Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation

Nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite have been receiving increasing attention as mediators of nitric oxide toxicity. Indeed, nitrated and nitrosated compounds have been detected in biological fluids and tissues of healthy subjects and in higher yields in patients under inflammatory or infectious conditions as a consequence of nitric oxide overproduction. Among them, nitrated lipids have been detected in vivo. Here, we confirmed and extended previous studies by demonstrating that nitrolinoleate, chlolesteryl nitrolinoleate, and nitrohydroxylinoleate induce vasorelaxation in a concentration-dependent manner while releasing nitric oxide that was characterized by chemiluminescence-and EPR-based methodologies. As we first show here, diffusible nitric oxide production is likely to occur by isomerization of the nitrated lipids to the corresponding nitrite derivatives that decay through homolysis and/or metal ion/ascorbate-assisted reduction. The homolytic mechanism was supported by EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid that trapped a lipid-derived radical during nitrolinoleate decomposition. In addition to provide a mechanism to explain nitric oxide production from nitrated lipids, the results support their role as endogenous sources of nitric oxide that may play a role in endothelium-independent vasorelaxation.     

Deoxynivalenol and 15-monoacetyl deoxynivalenol production by Fusarium graminearum R6576 in liquid media

Growth and toxigenesis by Fusarium graminearum R6576, were compared in four liquid media. Parameters monitored during the fermentation were deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON) production, fungal mass, carbohydrate utilization, and pH. Factors which were varied included basal medium composition, corn steep liquor (CSL) concentration, sucrose concentration and ammonium tartrate concentration. Growth in modified Fries medium resulted in only low levels of DON (0.25 mg/ L) and 15-ADON (0.25 mg/ L) after 20 days. Addition of 4% CSL to modified Fries medium raised the 20 day DON yield to 16.5 mg/ l. Increasing the sucrose concentration in modified Fries medium amended with 4% CSL resulted in increased mycelial dry weight but decreased levels of DON. Concentrations of ammonium tartrate greater than 1% in modified Fries amended with 4% CSL greatly reduced DON yield. Use of glucose-yeast extract-peptone (GYEP) for toxin production resulted in higher yields of 15-ADON (14.0 mg/ L) than DON (5.5 mg/ L) after 20 days. However, supplementation of GYEP with 4% CSL resulted primarily in DON production (4.5 mg/ L) after 20 days. In general, qualitative and quantitative production of DON and 15-ADON by Fusarium graminearum R6576 were dependent on the composition of the complex liquid medium.     

Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.     

Cholesteryl nitrolinoleate,a nitrated lipid present in human blood plasma and lipoproteins

Nitric oxide (*NO) and *NO-derived reactive species (e.g., peroxynitrite anion, nitrogen dioxide radical) react with lipids containing unsaturated fatty acids to generate nitrated species. In the present work, we synthesized, characterized, and detected a nitrated derivative of cholesteryl linoleate (Ch18:2) in human blood plasma and lipoproteins using a high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry method. It was synthesized by a reaction of Ch18:2 with nitronium tetrafluoroborate, yielding a species with m/z 711, which is characteristic of the cholesteryl nitrolinoleate (Ch18:2NO2) ammonium adduct. The presence of the nitro group was confirmed by using [15N]nitrite, which gave a product with m/z 712, with the same chromatographic and spectrometric characteristics of those of m/z 711. Furthermore, a C-NO2 structure was also demonstrated in Ch18:2NO2 by infrared analysis (Vmax 1549, 1374 cm-1). A stable product with m/z of 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standard, was found in human blood plasma and lipoproteins of normolipidemic subjects. The presence of this novel nitrogen-containing lipid product in human plasma and lipoproteins could represent a potential indicator of the oxidative/nitrative roles that *NO or its metabolites play during in vivo lipid oxidation, generating a compensatory mechanism of protection in vascular disease.     

Synthesis and in vitro anti-HCV activity of beta-D- and 1-2'-deoxy-2'-fluororibonucleosides

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2'-fluoro group was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely beta-D-2'-deoxy-2',5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As beta-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2'-fluoro were combined into one molecule, yielding beta-D-2'-deoxy-2'-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

The Modulatory Effect of Metabotropic Glutamate Receptor Type-1α on Spike-Wave Discharges in WAG/Rij Rats

Modulatory function of metabotropic glutamate type 1 (mGlu1) receptors plays a crucial role in the pathophysiology of some neurological disorders, including schizophrenia and epilepsy. In this study, the expression of mGlu1α receptors in the thalamic nuclei was assessed during development of absence seizures in the WAG/Rij rats, a valid genetic animal model of absence epilepsy. In addition, the effect of pharmacological modulation of mGlu1α receptors in the laterodorsal (LD) nucleus of the thalamus on the characteristic features of bioelectrical brain activities in the WAG/Rij rats was assessed. The expression of mGlu1α receptors in the LD was assessed in four experimental groups of both WAG/Rij and Wistar rats with 2 and 6 months of age. Agonist and antagonist of mGlu1α receptors were infused in LD in the six months old WAG/Rij (epileptic) rats. The protein level of mGlu1α receptors in the thalamus of the 6-month-old WAG/Rij rats was lower than non-epileptic animals. In addition, the distribution of mGlu1α receptors in different thalamic nuclei was lower in the 6-month-old WAG/Rij compared to age-matched Wistar rats. The gene expression of mGlu1α receptor was also significantly lower in 6-month-old WAG/Rij rats in the LD compared to other animal groups. The microinjection of mGlu1α receptors agonist and antagonist in the LD reduced the duration of spike-wave discharges (SWDs) and increased the amplitude and duration of SWDs, respectively, in 6-month-old WAG/Rij rats. The alterations of mGlu1α receptors expression in the thalamus of epileptic WAG/Rij rats as well as its modulatory effects in the generation of SWDs suggest the potential of mGlu1 receptors as a therapeutic target in absence epilepsy.     

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO₂), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO₂ was capable to deliver free radical nitric oxide (^·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO₂ for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO₂ were observed in vivo by intravital microscopy assays. LNO₂ decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO₂ reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO₂ on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO₂ involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO₂ inhibited adhesion molecules expression and promoted ^·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.