Members of the endocannabinoid system are distinctly regulated in inflammatory bowel disease and colorectal cancer.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

A rare eicosanoid precursor analogue, sciadonic acid (5Z,11Z,14Z-20:3), detected in vivo in hormone positive breast cancer tissue.

Numerous genetic alterations of HSA 11q13 are found frequently in several cancer types, including breast cancer (BC). The 11q13 locus harbors FADS2 encoding Δ6 desaturation which is not functional in several cancer cell lines, including hormone positive MCF7 BC cells. In vitro, the non-functional FADS2 activity unmasks 18:2n-6 elongation to 20:2n-6 and Δ5 desaturation by FADS1 to yield 5Z,11Z,14Z-20:3 (sciadonic acid) rather than 5Z,8Z,11Z,14Z-20:4 (arachidonic acid). In this pilot study we aimed to determine whether 5,11,14-20:3 appears in vivo in hormone positive human BC tissue. Fatty acids were profiled in surgically removed human breast tumor and adjacent normal tissue (n = 9). Sciadonic acid was detected in three of nine breast tumor samples and was below detect limits in normal breast tissue. The internal Δ8 double bond of arachidonic acid is required for normal eicosanoid synthesis but is missing in sciadonic acid. This pilot study demonstrates for the first time in vivo sciadonic acid in hormone positive BC tissue, warranting a larger survey study to further evaluate its appearance and the functional implications.

Human bone marrow mesenchymal stem cells secrete endocannabinoids that stimulate in vitro hematopoietic stem cell migration effectively comparable to beta-adrenergic stimulation.

Granulocyte colony-stimulating factor (G-CSF) is a well-known hematopoietic stem cell (HSC)-mobilizing agent used in both allogeneic and autologous transplantation. However, a proportion of patients or healthy donors fail to mobilize a sufficient number of cells. New mobilization agents are therefore needed. Endocannabinoids (eCBs) are endogenous lipid mediators generated in the brain and peripheral tissues and activate the cannabinoid receptors CB1 and CB2. We suggest that eCBs may act as mobilizers of HSCs from the bone marrow (BM) under stress conditions as beta-adrenergic receptors (Adrß). This study demonstrates that BM mesenchymal stem cells (MSCs) secrete anandamide (AEA) and 2-arachidonylglycerol (2-AG) and the peripheral blood (PB) and BM microenvironment contain AEA and 2-AG. 2-AG levels are significantly higher in PB of the G-CSF-treated group compared with BM plasma. BM mononuclear cells (MNCs) and CD34+ HSCs express CB1, CB2, and Adrß subtypes. CD34+ HSCs had higher CB1 and CB2 receptor expression in G-CSF-untreated and G-CSF-treated groups compared with MSCs. MNCs but not MSCs expressed CB1 and CB2 receptors based on qRT-PCR and flow cytometry. AEA- and 2-AG-stimulated HSC migration was blocked by eCB receptor antagonists in an in vitro migration assay. In conclusion, components of the eCB system and their interaction with Adrß subtypes were demonstrated on HSCs and MSCs of G-CSF-treated and G-CSF-untreated healthy donors in vitro, revealing that eCBs might be potential candidates to enhance or facilitate G-CSF-mediated HSC migration under stress conditions in a clinical setting.

Effects of tumour necrosis factor α upon the metabolism of the endocannabinoid anandamide in prostate cancer cells.

Tumour necrosis factor α (TNFα) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase-2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNFα treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acyl-phosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon γ treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE2-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFα treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.

