only slightly lower in resting than in growing cells. Both resting and growing cells contain similarly large concentrations of the viral proteins P(30) and P(12). Dimethyl sulfoxide induces hemoglobin synthesis in growing cells, but its effects on virus production appear to be indirect results of its action to inhibit cell growth and thus to delay entry of cells into the G resting state. Furthermore, variant cell lines were obtained with differing abilities to synthesize virus or hemoglobin. Some lines no longer produce infectious virus, although they all harbor murine leukemia virus genes which are expressed to varying extents. The major internal protein of these oncornaviruses, P(30), is synthesized in large amounts by all of the cell lines. These results suggest that Friend virus production is not coinduced with erythroid differentiation, as had been proposed, but rather is controlled by a cellular growth cycle.     

A rat brain fraction and different purified peroxidases catalyzing the formation of dopaminochrome from dopamine.

Dopaminochrome formation is catalyzed by commercially available purified peroxidases (EC 1.11.1.7) such as horseradish, lacto- and myelo-peroxidase using dopamine, hydrogen peroxide or promethazine sulfoxide as substrates. A rat brain fraction (RBF) catalyzes a similar reaction and its catalytic power increases after preincubation with hydrogen peroxide/ascorbic acid. The activity of both the purified enzymes and the RBF preparation is inhibited by carnosine and characterized by excess substrate inhibition. The enzymes recognize different substrates but show the highest affinity for dopamine. The RBF fraction is strongly buffered against oxidation by compounds such as glutathione and by bioreductive enzymes such as DT-diaphorase (EC 1.6.99.2) which can use as a substrate menadione or dopaminochrome. The rat brain dopamine peroxidizing activity appeared to be mostly bound to the synaptosomal fraction. The reaction catalyzed by the purified peroxidases was followed by electron spin resonance spectroscopy and, unlike that catalyzed by RBF, was shown to produce the signal of a transient dopamine-o-semiquinone radical.     

Regulator gene of phenyl oxidase activity in Drosophila melanogaster

A recessive lethal mutation l(2)hemo causing the occurrence of melanotic tumors in homozygous Drosophila larvae was found. The study of phenoloxidase (PO) activity revealed that the number of hemocytes with PO activity in homozygous larvae was significantly reduced (0.4 +/- 0.24%), compared to wild-type larvae (6.3 +/- 0.5%). On injury followed by injection with bacterial cells, the formation of melanotic thrombus did not occurred and hemocytes with PO activity were not recorded in homozygotes of line P103. Suppression of the activity of PO isozymes A1 and A3 was detected by means of electrophoretic analysis of homozygotes. According to gene mapping data, the localization of this mutation did not match any structural gene for known PO forms and is therefore related to a regulatory gene controlling the activity of the immune system of Drosophila.     

2-羟基-4-甲氧基苯甲醛缩苯胺对菜青虫酚氧化酶的抑制作用

为筛选对十字花科蔬菜害虫菜青虫Pieris rapae（L.）酚氧化酶具有高抑制活性的化合物,为寻找新型害虫控制剂提供线索,采用酶标仪微量法以室内合成、筛选的高活性化合物2-羟基-4-甲氧基苯甲醛缩苯胺为抑制剂,研究了其对菜青虫酚氧化酶的抑制活性及抑制类型。结果表明,供试化合物对菜青虫酚氧化酶的抑制中浓度（IC₅₀）为0.116 mmol/L;该化合物为典型的可逆非竞争型抑制剂,抑制常数（K_i）为1.96 mmol/L。该化合物直接对靶标酚氧化酶产生作用,而不是通过影响酶结构内的铜离子来产生作用的。     

Effect of antihistamines on anaphylactoid oedema in rats using a modified plethysmometer for measuring foot volume.

