Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.     

Factors influencing the regional haemodynamic responses to methanandamide and anandamide in conscious rats

Background and purpose:

In vitro evidence suggests that metabolism of anandamide by cyclooxygenase-2 (COX-2) may be more important when the primary metabolic pathway [i.e. fatty acid amide hydrolase (FAAH)] is inhibited. Thus, the first aim of the present study was to assess the effects of COX-2 and/or FAAH inhibition, on the cardiovascular actions of anandamide. The second aim was to compare the effects of anandamide with those of the metabolically stable analogue (i.e. methanandamide) and investigate mechanisms involved in responses to the latter in conscious rats.

Experimental approach:

Rats were chronically instrumented for recording blood pressure, heart rate and renal, mesenteric and hindquarters vascular conductances in the freely moving state.

Key results:

Inhibition of FAAH with URB597 (cyclohexycarbamic acid 3′-carbamoyl-biphenyl-3-yl-ester) augmented the haemodynamic actions of anandamide, but there was no effect of COX-2 inhibition with parecoxib, either in the absence or the presence of URB597. Methanandamide caused CB₁ receptor-mediated renal and mesenteric vasoconstriction and evoked β₂-adrenoceptor-mediated hindquarters vasodilatation.

Conclusions and implications:

No evidence for an involvement of COX-2 in the systemic cardiovascular actions of anandamide could be demonstrated. Vasoconstrictor actions of methanandamide were shown to involve CB₁ receptors, whereas no involvement of CB₁ receptors in such actions of anandamide has been shown. However, β₂-adrenoceptor-mediated hindquarters vasodilatation, independent of CB₁ receptors, observed here with methanandamide, has previously been seen with anandamide and differs from previous results with other synthetic cannabinoids for which the response was CB₁ receptor-dependent. Thus, mechanisms underlying the cardiovascular actions of endocannabinoids and synthetic analogues appear to be agonist-specific.     

The ontogeny of drug metabolism enzymes and implications for adverse drug events

Profound changes in drug metabolizing enzyme (DME) expression occurs during development that impacts the risk of adverse drug events in the fetus and child. A review of our current knowledge suggests individual hepatic DME ontogeny can be categorized into one of three groups. Some enzymes, e.g., CYP3A7, are expressed at their highest level during the first trimester and either remain at high concentrations or decrease during gestation, but are silenced or expressed at low levels within one to two years after birth. SULT1A1 is an example of the second group of DME. These enzymes are expressed at relatively constant levels throughout gestation and minimal changes are observed postnatally. ADH1C is typical of the third DME group that are not expressed or are expressed at low levels in the fetus, usually during the second or third trimester. Substantial increases in enzyme levels are observed within the first one to two years after birth. Combined with our knowledge of other physiological factors during early life stages, knowledge regarding DME ontogeny has permitted the development of robust physiological based pharmacokinetic models and an improved capability to predict drug disposition in pediatric patients. This review will provide an overview of DME developmental expression patterns and discuss some implications of the data with regards to drug therapy. Common themes emerging from our current knowledge also will be discussed. Finally, the review will highlight gaps in knowledge that will be important to advance this field.     

Endocannabinoid and serotonergic systems are needed for acetaminophen-induced analgesia

Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhibition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical pain tests, AM-251, a specific CB(1) receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB(1) receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB(1) receptors demonstrating an indirect involvement of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhibition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity of ACEA, a CB(1) receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5-HT receptors. We therefore propose that acetaminophen-induced analgesia could involve the following sequence: (1) FAAH-dependent metabolism of acetaminophen into AM404; (2) indirect involvement of CB(1) receptors by this metabolite; (3) endocannabinoid-dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain-suppressing serotonergic receptors.     

Effects of cocaine in 5-lipoxygenase-deficient mice

Summary. 5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine’s actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine’s actions. M. Maccarrone, H. Manev: These authors contributed equally as the senior authors. Correspondence: Hari Manev, Department of Psychiatry, The Psychiatric Institute, University of Illinois, 1601 West Taylor Street, MC912 Chicago, IL 60612, USA     

Localization of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression in mouse brain: A new perspective on N-acylethanolamines as neural signaling molecules

