Synthesis, biological evaluation, and molecular docking studies of deoxygenated C-glycosides as LpxC inhibitors.

The bacterial deacetylase LpxC is a promising target for the development of novel antibiotics being selectively active against Gram-negative bacteria. In chiral pool syntheses starting from d- and l-ribose, a series regio- and stereoisomeric monohydroxytetrahydrofuran derivatives was synthesized and tested for LpxC inhibitory and antibacterial activities. Molecular docking studies were performed to rationalize the obtained structure-activity relationships. The (2S,3R,5R)-configured 3-hydroxytetrahydrofuran derivative ent-8 ((2S,3R,5R)-N,3-Dihydroxy-5-(4-{[4-(morpholinomethyl)phenyl]ethynyl}phenyl)tetrahydrofuran-2-carboxamide) was found to be the most potent LpxC inhibitor (Ki = 3.5 µM) of the synthesized series of monohydroxytetrahydrofuran derivatives and to exhibit the highest antibacterial activity against E. coli BL21(DE3) and the D22 strain.

Consumption of gossypol increases fatty acid-amino acid conjugates in the cotton pests Helicoverpa armigera and Heliothis virescens.

Gossypol is a toxic sesquiterpene dimer produced by cotton plants which deters herbivory by insects and vertebrates. Two highly reactive aldehyde groups contribute to gossypol toxicity by cross-linking herbivore proteins. We identified another consequence of consuming gossypol in two insect pests of cotton: increased amounts of fatty acid-amino acid conjugates (FACs). Eight different FACs in the feces of larval Helicoverpa armigera and Heliothis virescens increased when larvae consumed artificial diet containing gossypol, but not a gossypol derivative lacking free aldehyde groups (SB-gossypol). FACs are produced by joining plant-derived fatty acids with amino acids of insect origin in the larval midgut tissue by an unknown conjugase, and translocated into the gut lumen by an unknown transporter. FACs are hydrolyzed back into fatty acids and amino acids by an aminoacylase (L-ACY-1) in the gut lumen. The equilibrium level of FACs in the lumen is determined by a balance between conjugation and hydrolysis, which may differ among species. When heterologously expressed, L-ACY-1 of H. armigera but not H. virescens was inhibited by gossypol; consistent with the excretion of more FACs in the feces by H. armigera. FACs are known to benefit the plant host by inducing anti-herbivore defensive responses, and have been hypothesized to benefit the herbivore by acting as a surfactant and increasing nitrogen uptake efficiency. Thus in addition to its direct toxic effects, gossypol may negatively impact insect nitrogen uptake efficiency and amplify the signal used by the plant to elicit release of volatile compounds that attract parasitoids.

Beneficial Changes in Rat Vascular Endocannabinoid System in Primary Hypertension and under Treatment with Chronic Inhibition of Fatty Acid Amide Hydrolase by URB597.

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB1 receptor (CB1R) expression were elevated. The CB1R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB1R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Retain Activity against Multidrug-Resistant Pseudomonas aeruginosa In Vitro and In Vivo.

Most antimicrobials currently in the clinical pipeline are modifications of existing classes of antibiotics and are considered short-term solutions due to the emergence of resistance. Pseudomonas aeruginosa represents a major challenge for new antimicrobial drug discovery due to its versatile lifestyle, ability to develop resistance to most antibiotic classes, and capacity to form robust biofilms on surfaces and in certain hosts such as those living with cystic fibrosis (CF). A precision antibiotic approach to treating Pseudomonas could be achieved with an antisense method, specifically by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs). Here, we demonstrate that PPMOs targeting acpP (acyl carrier protein), lpxC (UDP-(3-O-acyl)-N-acetylglucosamine deacetylase), and rpsJ (30S ribosomal protein S10) inhibited the in vitro growth of several multidrug-resistant clinical P. aeruginosa isolates at levels equivalent to those that were effective against sensitive strains. Lead PPMOs reduced established pseudomonal biofilms alone or in combination with tobramycin or piperacillin-tazobactam. Lead PPMO dosing alone or combined with tobramycin in an acute pneumonia model reduced lung bacterial burden in treated mice at 24 h and reduced morbidity up to 5 days postinfection. PPMOs reduced bacterial burden of extensively drug-resistant P. aeruginosa in the same model and resulted in superior survival compared to conventional antibiotics. These data suggest that lead PPMOs alone or in combination with clinically relevant antibiotics represent a promising therapeutic approach for combating P. aeruginosa infections.IMPORTANCE Numerous Gram-negative bacteria are becoming increasingly resistant to multiple, if not all, classes of existing antibiotics. Multidrug-resistant Pseudomonas aeruginosa bacteria are a major cause of health care-associated infections in a variety of clinical settings, endangering patients who are immunocompromised or those who suffer from chronic infections, such as people with cystic fibrosis (CF). Herein, we utilize antisense molecules that target mRNA of genes essential to bacterial growth, preventing the formation of the target proteins, including acpP, rpsJ, and lpxC We demonstrate here that antisense molecules targeted to essential genes, alone or in combination with clinically relevant antibiotics, were effective in reducing biofilms and protected mice in a lethal model of acute pneumonia.