High Ratios of C20:4n-6/C20:5n-3 and Thromboxane B2 /6-Keto-Prostaglandin F1α in Placenta Are Potential Risk Contributors for Neural Tube Defects: A Case-Control Study in Shanxi Province, China.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations. Folate supplementation can reduce the risk, but cannot prevent all NTDs, suggesting other reasons for folate-resistant NTDs. The present study assesses placental fatty acid composition, eicosanoids, and cytokines as risk factors for NTDs in a Chinese population with highly incident NTDs. METHODS: Seventy-seven aborted fetuses with NTDs during the third trimester were cases and 142 healthy newborns were controls. Placental fatty acid composition, eicosanoids, and cytokines were determined by standard methods. RESULTS: The placental C20:4n-6/C20:5n-3 and thromboxane B2 (TXB2 )/6-keto-prostaglandin F1α (6-keto-PGF1α ) ratios were significantly higher for cases than controls (p < 0.001 and 0.05, respectively). For the top versus the lowest tertiles of placental C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α , odds ratios for NTD occurrence were 3.79 (95% confidence interval, 1.60-8.96) (p for trend < 0.01) and 5.52 (95% confidence interval, 2.07-14.74) (p for trend < 0.001), respectively, adjusted for fetal sex as well as maternal age, occupation, parity, smoking, passive smoking, periconceptional folate supplementation, conception season, and tea drinking. The C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α ratios were positively correlated (r = 0.14; p < 0.05). The proportions of C18:2n-6, C18:3n-6, C20:3n-6, C18:3n-3, C20:3n-3, C20:5n-3, and C22:5n-3 were significantly lower in cases than controls, and all negatively associated with NTD occurrence (tertile-specific odds ratios); after adjustment for the potential confounders, these associations remained significant (p for trend < 0.05) except for C20:3n-3. CONCLUSION: High placental ratios of C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α are risk factors for neural tube defects.Birth Defects Research 109:550-563, 2017.© 2017 Wiley Periodicals, Inc.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

A simple method for simultaneous determination of N-arachidonoylethanolamine, N-oleoylethanolamine, N-palmitoylethanolamine and 2-arachidonoylglycerol in human cells.

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and 2-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for 2-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between 2 and 17 pmol mg(-1) protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.

A sensitive and accurate quantitative method to determine N-arachidonoyldopamine and N-oleoyldopamine in the mouse striatum using column-switching LC-MS-MS: use of a surrogate matrix to quantify endogenous compounds.

The transient receptor potential vanilloid 1 (TRPV1) channel, a nonselective Ca(2+) and Na(+) channel, is a molecular transducer of nociceptive stimuli. N-Arachidonoyl dopamine (NADA) and N-oleoyldopamine (OLDA), two unsaturated N-acyldopamines, are major activating endogenous TRPV1 ligands and their presence in mammalian brain tissue has been reported. However, the biological significance of NADA and OLDA remains unknown. To investigate their biological function in the nervous system, a sensitive and accurate quantitative method for determining endogenous NADA and OLDA in the brain is necessary. Thus, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to quantify NADA and OLDA in mouse striatum. Mouse cerebellum tissue in which neither NADA nor OLDA were detected was used as a surrogate matrix to prepare calibrators. NADA and OLDA were extracted from mouse brain tissue by solid-phase extraction and then filtered and analyzed by LC-MS-MS with electrospray ionization in the positive ion mode. The selectivity results and comparison of calibration curves prepared with mouse cerebellum and striatum established that the former was acceptable as the surrogate matrix of the latter for analyzing NADA and OLDA. The validation results of the matrix effect, linearity, precision, accuracy, and stability were satisfactory. The limits of detection and limits of quantification were 0.125 pg mg(-1) for both analytes. This method was sensitive and accurate enough to determine endogenous concentrations of these compounds in mouse striatum and will be very useful for further study of the biological functions of NADA and OLDA and other related factors in vivo.

A review of analytical methods for eicosanoids in brain tissue.

Eicosanoids are potent lipid mediators of inflammation and are known to play an important role in numerous pathophysiological processes. Furthermore, inflammation has been proven to be a mediator of diseases such as hypertension, atherosclerosis, Alzheimer's disease, cancer and rheumatoid arthritis. Hence, these lipid mediators have gained significant attention in recent years. This review focuses on chromatographic and mass spectrometric methods that have been used to analyze arachidonic acid and its metabolites in brain tissue. Recently published analytical methods such as LC-MS/MS and GC-MS/MS are discussed and compared in terms of limit of quantitation and sample preparation procedures, including solid phase extraction and derivatization. Analytical challenges are also highlighted.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1ß, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, ß-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Placental docosahexaenoic and arachidonic acids correlate weakly with placental polychlorinated dibenzofurans (PCDF) and are uncorrelated with polychlorinated dibenzo-p-dioxins (PCDD) or polychlorinated biphenyls (PCB) at delivery: a pilot study.

