No abstract

The isolation and total synthesis of the antimicrobial lipopeptide cerexin A1 is reported. This synthesis includes the preparation of orthogonally protected γ-hydroxylysine, utilizing a nitrile Reformatsky-type reaction as a key step to yield both diastereomers more efficiently than previously reported methods. The configuration of the β-hydroxyl in the lipid tail was determined by the use of a modified Ohrui-Akasaka approach. Furthermore, new cerexin analogues from Bacillus mycoides ATCC 21929 were isolated and characterized, revealing an ε-amino succinylation of a hydroxylysine residue that is unusual in a nonribosomal peptide synthetase product.

Vitelline membrane (VM) is a multilayered structure that surrounds the egg yolk serving to separate the yolk and the white. Due to its poor solubility in aqueous-based media, VM proteins and their biological properties have not been fully defined. In the current study, VM was hydrolyzed using different enzymes under the optimum hydrolysis conditions. Antioxidant and anti-inflammatory properties were evaluated in chemical and cellular models. Flavourzyme- and trypsin-treated samples showed the highest radical scavenging and ferric ion reducing effect (31% and 20 μM of Trolox equivalents/mg, respectively). In cellular studies, all VM hydrolysates were cyto-compatible and inhibited nitric oxide production by RAW264.7 macrophage cells significantly. Lipopolysaccharide-stimulated up-regulation of pro-inflammatory cytokines in RAW264.7 cells was suppressed by flavourzyme-treated VM. These results revealed that enzymatic hydrolysis of VM is a promising approach to produce peptides with several bioactivities (free radical scavenging, metal chelation, and anti-inflammatory) as valuable ingredients for cosmeceuticals and nutraceuticals.

Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism.

Background: The benefit of dietary n-3 polyunsaturated fatty acids (PUFAs) in the reduction of cardiovascular disease has been recognized for many years. Epoxydocosapentanoic acids (EDPs) are lipid mediators produced from the metabolism of docosahexaenoic acids (DHA) by cytochrome P450 epoxygenase. In this study, we investigated the mechanism of the effect of DHA and 19,20-EDP on H9c2 cells cultured under glycolytic and OXPHOS conditions. Methods: H9c2 cells were cultured in DMEM containing either normal (25 mM) or low (5.5 mM) glucose to change the metabolic state. Cells were treated with or without 19,20-EDP (1 μM), DHA (100 μM), MSPPOH (50 μM) or myriocin (1 μM) for 24 h. Cellular viability, caspase-3 activity, ATP production and autophagic response were assessed by fluorometric assay. Ceramide levels were measured by LC/MS after cell fractionation. Mitochondrial function was determined by assessing respiration and enzymatic activities of complex IV and citrate synthase. Results: H9c2 cells cultured under normal glucose conditions had lower respiration rates indicating a more glycolytic phenotype compared to cells under low glucose conditions (OXPHOS). DHA and 19,20-EDP decreased cell viability, ATP production and mitochondrial function. Inhibition of de novo synthesis of ceramide by myriocin blocked the cell injury effects. Moreover, DHA and 19,20-EDP increased caspase-3. Importantly, MSPPOH, a CYP epoxygenase inhibitor, attenuated DHA-induced effects in cells treated under normal glucose conditions, indicating the CYP-derived metabolite, 19,20-EDP, is the active lipid mediator. Conclusion: Our data suggest that 19, 20-EDP induces a cell death associated with autophagy due to ceramide-induced mitochondrial dysfunction under normal glucose conditions. Thus reflecting a differential response where 'aerobic glycolytic' states are more susceptible to effects observed in conditions of cells under more 'oxidative phosphorylation' states. Thus, 19,20-EDP has different effects on H9c2 cell survival depending on the metabolic state.