Eradication of Helicobacter pylori through the inhibition of urease and peptide deformylase: Computational and biological studies.

This work tested anti- Helicobacter pylori, free radicals scavenging and toxicity property as well as chemical constituents in the extract of chloroform (CE) and ethyl acetate (EAE) from the pedicel of Diospyros kaki L. (PDK-CE and PDK-EAE). There were 33 and 36 chemical constituents respectively in the extracts of PDK-CE and PDK-EAE, belonging to the fatty acids methyl ester, fatty acids, and stearic acids, as revealed by Gas Chromatography-Mass Spectrometry (GC-MS). The extracts did not exhibit any toxicity on NIH3T3 cells, but they significantly showed scavenging of NO, DPPH, and H2O2 free radicals. The extracts displayed in vitro anti-H. pylori activity. PDK-CE had the maximum inhibitory zone at a minimal inhibitory concentration (MIC) of 10 µg. ml-1 and the extract also triggered the cellular damage in the bacteria. PDK-CE extract had a high urease inhibitory activity (IC50 value of 8.5 µg). Further, in silico studies was performed by using 41 compounds against H. pylori urease (HPU) and H. pylori peptide deformylase (HPPD). The score value was the maximum (-19.58 kcal/mol) against HPU with 17-(5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol, while the score value was the maximum (-14.51 kcal/mol) against HPPD with hexadecanoic acid. The results demonstrated the importance of the pedicel extracts in future pharmaceutical drug development against H. pylori infections.

The Pharmacological Inhibition of Fatty Acid Amide Hydrolase Prevents Excitotoxic Damage in the Rat Striatum: Possible Involvement of CB1 Receptors Regulation.

The endocannabinoid system (ECS) actively participates in several physiological processes within the central nervous system. Among such, its involvement in the downregulation of the N-methyl-D-aspartate receptor (NMDAr) through a modulatory input at the cannabinoid receptors (CBr) has been established. After its production via the kynurenine pathway (KP), quinolinic acid (QUIN) can act as an excitotoxin through the selective overactivation of NMDAr, thus participating in the onset and development of neurological disorders. In this work, we evaluated whether the pharmacological inhibition of fatty acid amide hydrolase (FAAH) by URB597, and the consequent increase in the endogenous levels of anandamide, can prevent the excitotoxic damage induced by QUIN. URB597 (0.3 mg/kg/day × 7 days, administered before, during and after the striatal lesion) exerted protective effects on the QUIN-induced motor (asymmetric behavior) and biochemical (lipid peroxidation and protein carbonylation) alterations in rats. URB597 also preserved the structural integrity of the striatum and prevented the neuronal loss (assessed as microtubule-associated protein-2 and glutamate decarboxylase localization) induced by QUIN (1 µL intrastriatal, 240 nmol/µL), while modified the early localization patterns of CBr1 (CB1) and NMDAr subunit 1 (NR1). Altogether, these findings support the concept that the pharmacological manipulation of the endocannabinoid system plays a neuroprotective role against excitotoxic insults in the central nervous system.