N-acylethanolamines (NAEs) are membrane-derived lipids that are utilized as signaling molecules in the nervous system (e.g., the endocannabinoid anandamide). An N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) that catalyzes formation of NAEs was recently identified as a member of the zinc metallohydrolase family of enzymes. NAPE-PLD(-/-) mice have greatly reduced brain levels of long-chain saturated NAEs but wild-type levels of polyunsaturated NAEs (e.g., anandamide), suggesting an important role for NAPE-PLD in the biosynthesis of at least a subset of endogenous NAEs in the mammalian nervous system. To provide a neuroanatomical basis for investigation of NAPE-PLD function, here we have analyzed expression of NAPE-PLD in the mouse brain using mRNA in situ hybridization and immunocytochemistry. NAPE-PLD(-/-) mice were utilized to establish the specificity of probes/antibodies used. The most striking feature of NAPE-PLD expression in the brain was in the dentate gyrus, where a strong mRNA signal was detected in granule cells. Accordingly, immunocytochemical analysis revealed intense NAPE-PLD immunoreactivity in the axons of granule cells (mossy fibers). Intense NAPE-PLD immunoreactivity was also detected in axons of the vomeronasal nerve that project to the accessory olfactory bulb. NAPE-PLD expression was detected in other brain regions (e.g., hippocampus, cortex, thalamus, hypothalamus), but the intensity of immunostaining was weaker than in mossy fibers. Collectively, the data obtained indicate that NAPE-PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. We suggest that NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons.     

The effects of perinatal/juvenile heptachlor exposure on adult immune and reproductive system function in rats.

This study was performed to determine if developmental exposure of rats to heptachlor (H) during the last half of gestation through puberty adversely affects adult functioning of the immune and reproductive systems. Time-bred pregnant female Sprague-Dawley rats were dosed by gavage with H (0, 30, 300, or 3000 microg/kg/day) from gestation day (GD) 12 to postnatal day (PND) 7, followed by direct dosing of the pups with H through PND 42. Separate groups of rats were evaluated with a battery of immune function tests, while other groups of rats were evaluated for reproductive development and function. Additional groups of rats were euthanized at the end of the dosing period for histological analyses of major organ systems. Some dams and PND 7 pups were euthanized; milk, plasma, fat and/or tissues were assayed for H and heptachlor epoxide B (HEB), a major metabolite of H. The amount of H and HEB found in milk, blood, fat, and tissues was proportional to the dose of H administered. There were no effects on the number or survival of pups born to H-exposed dams nor to pups exposed postnatally. There were no effects on the number of treated dams delivering litters or on litter size, nor were there any effects on any of the reproductive end points examined in the F(0) or F(1) rats. There were no effects of H exposure on lymphoid organ weights, splenic natural killer (NK) cell activity, and splenic lymphoproliferative (LP) responses to mitogens and allogeneic cells in a mixed lymphocyte response (MLR) assay at 8 weeks of age. H exposure did not alter delayed or contact hypersensitivity at 10 or 17 weeks of age, respectively. However, the primary IgM antibody response to sheep red blood cells (SRBCs) was suppressed in a dose-dependent manner in males, but not females, at 8 weeks of age. The percentage of B lymphocytes (OX12(+)OX19(-)) in spleen was also reduced in the high-dose males. The anti-SRBC IgM response was reduced only in males exposed to 30 microg H/kg/day in a separate group of rats 21 weeks of age. In these same rats, at 26 weeks of age, the secondary IgG antibody response to SRBCs was suppressed in all of the H-exposed males, but not females. These data indicate that perinatal exposure of male rats to H results in suppression of the primary IgM and secondary IgG anti-SRBC responses. Suppression of these antibody responses persisted for up to 20 weeks after the last exposure to H, at a total exposure of approximately 1500 microg H/kg/rat.     

荧光PCR-熔解曲线法检测新疆回族人群CYP2C9和VKORC1基因多态性研究

目的应用荧光PCR-熔解曲线法检测新疆回族人群细胞色素P450酶2C9(CYP2C9)和维生素K环氧化物还原酶复合体亚单位1(VKORC1)基因多态性,研究新疆回族人群CYP2C9和VKORC1基因分布及基因突变频率情况,并评价荧光PCR-熔解曲线法临床使用的适用性。方法采用荧光PCR-熔解曲线法和测序法对比检测CYP2C9*2、CYP2C9*3和VKORC1(-1639G/A)的基因多态性。结果 228例新疆回族人群中,共检测出CYP2C9*1/*1标本199例,CYP2C9*1/*2标本2例,CYP2C9*1/*3标本26例,CYP2C9*3/*3标本1例,未检测出CYP2C9*2/*2和CYP2C9*2/*3标本。VKORC1(-1639G/A)检测出2种等位基因G和A,其中VKORC1-1639G/G型标本检出2例,VKORC1-1639G/A型标本检出39例,VKORC1-1639A/A型标本检出187例,与测序法对比检测比较,荧光PCR-熔解曲线法与测序法检测结果完全一致。结论新疆回族人群同样具有CYP2C9基因*2、*3位点和VKORC1基因-1639位点的多态性,其发生频率与新疆维吾尔族及其他地域人群的具有一定差异,所采用的荧光PCR-熔解曲线法用于基因多态性检测可满足临床检测要求。