Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC) can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up-and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PSinduced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.cell cycle | shear | bioinformatics | gene regulation | mechanotransduction H emodynamic forces, e.g., stretch and shear stress, act constantly on the vascular endothelial cells (ECs) to modulate EC signaling, gene expression, and physiological functions (1). Atherosclerotic lesions in the arterial tree are found mainly at branch points, where blood flow is disturbed with a limited forwarding direction, but are generally spared at the straight parts of the arterial tree, where the flow is laminar with a large forwarding direction (2). Exposure of ECs to 24 h of steady laminar shear flow at 12 dyn/cm 2 (approximating the hemodynamic force in straight parts of arteries) leads to antiproliferative (3) and antiinflammatory (4) responses. In contrast, ECs exposed to disturbed flow, mimicking the hemodynamic force at branch points, exhibit opposite responses (5, 6). The laminar shear-induced EC growth arrest involves the expression of CDK inhibitors (e.g., p21 cip , p27 kip ), tumor suppressor p53, and retinoblastoma (Rb) hypophosphorylation (3, 7). Whereas there is considerable knowledge on mechanotransduction at protein and mRNA levels, there is little information on the role of microRNAs (miRs) in this process.miRs are small noncoding RNAs (∼21-25 nucleotides) that regulate gene expression by binding to target mRNAs to cause their degradation or translational repression (8). It is estimated that miRs regulate ∼30% of human protein-coding genes. More than 800 miRs have been identified in the human genome and registered in the Sanger miRBase. These small RNAs provide a powerful mechanism for posttranscriptional cont...

7-Carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg2+-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the first crystal structure of a QueE, along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg2+, which directly coordinates to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified (β6/α3) protein core and an expanded cluster-binding motif CX14CX2C.

Objective Pancreatic cysts are commonly detected in patients undergoing pancreatic imaging. Better approaches are needed to characterize these lesions. In this study we evaluated the utility of detecting mutant DNA in secretin-stimulated pancreatic juice. Design Secretin-stimulated pancreatic juice was collected from the duodenum of 291 subjects enrolled in Cancer of the Pancreas Screening trials at 5 US academic medical centers. The study population included subjects with a familial predisposition to pancreatic cancer who underwent pancreatic screening, and disease controls with normal pancreata, chronic pancreatitis, sporadic IPMN, or other neoplasms. Somatic GNAS mutations (reported prevalence; ~66% of IPMNs) were measured using high-resolution digital melt-curve analysis and pyrosequencing. Results GNAS mutations were detected in secretin-stimulated pancreatic juice samples of 50 of 78 familial and sporadic cases with IPMN(s) (64.1%), 15 of 33 (45.5%) with only diminutive cysts (<5mm), but none of 57 disease controls. GNAS mutations were also detected in 5 of 123 screened subjects without a pancreatic cyst. Among 97 subjects who had serial pancreatic evaluations, GNAS mutations detected in baseline juice samples predicted subsequent emergence or increasing size of pancreatic cysts. Conclusion Duodenal collections of secretin-stimulated pancreatic juice from patients with IPMNs have a similar prevalence of mutant GNAS to primary IPMNs, indicating these samples are an excellent source of mutant DNA from the pancreas. The detection of GNAS mutations before an IPMN is visible suggests that pancreatic juice analysis has potential to help in the risk stratification and surveillance of patients undergoing pancreatic screening.

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Objective-Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results-AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPK␣2 knockout mice when compared with wild-type control mice. Key Words: shear stress Ⅲ endothelial cells Ⅲ KLF2 Ⅲ AMPK Ⅲ eNOS V ascular endothelial cells (ECs) in the arterial tree are subjected to shear stress resulting from the flow of blood. ECs exposed to laminar flow patterns are spared from early lesions of atherosclerosis. In contrast, ECs at the arterial bifurcations and curvatures, where disturbed flow patterns exist, are susceptible to the development of atherosclerotic lesions. There is ample evidence indicating that the different patterns of shear stress associated with laminar versus disturbed flows play a significant role in regulating endothelial phenotype and vascular homeostasis. Conclusion-TheKrü ppel-like factor 2 (KLF2) is a transcription factor whose expression is flow-dependent in vitro and in vivo and has been shown to regulate various EC functions including inflammation, thrombosis, proliferation, and vascular tone. 1-3 KLF2 belongs to the family of KLF zinc-finger transcription factors that are important regulators of cell differentiation and development. 4 Using microarray analysis, Dekker et al demonstrated that KLF2 is upregulated by prolonged laminar shear stress and that it is expressed in ECs of atherosclerosis-resistant regions of the human aorta. 1 Subsequent work by Lingrel and colleagues demonstrated that laminar shear stress induces transcriptional activation of KLF2 in ECs. 5 Our previous studies showed that KLF2 was induced by the atheroprotective flow, but not by atheroprone flow. 3,6 KLF2 has been implicated in mediating the antiinflammatory effects of flow, presumably by inhibiting proinflammatory transcription factors, such as ATF2, 7 AP-1, and NFB, 8 which in turn regulate gene expression.Functioning as a "fuel gauge" in multiple organ systems, AMPK is also important in the vessel wall. Nagata et al showed that hypoxia activates AMPK in human umbilical vein ECs (HUVECs), as indicated by increased phosphorylation at Thr-172 of the AMPK␣ subunit. 9 The suppression of AMPK signaling by a dominant-negative...

TYW1 catalyzes the condensation of N-methylguanosine with two carbon atoms from an unknown second substrate to form 4-demethylwyosine, which is a common intermediate in the biosynthesis of all of the hypermodified RNA bases related to wybutosine found in eukaryal and archaeal tRNAPhe. Of potential substrates examined, only incubation with pyruvate resulted in formation of 4-demethylwyosine. Moreover, incubation with C1, C2, C3, or C1,2,3-13C-labeled pyruvate showed that C2 and C3 are incorporated while C1 is not. The mechanistic implications of these results are discussed in the context of the structure of TYW1.

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