Effects of the antipsychotic paliperidone on stress-induced changes in the endocannabinoid system in rat prefrontal cortex.

Objectives There is a need to explore novel mechanisms of action of existing/new antipsychotics. One potential candidate is the endocannabinoid system (ECS). The present study tried to elucidate the effects of the antipsychotic paliperidone on stress-induced ECS alterations. Methods Wister rats were submitted to acute/chronic restraint stress. Paliperidone (1 mg/kg) was given prior each stress session. Cannabinoid receptors and endocannabinoids (eCBs) synthesis and degradation enzymes were measured in prefrontal cortex (PFC) samples by RT-PCR and Western Blot. Results In the PFC of rats exposed to acute stress, paliperidone increased CB1 receptor (CB1R) expression. Furthermore, paliperidone increased the expression of the eCB synthesis enzymes N-acylphosphatidylethanolamine- hydrolysing phospholipase D and DAGLα, and blocked the stress-induced increased expression of the degrading enzyme fatty acid amide hydrolase. In chronic conditions, paliperidone prevented the chronic stress-induced down-regulation of CB1R, normalised DAGLα expression and reverted stress-induced down-regulation of the 2-AG degrading enzyme monoacylglycerol lipase. ECS was analysed also in periphery. Acute stress decreased DAGLα expression, an effect prevented by paliperidone. Contrarily, chronic stress increased DAGLα and this effect was potentiated by paliperidone. Conclusions The results obtained described a preventive effect of paliperidone on stress-induced alterations in ECS. Considering the diverse alterations on ECS described in psychotic disease, targeting ECS emerges as a new therapeutic possibility.

Epigallocatechin-3-gallate ameliorates erectile function in aged rats via regulation of PRMT1/DDAH/ADMA/NOS metabolism pathway.

Aging-related ED is predominantly attributed to neurovascular dysfunction mediated by NO suppression and increased oxidative stress in penis. The alterations of protein arginine methyltransferases 1 (PRMT1)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/NO synthase (NOS) pathway regulate NO production in the vascular endothelium. Epigallocatechin-3-gallate (EGCG) is one of the most abundant and antioxidative ingredients isolated from green tea. In the present study, 40 Sprague-Dawley rats were randomly distributed into four groups: one young rat group and three aged rat groups treated with daily gavage feedings of EGCG at doses of 0, 10 mg kg-1 and 100 mg kg-1 for 12 weeks, respectively. Erectile function was assessed by electrical stimulation of the cavernous nerves with intracavernous pressure (ICP) measurement. After euthanasia, penile tissue was investigated using Western blot and ELISA to assess the PRMT1/DDAH/ADMA/NOS metabolism pathway. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected by colorimetry. We also evaluated smooth muscle contents. The ratio of maximal ICP and mean systemic arterial pressure (MAP) was markedly higher in EGCG-treated aged rats than in untreated aged rats. We found that DDAH1 and DDAH2 were expressed in cavernosal tissue, and they were downregulated in corpora of aged rats. The administration of EGCG upregulated the expression and activity of DDAH. In contrast, EGCG treatment downregulated the expression of PRMT1 and ADMA content. Moreover, EGCG-treated rats showed an improvement in smooth muscle expression, the ratio of smooth muscle cell/collagen fibril, SOD activity, and MDA levels when compared with untreated aged rats.

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis.

Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance.

Differential effects of single versus repeated alcohol withdrawal on the expression of endocannabinoid system-related genes in the rat amygdala.

BACKGROUND: Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control. METHODS: The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)-exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH-free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal. RESULTS: Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB(1) , CB(2) , and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g., CB(1) , CB(2) , GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g., diacylglicerol lipase-α/ß) were found in any condition. CONCLUSIONS: These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission.

Photosynthetic Bradyrhizobium Sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene-dependent interaction with Aeschynomene plants.

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.

The therapeutic potential of targeting endogenous inhibitors of nitric oxide synthesis.