This study reports the quantitative effect of the H1--and H2--receptor antagonists on dextran-induced anaphylactoid oedema in rats. The findings indicate that mepyramine, promethazine and chlorpromazine which are H1--receptor antagonists significantly inhibited this anaphylactoid oedema. While on the other hand burimamide an H2--receptor antagonist at doses below 500 micrograms/kg inhibit dextran-induced oedema but at higher doses enhances oedema formation in the test rats. E.D50 values obtained for mepyramine, chlorpromazine and promethazine are 5.01 mg/kg, 0.36 mg/kg, 1.78 mg/kg respectively. The dual effects of burimamide on dextran-induced oedema merits further investigation and confirmation with the aid of other H1--and/or H2--receptor systems. A modification of the plethysmometric method of Buttle et. al. (1957) is also described.     

Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant

Because of increased concern about surface water eutrophication from nutrient-enriched agricultural runoff, many swine producers are encouraged to decrease application rates of waste-based P. Precipitation and subsequent removal of magnesium ammonium phosphate (MgNH(4)PO(4) x 6H(2)O), commonly known as struvite, is a promising mechanism for N and P removal from anaerobic swine lagoon effluent. The objectives of this research were to (i) quantify the effects of adjusting pH and Mg:P ratio on struvite precipitation and (ii) determine the rate constant pH effect for struvite precipitation in anaerobic swine lagoon liquid. Concentrations of PO(4)-P in liquid from two anaerobic swine lagoons were determined after 24 h of equilibration for a pH range of 7.5-9.5 and Mg:P ratios between 1:1 and 1.6:1. Struvite formation reduced the PO(4)-P concentration in the effluents to as low as 2 mgl(-1). Minimum concentrations of PO(4)-P occurred between pH 8.9 and 9.25 at all Mg:P ratios. Struvite precipitation decreased PO(4)-P concentrations by 85% within 20 min at pH 9.0 for an initial Mg:P ratio of 1.2:1. The rate of PO(4)-P decrease was described by a first-order kinetic model, with rate constants of 3.7, 7.9, and 12.3 h(-1) at pH 8.4, 8.7 and 9.0 respectively. Our results indicate that induced struvite formation is a technically feasible method to remove N and P from swine lagoon liquid and it may allow swine producers to recover nutrients for off-farm sale.     

Histamine-induced hyperphosphatemia and hypocalcemia in the laying hen

The effects of histamine on plasma inorganic phosphorus (Pi) and total calcium values were studied in laying hens during egg formation and in non-laying hens. Histamine induced hyperphosphatemia and slight hypocalcemia during eggshell calcification, whereas the most pronounced hypocalcemia was observed during the early stages of egg formation. The histamine-induced hyperphosphatemia was completely inhibited by cimetidine but only partly by promethazine. However, the histamine-induced hypocalcemia was totally inhibited by cimetidine and promethazine.     

A Regulatory Gene for Phenoloxidase Activity in Drosophila melanogaster

A recessive lethal mutationl(2)hemocausing the occurrence of melanotic tumors in homozygous Drosophilalarvae was found. The study of phenoloxidase (PO) activity revealed that the number of hemocytes with PO activity in homozygous larvae was significantly reduced (0.4 ± 0.24%), compared to wild-type larvae (6.3 ± 0.5%). On injury followed by injection with bacterial cells, the formation of melanotic thrombus did not occurred and hemocytes with PO activity were not recorded in homozygotes of line P103. Suppression of the activity of PO isozymes A₁and A₃was detected by means of electrophoretic analysis of homozygotes. According to gene mapping data, the localization of this mutation did not match any structural gene for known PO forms and is therefore related to a regulatory gene controlling the activity of the immune system ofDrosophila.     

Inhibition of chemically induced erythrodifferentiation in friends erythroleukemia cells by d,1-Propranolol

Dimethyl sulfoxide (2%), hexamethylene bisacetamide (4mM) and butyric acid (2mM) were potent inducers of erythrodifferentiation in Friend erythroleukemia cell lines, 5–18 and C19TK. Hydrocortisone (1μM) markedly inhibited dimethyl sulfoxide induced hemoglobin production in both 5–18 and C19TK cells. d,1-Propranolol (25–50μM) markedly inhibited both dimethyl sulfoxide and hexamethylene bisacetamide induced erythrodifferentiation in 5–18 cells but not in C19TK cells. Addition of either hydrocortisone or propranolol as late as 48 hrs after dimethyl sulfoxide addition still resulted in significant inhibition of hemoglobin synthesis in 5–18 cells. Although the mechanism of action of propranolol is not known, modulation of the β adrenergic receptor is apparently not involved since practolol failed to inhibit either dimethyl sulfoxide or hexamethylene bisacetamide induced erythrodifferentiation in 5–18 cells nor did isoproternol induce hemoglobin synthesis.     

Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides, which elicit toxicity following biotransformation to the potent acetylcholinesterase inhibitors, paraoxon (PO) and chlorpyrifos oxon (CPO). Both oxons have also been shown to interact directly with muscarinic receptors coupled to inhibition of adenylyl cyclase. Immature animals are more sensitive than adults to the acute toxicity of PS and CPF but little is known regarding possible age-related differences in interactions between these toxicants and muscarinic receptors. We compared the inhibition of forskolin-stimulated cAMP formation by PO and CPO (1 nM-1 mM) in vitro in brain slices from 7-, 21-, and 90-day-old rats to the effects of well-known muscarinic agonists, carbachol and oxotremorine (100 microM). Both agonists inhibited cAMP formation in tissues from all age groups and both were more effective in adult and juvenile (20-26% inhibition) than in neonatal (12-13% inhibition) tissues. Atropine (10 microM) completely blocked agonist-induced inhibition in all cases. PO maximally inhibited (37-46%) cAMP formation similarly in tissues from all age groups, but atropine blocked those effects only partially and only in tissues from 7-day-old rats. CPO similarly inhibited cAMP formation across age groups (27-38%), but ATR was partially effective in tissues from all three age groups. Both oxons were markedly more potent in tissues from younger animals. We conclude that PO and CPO can directly inhibit cAMP formation through muscarinic receptor-dependent and independent mechanisms and that the developing nervous system may be more sensitive to these noncholinesterase actions.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

注射亲水性硅珠对棉铃虫血浆酚氧化酶活性的影响

昆虫通过细胞免疫和体液免疫的协同作用对入侵的异物做出防御反应。在不同时间向棉铃虫体内注射亲水性硅珠后,测定了血浆中酚氧化酶（PO）的活性,同时研究了不同抑制剂和激活剂对注射硅珠后的酚氧化酶活性的影响。结果表明,注射亲水性硅珠后,棉铃虫血浆中酚氧化酶的活性明显升高。分别以牛胰蛋白酶和昆布多糖作为酚氧化酶原（proPO）的激活剂,发现两者都可激活注射硅珠后血浆中的proPO。以牛胰蛋白酶激活时,随着注射硅珠后时间的延长,PO活性逐渐增高;而用昆布多糖激活后PO活性也明显升高,但注射硅珠后不同时间proPO被昆布多糖激活的情况基本相似。这些结果表明,在异物入侵后酚氧化酶原有很大程度的积累,并能被激活,协同细胞免疫抵御异物入侵。P-NPGB和PTU几乎能完全抑制酚氧化酶的活性。     

Distinct effects of Na+ and Li+ on the induction of morphological changes in A431 cells mediated by epidermal growth factor

Epidermal growth factor (EGF) induced the formation of thin sheetlike extensions (lamellipodia) and filamentous extensions at the edges of colonies of A431 cells. To determine the necessary processes for the induction of the morphological changes mediated by EGF, the effects of a variety of ions on these changes were examined. In a NaCl solution supplemented with CaCl2, MgCl2 and glucose, no EGF-induced morphological changes were observed. However, when the NaCl was replaced with LiCl, fingerlike extensions were formed, but sheetlike extensions were not. Addition of vanadate to the NaCl solution also induced fingerlike extensions in cells treated with EGF. In contrast, sheetlike lamellipodia were formed in EGF-treated cells by the addition of K+ or PO4(3-) to the NaCl solution or by the addition of PO4(3-) to the LiCl solution. These findings indicate that Li+, K+, PO4(3-) and vanadate are involved in the processes of EGF-induced morphological changes. Since vanadate and Li+ have been shown to inhibit phosphatases, an EGF-dependent phosphorylation step may play an important role in the induction of the morphological changes observed.