Long chain polyunsaturated fatty acids (LC-PUFA), ARA (arachidonic acid, 20:4n-6) and DHA (docosahexaenoic acid, 22:6n-3) have positive effects and environment pollutants, polychlorinated dibenzo-p-dioxins/dibenzofurans(PCDD/F) and polychlorinated biphenyls (PCB) have negative effects on neural development during early life. Placental dioxin/PCB serves as markers for cumulative exposure to fetus. Fatty acid composition of placenta depends on nutrient supply during pregnancy, serving as indicators for fetal ARA and DHA accretion. This study investigated correlation between placental PCDD/F and PCB toxic equivalent (TEQ) and LC-PUFA in 34 pregnant women from Taiwan. Placental PCDF TEQ were inversely correlated with placental ARA (p=0.020), C20:3n-6 (p=0.01), C22:4n-6 (p=0.04), C22:5n-6 (p<0.01) and with DHA (p=0.03), but ARA and DHA did not vary with PCDD, dioxin-like and indicator PCB. After adjustment for age and body mass index, a one-unit PCDF TEQ increase was associated with 1.021%w/w and 0.312%w/w decreases in ARA (ß=-1.021, p=0.03) and DHA (ß=-0.312, p=0.03). Since ARA and DHA were unrelated to three classes of toxins, and a weak negative association was found with PCDF, these data provide no basis for discouraging marine fish consumption during pregnancy for Taiwan women on the basis of these organics. Pregnant women should consume fish for its unique package of nutrients while avoiding few species with high organic pollutant or mercury contamination.

Reducing acyl migration during purification of 2-arachidonoylglycerol from biological samples before gas chromatography mass spectrometry analysis.

Endocannabinoid 2-arachidonoylglycerol (2-AG) regulates several important physiological processes in the brain. 2-AG is commonly quantified by gas chromatography mass spectrometry after an initial purification step. The most precise and rapid purification utilizes C(18) solid-phase extraction, but quantification problems can arise with acyl migration from 2-AG to 1-arachidonoylglycerol. We found that extraction with methanol promoted this migration, but acetone and diethyl ether (Et(2)O) did not. Acetone and Et(2)O were used to develop a purification method for the direct determination of 2-AG.

Localization of an endocannabinoid system in the hypophysial pars tuberalis and pars distalis of man.

The hypophysial pars tuberalis (PT) acts as an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis. Recently, we have identified an endocannabinoid system in the PT of hamsters and provided evidence that 2-arachidonoylglycerol is a messenger molecule that appears to play an essential role in seasonal reproduction and prolactin release by acting on the cannabinoid receptors in the PD. We now demonstrate the enzymes involved in endocannabinoid synthesis and degradation, namely sn-1-selective diacylglycerol lipase α, N-acylphosphatidylethanolamine-specific phospholipase D, and monoacylglycerol lipase, in the PT of man by means of immunohistochemistry. High-performance liquid chromatography coupled with tandem mass spectrometry revealed 2-arachidonoylglycerol and other endocannabinoids in the human PT. Furthermore, we detected the expression of the cannabinoid receptor 1 (CB1), a primary receptor for endocannabinoids, in the PD. Double-immunofluorescence staining for CB1 and various hypophysial hormones or S-100, a marker for folliculostellate (FS) cells, revealed that CB1 immunoreactivity was mainly localized to corticotrophs and FS-cells. A limited number of lactotrophs and somatotrophs also showed CB1 immunoreactivity, which was however absent from gonadotrophs and thyrotrophs. Our data thus indicate that the human PT comprises an endocannabinoid system, and that corticotrophs and FS-cells are the main target cells for endocannabinoids. The functional significance of this newly discovered pathway remains to be elucidated in man; it might be related to the control of stress responses and/or reflect a remnant seasonal control of hypophysial hormonal secretion.

Erythrocyte fatty acids and prostate cancer risk: a comparison of methods.

The role of fatty acids (FA) in prostate carcinogenesis is unclear. Interest in the inter-relationship among different types of FA has resulted in new analytic approaches to FA and their role in cancer development. We evaluated the association between erythrocyte FA and prostate cancer in 127 prostate cancer patients and 183 screen negative controls. We present three approaches to the analyses of the FA and prostate cancer association; (1) individual or common groups of FA, (2) biologically meaningful FA ratios and (3) principal components analysis. Monounsaturated FA and the alpha-linolenic:eicosapentaenoic ratio were associated with reduced risk of prostate cancer. However, Factor 1, which was strongly correlated with some long chain saturated FA, was associated with an increased risk of prostate cancer. We provide an example of modeling FA and their inter-relationships on the risk of prostate cancer. Comparing three approaches suggests the importance of considering the impact of the entire fatty acid profile in disease prevention.

Participation of the endocannabinoid system in lipopolysaccharide-induced inhibition of salivary secretion.