Asymmetric dimethylarginine (ADMA)--a naturally occurring amino acid that is a product of protein breakdown--is released into the cytoplasm following the post-translational methylation of arginine residues within proteins and the subsequent proteolysis of these arginine-methylated proteins. ADMA inhibits all three isoforms of nitric oxide synthase and therefore has the potential to produce diverse biological effects, particularly in the cardiovascular system. In addition to its renal clearance, endogenously produced ADMA is metabolized to L-citrulline and dimethylamine by the dimethylarginine dimethylaminohydrolase (DDAH) enzymes. Pharmacological modification of DDAH has therefore been proposed as a mechanism for manipulating endogenous ADMA concentrations and regulating the production of nitric oxide in situations where alterations in nitric oxide signalling have been shown to contribute to pathophysiology. This review describes the biology of ADMA and the potential therapeutic utility of manipulating DDAH activity.

Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing.

OBJECTIVES: To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates. METHODS: 33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present. RESULTS: Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughout the pncA gene (one isolate did not show any mutation). Of the 30 phenotypically susceptible isolates, 21 were wild types whilst nine isolates showed the presence of a silent mutation C-T at codon 195. Fifteen mutations found in this study has not been described earlier. Not a single isolate of M. bovis was detected among PZA resistant M. tuberculosis complex isolates. CONCLUSION: MGIT 960 showed better concordance with sequencing results in comparison with Wayne assay. In present study, a high proportion (85%) of MDR-TB isolates from patients receiving anti-TB treatment were found to be resistant to PZA.

Endocannabinoid modulation of amphetamine sensitization is disrupted in a rodent model of lesion-induced dopamine dysregulation.

We tested the hypothesis that increased dopaminergic sensitivity induced by olfactory bulbectomy is mediated by dysregulation of endocannabinoid signaling. Bilateral olfactory bulbectomy induces behavioral and neurobiological symptomatology related to increased dopaminergic sensitivity. Rats underwent olfactory bulbectomy or sham operations and were assessed 2 weeks later in two tests of hyperdopaminergic responsivity: locomotor response to novelty and locomotor sensitization to amphetamine. Amphetamine (1 mg/kg i.p.) was administered to rats once daily for 8 consecutive days to induce locomotor sensitization. URB597, an inhibitor of the anandamide hydrolyzing enzyme fatty-acid amide hydrolase (FAAH), was administered daily (0.3 mg/kg i.p.) to sham and olfactory bulbectomized (OBX) rats to investigate the impact of FAAH inhibition on locomotor sensitization to amphetamine. Pharmacological specificity was evaluated with the CB(1) antagonist/inverse agonist rimonabant (1 mg/kg i.p). OBX rats exhibited heightened locomotor activity in response to exposure either to a novel open field or to amphetamine administration relative to sham-operated rats. URB597 produced a CB(1)-mediated attenuation of amphetamine-induced locomotor sensitization in sham-operated rats. By contrast, URB597 failed to inhibit amphetamine sensitization in OBX rats. The present results demonstrate that enhanced endocannabinoid transmission attenuates development of amphetamine sensitization in intact animals but not in animals with OBX-induced dopaminergic dysfunction. Our data collectively suggest that the endocannabinoid system is compromised in olfactory bulbectomized rats.

Dimethylarginine dimethylaminohydrolase regulation: a novel therapeutic target in cardiovascular disease.

Asymmetric dimethylarginine (ADMA), an endogenous methylated form of the amino acid L-arginine, inhibits the activity of the enzyme endothelial nitric oxide synthase, with consequent reduced synthesis of nitric oxide. ADMA is metabolised to L-citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The modulation of DDAH activity and expression plays a pivotal role in regulating intracellular ADMA concentrations, with important effects on vascular homeostasis. For example, impairment in DDAH activity, resulting in elevated ADMA concentrations and reduced nitric oxide synthesis, can promote the onset and progression of atherosclerosis in experimental models. This review discusses the current role of ADMA and DDAH in vascular health and disease, the techniques used to assess DDAH activity and expression, and the results of recent studies on pharmacological and biological agents modulating DDAH activity and expression. Suggestions for future basic and clinical research directions are also discussed.

Expression and function of fatty acid amide hydrolase in prostate cancer.

