Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

Genetic polymorphism of KIR2DL4 results in alleles with either 9 or 10 consecutive adenines in exon 6, which encodes the transmembrane domain. "10A" alleles encode a membrane-expressed receptor that is constitutively expressed on resting CD56bright NK cells and on CD56 dim cells after culture. However, in some individuals with the 10A allele, KIR2DL4 cannot be detected on their resting CD56 bright NK cells. "9A" alleles have been predicted to encode a secreted receptor due to the splicing out of the transmembrane region. In this publication, we show that those individuals with a 10A allele who lack detectable KIR2DL4 on CD56 bright NK cells express a KIR2DL4 receptor in which the D0-domain is excised. This D-D0 receptor cannot be detected by the available anti-KIR2DL4 monoclonal antibodies. In such individuals, KIR2DL4 becomes detectable on cultured NK cells due to up-regulation of the full-length KIR2DL4 transcript. In all individuals with 10A alleles, KIR2DL4 ceases to be expressed at the cell surface 16 days after activation, despite the maintenance of maximal levels of KIR2DL4 mRNA transcription, suggesting the existence of a negative regulator of cell surface expression. Finally, we show that the 9A allele can produce a secreted KIR2DL4 receptor.

Processing of membrane-bound transcription factors such as sterol regulatory element-binding proteins (SREBPs) and the ER-stress response factor ATF6, and glycoproteins of some hemorrhagic fever viruses are initiated by the proprotein convertase SKI-1/S1P. So far, no cellular protein-based inhibitor of the hydrophobicamino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or ␣ 1 -PDX, a variant of ␣ 1 -antitrypsin (␣ 1 -AT) exhibiting an RIPR 358 sequence at the reactive site loop, were shown to potently inhibit these secretory proteinases. Accordingly, we tested the SKI-1-inhibitory potential of various point mutants of either the 198 amino acid preprosegment of SKI-1-(1-198) or ␣ 1 -AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the ␣ 1 -AT reactive site loop variants RRVL 358 , RRYL 358 and RRIL 358 are the best specific cellular inhibitors of SKI-1. The observed inhibition of the processing of endogenous SREBP-2, exogenous ATF6 and a PDGF-A (RRLL 86 ) variant were >55% and reach ϳ80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these ␣ 1 -AT variants, but not with wild-type ␣ 1 -AT or ␣ 1 -PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein.Proteins and peptides that are biologically active are often generated by intracellular limited proteolysis of inactive precursors. The mammalian proprotein convertases (PCs) 1 of the secretory pathway are calcium-dependent serine proteinases related to bacterial subtilisin that cleave various precursors at the general consensus motif (K/R)(X) n (K/R)2, where n ϭ 0, 2, 4, or 6 and X is any amino acid (1-3). The PC family counts seven basic amino acid-specific kexin-like convertases: furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC (4). The eighth member is the recently discovered pyrolysin-like SKI-1/S1P that cleaves at the consensus motif (R/K)X(hydrophobic)Z2, where Z is variable (5), while the last member (6, 7) NARC-1 cleaves the sequence VFAQ 153 2 in its prosegment (8, 9). 2 More PCs contain an N-terminal signal sequence, followed by a prosegment, a catalytic domain and a P-domain. In addition, PCs possess a C-terminal segment that varies between the different members. The critical role of PCs in the proteolytic maturation of multiple proproteins, their implication in various pathologies (1, 10, 11), and their unidentified specific and/or redundant functions, make them attractive targets for the development of potent and selective inhibitors. The various successful approaches include: active site-directed chloromethyl ketone inhibitors (12, 13), reversible peptide-based inhibitors (14 -17), plant derivatives (18), and several engineered variants of protein-based inhibitors that possess a furin-like motif. These include ␣2-macroglobulin (␣2-MF) (19), ␣ 1 -antitrypsin (␣ 1 -AT) Portland (␣ 1 -PDX) (20 -22), proteinase i...

Epigallocatechin gallate (EGCG), the major polyphenol in green tea, acutely stimulates production of nitric oxide (NO) from vascular endothelium to reduce hypertension, and improve endothelial dysfunction in SHR rats. Herein, we explored additional mechanisms whereby EGCG may mediate beneficial cardiovascular actions. When compared with vehicle-treated controls, EGCG treatment (2.5 μM, 8 h) of human aortic endothelial cells (HAEC) caused a ~3-fold increase in hemeoxygenase-1 (HO-1) mRNA and protein with comparable increases in HO-1 activity. This was unaffected by pre-treatment of cells with wortmannin, LY294002, PD98059, or L-NAME (PI 3-kinase, MEK, and NO synthase inhibitors, respectively). Pre-treatment of HAEC with SB203580 (p38 MAPK inhibitor) or siRNA knockdown of p38 MAPK completely blocked EGCG-stimulated induction of HO-1. EGCG treatment also inhibited TNF-α-stimulated expression of VCAM-1 and decreased adhesion of monocytes to HAEC. siRNA knockdown of HO-1, p38 MAPK, or Nrf-2 blocked these inhibitory actions of EGCG. In HAEC transiently transfected with a human HO-1 promoter luciferase reporter (or an isolated Nrf-2 responsive region), luciferase activity increased in response to EGCG. This was inhibitable by SB203580 pre-treatment. EGCG-stimulated expression of HO-1 and Nrf-2 was blocked by siRNA knockdown of Nrf-2 or p38 MAPK. Finally, liver from mice chronically treated with EGCG had increased HO-1 and decreased VCAM-1 expression. Thus, in vascular endothelium, EGCG requires p38 MAPK to increase expression of Nrf-2 that drives expression of HO-1 resulting in increased HO-1 activity. Increased HO-1 expression may underlie anti-inflammatory actions of EGCG in vascular endothelium that may help mediate beneficial cardiovascular actions of green tea.