OBJECTIVE: The aim of the present paper was to assess whether lipopolysaccharide (LPS)-induced inhibition of salivary secretion involves the activation of the endocannabinoid system and the participation of tumor necrosis factor (TNF)alpha in the submandibular gland. DESIGN: Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid receptor antagonists, AM251 and AM630, respectively, injected into the submandibular gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on metacholine-induced salivary secretion. To assess the participation of TNFalpha on LPS inhibitory effects, salivary secretion was studied in LPS treated rats after the intraglandular injection of etanercept, a soluble form of TNF receptor which blocks TNFalpha action. Finally, to evaluate the possible interplay between endocannabinoids and TNFalpha on the submandibular gland function reduced during LPS challenge, the salivary secretion was studied after the intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630. RESULTS: AM251 and AM630, injected separately or concomitantly, partially prevented LPS-induced inhibition of salivation. Also, anandamide synthase activity was increased in submandibular glands extracted from rats 3h after LPS injection, suggesting that the endocannabinoid system was activated in response to this challenge. On the other hand, etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFalpha injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251 and AM630 injected concomitantly. CONCLUSION: The present results demonstrate the participation of the endocannabinoid system and TNFalpha on salivary responses during systemic inflammation induced by LPS.

The relationship between plasma levels of the endocannabinoid, anandamide, sex steroids, and gonadotrophins during the menstrual cycle.

OBJECTIVE: To further investigate the relationship between plasma anandamide (AEA), sex steroids, and gonadotrophins to improve our understanding of how AEA may be involved in human fertility. DESIGN: Cross-sectional and longitudinal study. SETTING: University Hospital of Leicester NHS Trust, Leicester Royal Infirmary. PATIENT(S): Healthy premenopausal and postmenopausal volunteers. INTERVENTION(S): UPLC-MS/MS-measured plasma AEA and ELISA-measured serum FSH, LH, estradiol, and progesterone levels at five different phases of the menstrual cycle and postmenopause. MAIN OUTCOME MEASURE(S): Plasma AEA, serum steroids and gonadotrophins. RESULT(S): Changes in AEA levels were similar in the two cohorts. The mean +/- SEM levels in the early follicular phase (0.89 +/- 0.06) for the cross-sectional cohort and the longitudinal cohort (0.73 +/- 0.03) were higher than those in the late follicular phase (0.77 +/- 0.09 cross-sectional; 0.63 +/- 0.08 longitudinal). The highest AEA levels were measured at ovulation (1.38 +/- 0.14 and 1.33 +/- 0.16) and the lowest level was measured in the late luteal phase (0.66 +/- 0.07 and 0.56 +/- 0.06). There was a statistically significant positive correlation between AEA, estradiol (P=0.0015), LH (P<0.0001) and FSH levels but not progesterone (P=0.022). CONCLUSION(S): Peak plasma AEA occurred at ovulation and positively correlated with estradiol and gonadotrophin levels suggesting that these may be involved in the regulation of AEA levels.

Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Lipid formation and gama-linolenic acid production by Mucor circinelloides and Rhizopus sp., grown on vegetable oil / Formação de lipídeos e produção de ácido gama-linolênico por Mucor circinelloides e Rhizopus sp., em óleos vegetais

The fungi strains were tested in Bioscreen automated system to select the best nutritional source. Following, shaking submserse cultures were studied in media containing sole carbon or nitrogen source. The growth of these strains improved in media containing vegetable oil, with high concentration of lipids. The high concentration of γ-linolenic acid was obtained with M. circinelloides in culture containing sesame oil.

Linhagens de fungos foram testadas em sistema automatizado Bioscreen para selecionar melhor fonte nutricional. Em seguida, foram estudadas culturas submersas em meios contendo uma única fonte de carbono e de nitrogênio. As linhagens contendo alta concentração de lipídeos tiveram melhor crescimento em meio contendo óleos de gergelim ou de dendê. Maior concentração de ácido γ-linolênico foi obtida com M. circinelloides nas culturas em óleo de gergelim.

A complex between 6-iodolactone and the peroxisome proliferator-activated receptor type gamma may mediate the antineoplastic effect of iodine in mammary cancer.

Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices.

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means+/-standard deviations: 0.68+/-0.29 and 0.64+/-0.28 nM, respectively), from pregnant women at term (1.37+/-0.42 nM), and from umbilical vein (1.26+/-0.33 nM) and umbilical artery (1.14+/-0.35nM), in milk (0.12+/-0.05 nM) and from amniotic (0.03+/-0.02 nM), peritoneal (0.93+/-0.27 nM), follicular (1.17+/-0.51 nM), and ovarian cyst (0.32+/-0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n=15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.