This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [ 32 P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.Endothelial nitric-oxide synthase (eNOS 1 or type III NOS) is one of three isoenzymes that converts L-arginine to L-citrulline and nitric oxide (NO). Endothelial cells synthesize NO tonically and increase NO production in response to agonists and increased fluid shear stress (FSS). Endothelial NO contributes to blood vessel homeostasis by regulating vessel tone (1), cell growth (2), platelet aggregation (3), and leukocyte binding to endothelium (4). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (5-7). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (8), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (9, 10) binding motif (11), located near the domain that binds Ca 2ϩ /calmodulin. Recent studies suggest that the activity of eNOS is regulated in a reciprocal manner through caveolin-1 inhibition and Ca 2ϩ /calmodulin stimulation (12-14).Increased FSS stimulates an increase in free intracellular calcium [Ca 2ϩ ] i from intracellular stores (15, 16) leading to a Ca 2ϩ /calmodulin-dependent increase in eNOS activity. However, recent investigations show that increases in [Ca 2ϩ ] i do not fully explain the rapid rise in NO production in response to FSS (17). Exposure of bovine aortic endothelial cells (BAEC) to 25 dynes/cm 2 FSS for 30 s caused a 7-fold rise in NO production and a corresponding 2-fold increase in eNOS phosphorylation, whereas the calcium ionophore A23187 neither caused rapid NO production...

We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.

MicroRNAs have recently been identified as important regulators of gene expression at the post-transcriptional level. While it has clearly been established that microRNAs influence the ontogeny of several immune cell lineages, the role of individual microRNAs during natural killer (NK) cell development has not been described. Here, we show that miR-181 expression levels have a profound impact on the development of human NK cells from CD34+ hematopoietic progenitor cells (HPC) and IFN-γ production in primary CD56+ NK cells. We also demonstrate that nemo-like kinase (NLK), an inhibitor of Notch signaling, is a target of miR-181 in NK cells, and knockdown of NLK mirrors the developmental effect of miR-181 over-expression. We conclude that miR-181 promotes NK cell development, at least in part, through the suppression of NLK, providing an important link between microRNAs and Notch signaling.

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56bright NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56bright cells differentiate into CD56dim NK cells in vivo. The relationship between CD56dim and CD56bright proliferating cells indicates that proliferating CD56dim cells both self-renew and are derived from proliferating CD56bright NK cells. Our data suggest that some dying CD56dim cells become CD16+CD56− NK cells and that CD16−CD56low NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56dim NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8+ and CD8− T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56bright NK cells declined with age and the percentage of CD56dim NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.