Palmitoylethanolamide Treatment Reduces Blood Pressure in Spontaneously Hypertensive Rats: Involvement of Cytochrome P450-Derived Eicosanoids and Renin Angiotensin System

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

Comparison of forest above‐ground biomass from dynamic global vegetation models with spatially explicit remotely sensed observation‐based estimates

Gaps in our current understanding and quantification of biomass carbon stocks, particularly in tropics, lead to large uncertainty in future projections of the terrestrial carbon balance. We use the recently published GlobBiomass data set of forest above‐ground biomass (AGB) density for the year 2010, obtained from multiple remote sensing and in situ observations at 100 m spatial resolution to evaluate AGB estimated by nine dynamic global vegetation models (DGVMs). The global total forest AGB of the nine DGVMs is 365 ± 66 Pg C, the spread corresponding to the standard deviation between models, compared to 275 Pg C with an uncertainty of ~13.5% from GlobBiomass. Model‐data discrepancy in total forest AGB can be attributed to their discrepancies in the AGB density and/or forest area. While DGVMs represent the global spatial gradients of AGB density reasonably well, they only have modest ability to reproduce the regional spatial gradients of AGB density at scales below 1000 km. The 95th percentile of AGB density (AGB₉₅) in tropics can be considered as the potential maximum of AGB density which can be reached for a given annual precipitation. GlobBiomass data show local deficits of AGB density compared to the AGB₉₅, particularly in transitional and/or wet regions in tropics. We hypothesize that local human disturbances cause more AGB density deficits from GlobBiomass than from DGVMs, which rarely represent human disturbances. We then analyse empirical relationships between AGB density deficits and forest cover changes, population density, burned areas and livestock density. Regression analysis indicated that more than 40% of the spatial variance of AGB density deficits in South America and Africa can be explained; in Southeast Asia, these factors explain only ~25%. This result suggests TRENDY v6 DGVMs tend to underestimate biomass loss from diverse and widespread anthropogenic disturbances, and as a result overestimate turnover time in AGB.     

1H, 13C, and 15N resonance assignments of the N-terminal domain of human Tubulin Binding Cofactor C

Human Tubulin Binding Cofactor C (hTBCC) is a 346 amino acid protein composed of two domains, which is involved in the folding pathway of newly synthesized α and β-tubulins. The 3D structure of the 111-residue hTBCC N-terminal domain of the protein has not yet been determined. As a previous step to that end, here we report the NMR ¹H, ¹⁵N, and ¹³C chemical shift assignments at pH 6.0 and 25°C, based on a uniformly doubly labelled ¹³C/¹⁵N sample of the domain.     

Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1

AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.     

The cellular basis for immune interferon production in autoimmune MRL-Ipr/Ipr mice

Disturbances in immune interferon (IFN gamma) activity have been implicated in the development of human systemic lupus erythematosus (SLE) and the spontaneous disease sustained by autoimmune-prone mice. We therefore investigated the cellular basis for IFN gamma production in MRL-Ipr/Ipr mice and examined the relationship between synthesis of interleukin 2 (IL 2) and IFN gamma. In vitro IL 2 and IFN gamma production in 3 to 6-mo-old, autoimmune MRL-Ipr/Ipr and MRL-+/+ mice was compared with that seen in age- and sex-matched, immunologically normal CBA/J mice. 5 X 10(6) spleen cells were pulsed with 5 micrograms of concanavalin A (Con A), and the cellfree supernatant was assayed for IL 2 and IFN gamma activity at various times up to 72 hr. We found that peak levels of IL 2 in MRL mice were less than 10% of those in the CBA/J. Yet, production of IFN gamma by cells from the autoimmune and normal strains was quite comparable. The addition of murine IL 2 to optimally Con A-stimulated cells from the MRL-Ipr/Ipr or normal mice did not affect the subsequent peak production of IFN gamma. Although the primary producers of IFN gamma in cultures of normal mice bear the Lyt-2+ phenotype, the Lyt-1+2- T-cell subset was found to be the principal source of IFN gamma in the aged MRL-Ipr/Ipr. These data suggest that Lyt-1+ cells from MRL-Ipr/Ipr mice may be differentially responsive to the signal delivered by the same mitogenic lectin with respect to lymphokine production and may indicate a distorted commitment of such cells toward production of IFN gamma and repression of IL 2 synthesis. The relationship between hypoproduction of IL 2, this usual source of IFN gamma, and the autoimmune disease sustained by MRL-Ipr/Ipr mice remains unclear.     

The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.