The hydrolysis of endocannabinoids has profound effects on the function of the endocannabinoid signaling system in the regulation of prostate carcinoma cells. Prostate carcinoma cells exhibit a wide range of hydrolysis activity for 2-arachidonoylglycerol (2-AG), the major endocannabinoid. However, enzyme(s) responsible for 2-AG hydrolysis and their functions in prostate cancer have not been characterized. In this study, we demonstrated that fatty acid amide hydrolase (FAAH) was differentially expressed in normal and prostate carcinoma cells. In PC-3 cells, overexpression of FAAH resulted in increased FAAH protein, 2-AG hydrolysis, cell invasion and cell migration. Conversely, small-interfering RNA (siRNA) knockdown of FAAH in LNCaP cells decreased FAAH protein, 2-AG hydrolysis and cell invasion. Furthermore, CAY10401, a FAAH inhibitor, decreased cell invasion and it enhanced the reduction of invasion in FAAH siRNA-transfected LNCaP cells. Immunohistochemistry staining of commercial tissue microarrays (TMAs) demonstrated FAAH staining in 109 of 157 cores of prostate adenocarcinomas but weak staining in 1 of 8 cores of normal prostate tissues. These results suggest that FAAH regulates 2-AG hydrolysis and invasion of prostate carcinoma cells and is potentially involved in prostate tumorigenesis.

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase.

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.

Interaction of ligands for the peroxisome proliferator-activated receptor gamma with the endocannabinoid system.

BACKGROUND AND PURPOSE: There is good evidence that agents interacting with the endocannabinoid system in the body can also interact with the peroxisome proliferator-activated receptor gamma. The present study was designed to test whether the reverse is true, namely whether peroxisome proliferator-activated receptor gamma ligands have direct effects upon the activity of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase. EXPERIMENTAL APPROACH: Fatty acid amide hydrolase activity was measured in rat brain homogenates, C6 glioma and RBL2H3 basophilic leukaemia cells. Cellular uptake of anandamide was also assessed in these cells. KEY RESULTS: Peroxisome proliferator-activated receptor gamma activators inhibited the metabolism of the endocannabinoid anandamide in rat brain homogenates with an order of potency MCC-555 > indomethacin approximately ciglitazone approximately 15-deoxy-Delta(12,14)-prostaglandin J(2) approximately pioglitazone > rosiglitazone > troglitazone. The antagonists BADGE, GW9662 and T0070907 were poor inhibitors of anandamide hydrolysis. The inhibition by ciglitazone was competitive and increased as the pH of the assay buffer was decreased; the K(i) value at pH 6.0 was 17 microM. In intact C6 glioma cells assayed at pH 6.2, significant inhibition of anandamide hydrolysis was seen at 3 microM ciglitazone, whereas 100 microM was required to produce significant inhibition at pH 7.4. Ciglitazone also interacted with monoacylglycerol lipase as well as with cannabinoid CB(1) and CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: Ciglitazone may be useful as a template for the design of novel dual action anti-inflammatory agents which are both inhibitors of fatty acid amide hydrolase and agonists at the peroxisome proliferator-activated receptor gamma.

Effects of hydroxypropyl cyclodextrins on the reactivation of SDS-denatured aminoacylase.

Cyclodextrins are natural-occurring circular oligosaccharides with an internal hydrophobic cavity and external hydrophilic edges. Because cyclodextrins bind with protein aromatic residues, they can prevent protein aggregation, and their ability to bind with detergents enables them to act as stripping reagents to release proteins from protein-detergent complexes. In this research, we investigated the effects of three hydroxypropyl cyclodextrins (HPCDs) on the refolding of aminoacylase from SDS-denatured states. It was found that the three HPCDs could effectively assist aminoacylase reactivation though they have different abilities. HP-gamma-CD, which has the largest cavity among the three HPCDs, was the most efficient one. Spectroscopic results further indicated that the secondary structure recovery of aminoacylase could be completed with the help of low concentrations of HPCDs. However, the activity of the released protein could not fully recover even though high concentrations of HPCDs were used. The concentration-dependent effects of HPCDs also indicated that cyclodextrins could also act as folding assistants in addition to acting as stripping reagents during the refolding of detergent-denatured proteins.