TNF-alpha contributes to the elevated TGF-beta expression demonstrated in the adipose tissue of obese mice. A potential role for TGF-beta in the increased PAI-1 and vascular pathologies associated with obesity/NIDDM is suggested.

Obesity is associated with a cluster of abnormalities, including hypertension, insulin resistance, hyperinsulinemia, and elevated levels of both plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor ␤ (TGF-␤). Although these changes may increase the risk for accelerated atherosclerosis and fatal myocardial infarction, the underlying molecular mechanisms remain to be defined. Although tumor necrosis factor ␣ (TNF-␣) has been implicated in the insulin resistance associated with obesity, its role in other disorders of obesity is largely unknown. In this report, we show that in obese (ob͞ob) mice, neutralization of TNF-␣ or deletion of both TNF receptors (TNFRs) results in significantly reduced levels of plasma PAI-1 antigen, plasma insulin, and adipose tissue PAI-1 and TGF-␤ mRNAs. Studies in which exogenous TNF-␣ was infused into lean mice lacking individual TNFRs indicate that TNF-␣ signaling of PAI-1 in adipose tissue can be mediated by either the p55 or the p75 TNFR. However, TNF-␣ signaling of TGF-␤ mRNA expression in adipose tissue is mediated exclusively via the p55 TNFR. Our results suggest that TNF-␣ is a common link between the insulin resistance and elevated PAI-1 and TGF-␤ in obesity. The chronic elevation of TNF-␣ in obesity thus may directly promote the development of the complex cardiovascular risk profile associated with this condition.

Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates αIIbβ3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1–guanosine triphosphate–interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to αIIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and αIIbβ3 activation. However, one talin mutant (L325R) is recruited to αIIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated αIIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and αIIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to αIIbβ3 by RIAM mediates agonist-induced αIIbβ3 activation, with implications for hemostasis and thrombosis.

Genomic DNA is replicated by two DNA polymerase molecules: one works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through synthesis of Okazaki fragments 1, 2 . Considering the lagging-strand polymerase has to recycle after every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase 3-5 , it is not understood how the two strands are synthesized with the same net rate [6][7][8][9] . Here, we show with the T7 replication proteins 10, 11 that RNA primers are made on the fly during ongoing DNA synthesis, and the leading-strand T7 replisome does not pause during primer synthesis contrary to previous reports 12,13 . Instead, the leading-strand polymerase remains limited by the speed of the helicase 14 , so synthesizes DNA at a slower rate than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; hence, the nascent lagging-strand template organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provides three synergistic mechanisms of coordination: 1) Primers are made concomitantly with DNA synthesis; 2) the priming-loop assures efficient primer utilization and hand-off to the polymerase; 3) the laggingstrand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.To investigate the functional cooperativity between the enzymatic activities of the T7 replication complex, we measured the kinetics of DNA unwinding, DNA synthesis, and primer synthesis on synthetic replication fork substrates with and without the T7 primingsequence (3'-CTGGG, Supplementary Table 1). Efficient synthesis of RNA primers from 2mer to 5mer by T7 replisome (T7 gp4 and T7 DNA polymerase) was observed on the priming fork ( Fig. 1a and Supplementary Fig. 1) with half-life of ~0.5 s and yield >60% (Fig. 1a, right). T7 gp4 alone also makes RNA primers on this priming fork, but at ~10-fold * Correspondence: patelss@umdnj.edu and tjha@illinois.edu.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions M.P. purified T7 gp5, T7 gp4 and constructed DNA substrates for the priming loop studies, and obtained and analyzed all the ensemble DNA synthesis and primer synthesis experiments; S.S. developed robust single molecule assays for observing DNA unwinding and priming loop formation, and obtained and analyzed all single molecule data; I.D. and G.P. performed the ensemble unwinding experiments; M.P, S.S., S.S.P., and T.H designed the experiments, analyzed the data, and wrote the manuscript. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 December 17. Supplementary Fig. 1), consistent with polymerase assistance of the helicase rate 14 . An average 46% yield of primer synthesis with fork...

Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. We previously reported that plasminogen activator inhibitor 1 (PAI-1) is dramatically up-regulated in the plasma and adipose tissues of genetically obese mice. This change may disturb normal hemostatic balance and create a severe hypofibrinolytic state. Here we show that tissue factor (TF) gene expression also is significantly elevated in the epididymal and subcutaneous fat pads from ob͞ob mice compared with their lean counterparts, and that its level of expression in obese mice increases with age and the degree of obesity. Cell fractionation and in situ hybridization analysis of adipose tissues indicate that TF mRNA is increased in adipocytes and in unidentified stromal vascular cells. Transforming growth factor ␤ (TGF-␤) is known to be elevated in the adipose tissue of obese mice, and administration of TGF-␤ increased TF mRNA expression in adipocytes in vivo and in vitro. These observations raise the possibility that TF and TGF-␤ may contribute to the increased cardiovascular disease that accompanies obesity and related non-insulin-dependent diabetes mellitus, and that the adipocyte plays a key role in this process. The recent demonstration that TF also inf luences angiogenesis, cell adhesion, and signaling suggests that its exact role in adipose tissue physiology͞pathology, may be complex.