BG-4, a novel bioactive peptide from momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages.

BACKGROUND: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. PURPOSE: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated from the seed of bitter melon. METHODS: Differentiated THP-1 human macrophages were pre-treated with BG-4 and stimulated with lipopolysaccharide. Pro-inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. The mechanism of action involving activation of NF-κB and phosphorylation of ERK and STAT3 was measured by western blot and immunofluorescence. The production of intracellular reactive oxygen species was evaluated by fluorescence microscopy and fluorescence spectrophotometry. RESULTS: BG-4 dose dependently reduce the production of pro-inflammatory cytokines IL-6 and TNF-α. The ability of BG-4 to reduce production of cytokines are associated with reduced phosphorylation of ERK and STAT3 accompanied by reduced nuclear translocation of p65 NF-κB subunit. The mechanism of action is reduction of LPS-induced production of intracellular reactive oxygen species. CONCLUSION: Our results demonstrated the ability of BG-4, a novel peptide from the seed of bitter melon, to exert anti-inflammatory action. This could explain the traditional use of bitter melon against diseases associated with aberrant and uncontrolled inflammation.

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein's conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Phytochemical concentrations and biological activities of Sorghum bicolor alcoholic extracts.

Sorghum is an important cereal with reported health benefits. The objectives of this study were to measure the biological activities of alcoholic extracts of ten sorghum varieties and to determine the association between the color of the extracts and their biological activities. Variation on concentrations of bioactives among sorghum varieties was observed with ethanolic extracts giving higher concentrations than methanolic extracts. The color of the extracts significantly correlated with the concentrations of bioactives and with nitric oxide scavenging activity. Freeze-dried ethanol extract is more potent than freeze-dried methanol extract and caused cytotoxicity to A27801AP and PTX-10 OVCA with ED50 values of 0.69 and 1.29 mg mL(-1), respectively. Pre-treatment of OVCA with ethanol extract led to chemosensitization to paclitaxel and the proliferation and colony formation of OVCA cells were reduced by 14.7 to 44.6% and 36.4 to 40.1%, respectively. Sorghum is a potential source of colorants with health promoting properties. This is the first report on the capability of sorghum alcoholic extracts to cause cytotoxicity and chemosensitize ovarian cancer cells in vitro.

Defining topological features of membrane proteins by nanoelectrospray ionisation mass spectrometry.

The D-galactose-H(+) symport protein, GalP, of Escherichia coli is the bacterial homologue of the human glucose transport protein, GLUT1. Here we demonstrate that mass spectrometry can be used to map modification by covalently bound reagents, and also to detect structural changes in the GalP protein that occur upon substrate binding. The small thiol-group-specific reagent N-ethylmaleimide (NEM) was used to modify the cysteine residues in GalP(His)(6) both alone and in the presence of D-glucose, a known substrate. Employing a mixture of proteolysis and thermal degradation methods, the three cysteine residues were found to undergo sequential reactions with NEM, with Cys374 being modified first, followed by Cys389 and finally Cys19, thus indicating their different accessibilities within the three-dimensional structure of the protein. Prior binding of the substrate D-glucose to the protein protected Cys19 and Cys374 against NEM modification, but not Cys389. Cys374 had been expected to be shielded by D-glucose binding while Cys389 had been expected to be unaffected, consistent with their proposed respective locations in the vicinity of, and distant from, the sugar binding site. However, the inaccessibility of Cys19 was unexpected and suggests a structural change in the protein promoted by D-glucose binding which changes the proximity of Cys19 with respect to the D-glucose-binding site.