Synthetic spatially graded Rac activation drives cell polarization and movement

Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.     

骨吸收抑制剂对卵巢切除骨折大鼠脂类代谢及骨钙素的影响

目的 研究骨吸收抑制剂对卵巢切除大鼠脂类代谢及骨钙素的影响.方法 雌性SD大鼠共140只,随机分为5组,每组28只.3月龄时选取其中4组大鼠行双侧卵巢切除建立绝经后骨质疏松症模型(OVX组、OVX+ EE2组、OVX+ Rlx组、OVX+ Aln组),另一组行假手术(Sham组).OVX组及Sham组皮下注射生理盐水,余下3组分别注射阿仑膦酸钠(Aln)、雷洛昔芬(Rlx)、雌激素(EE2),每周注射5次.分别在卵巢切除术后4周、10周及20周测定大鼠血脂、骨钙素及相关生化指标.结果 OVX组与Sham组相比体重增加,总胆固醇升高,甘油三脂降低,差异有统计学意义.应用雌激素及雷洛昔芬可有效调节卵巢切除导致的脂代谢紊乱,表现为体重下降及总胆固醇水平降低,差异有统计学意义(P＜0.05).不同时期OVX组骨钙素水平高于Sham组,差异有统计学意义(P＜0.05),其中阿仑膦酸钠组血清骨钙素水平最低.随着时间推移,Sham组及OVX组骨钙素呈现先上升再下降趋势,而OVX+EE2组、OVX+ Rlx组及OVX+ Aln组骨钙素表现为持续降低.结论 骨吸收抑制剂能够降低骨钙素水平,从而降低卵巢切除导致的高骨转换率,防止骨量丢失.此外,雌激素及雷洛昔芬在预防骨丢失的基础上还能够有效调节卵巢切除引起的脂类代谢紊乱.     

Rare case of a rudimentary medial metatarsal non-ossified structure

A 9-year-old boy presented with a rudimentary medial metatarsal non-ossified structure. We considered his condition to be classified as hypoplastic medial member type in the metatarsal type of medial ray polydactyly. When it was considered as polydactyly, it had the longest delay of ossification among reported cases.     

Disruption of blood flow in the outflow graft is a valuable marker for detecting suction events in patients with continuous-flow left ventricular assist devices

Journal of Echocardiography -     

A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease

BACKGROUND: Although some reports have indicated that acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) can predict the prognosis in patients with acute coronary syndrome, the value of these markers in patients with stable coronary artery disease (CAD) still remains obscure. Therefore, our aim was to determine the prognostic value of inflammatory markers in patients with stable coronary artery disease. METHODS AND RESULTS: We conducted a prospective cohort study in 140 consecutive patients with stable coronary artery disease who had at least one coronary stenosis more than 50% in diameter seen on diagnostic coronary angiography (CAG). We determined serum levels of the SAA/LDL complex as a new marker in addition to CRP and SAA. Serum levels of the SAA/LDL complex were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). End-points were defined as cardiac death, myocardial infarction, cerebral infarction, and coronary revascularization. End-point events occurred in 21 patients (2 death from myocardial infarction, 2 cerebral infarction, and 17 revascularization). Age (year) (OR = 1.14, CI: 1.05-1.25), diabetes mellitus (OR = 3.50, CI: 1.08-11.40), triglyceride (10mg/dl) (OR = 1.12, CI: 1.01-1.23) and SAA/LDL complex (10 microg/ml) (OR = 2.32, CI: 1.05-4.70) were independently related to the events. A reconstitution experiment suggested that the SAA/LDL complex is derived by oxidative interaction between SAA and lipoproteins. CONCLUSIONS: The SAA/LDL complex reflects intravascular inflammation directly and can be a new marker more sensitive than CRP or SAA for prediction of prognosis in patients with stable coronary artery disease.     

Inhibitory effect of 9-amino-1,2,3,4-tetrahydroacridine (THA) on the potassium current of rabbit sinoatrial node

STUDY OBJECTIVE - To examine the effect of 9-amino-1,2,3,4-tetrahydroacridine (THA), a compound similar to the K+ blocker 4-aminopyridine, on potassium channels in the sinoatrial node. DESIGN - The pacemaking portion of rabbit sinoatrial nodes was studied using the double microelectrode voltage clamp method in the presence of THA at various concentrations. MEASUREMENTS AND RESULTS - Above 1 mumol.litre-1, THA prolonged the spontaneous cycle length and the transmembrane action potential duration at 50% repolarisation. Above 10 mumol.litre-1, the compound also decreased the maximum rate of rise, the action potential amplitude, and the rate of diastolic depolarisation. Under voltage clamp conditions, THA reduced the time dependent K+ current (IK) in a dose dependent manner. Neither the decay process of IK nor its activation process were altered by THA. CONCLUSIONS - THA depresses sinoatrial node IK without changing its kinetics. Thus it may inhibit the open state of the potassium channels.     

Plasma concentrations and coronary vasodilation after sublingual and intracoronary administration of isosorbide dinitrate

To investigate the relationship between plasma levels and coronary vasodilation after administration of isosorbide dinitrate (ISDN), the plasma concentration and diarneters of six segments of the left coronary artery were measured before and after sublingual (SL) ISDN (5 mg) and left intracoronary (IC) administration of ISDN (3 mg) in 12 patients. After SL-ISDN, the systolic aortic pressure decreased with no significant concomitant changes in heart rate or diastolic aortic pressure. After IC-ISDN, all hemodynamic parameters showed significant changes, and these were greater after IC-ISDN than those after SL-ISDN. The individual mean vasodilation of six segments induced by SL- and IC-ISDN, were 23± 9 and 35± 11% (p<0.01), respectively. Before SL-ISDN, ISDN was not detected in plasma. After SL- and IC-ISDN, however, the plasma values of the ISDN were 36.1± 53.3 and 101.5± 90.0 ng/ml (p<0.01), respectively. Thus, both coronary vasodilative responses and plasma ISDN levels after IC-ISDN were significantly greater than those after SL-ISDN. However, neither the individual mean coronary vasodilation nor the hemodynamic changes correlated significantly with plasma ISDN levels. Consequently, with administration of the same dose, the coronary vasodilative response to ISDN did not correlate with plasma levels. Furthermore, IC-ISDN dilates coronary arteries more effectively than SL-ISDN.     

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.Activation-induced cytidine deaminase (AID) is essential for three different genetic events: class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM), which contribute to Ig gene diversification (1–5). Although AID generates single-strand breaks (SSBs) in the Ig genes, subsequent repair steps for CSR and GC are similar to each other but are distinct from SHM in their mechanistic properties, i.e, in (i) generation of the double-strand breaks (DSBs), (ii) recombination, and (iii) the requirement for uracil-DNA-glycosylase (UNG) for the pairing of the DSB ends (6–10). Despite the similarities between GC and CSR, their repair mechanisms have distinct features: CSR recombination requires nonhomologous end joining (NHEJ), and GC depends on homologous recombination (HR). During CSR, DSB ends normally are joined by classical NHEJ (C-NHEJ), which requires specific repair proteins such as Ku80, XRCC4, or DNA ligase IV (11, 12). In the absence of C-NHEJ, a back-up end-joining pathway called “alternative end joining” (A-EJ), which is reported to be slower and also more error prone than C-NHEJ, joins the broken DSBs ends (13). On the other hand, HR, the most common form of homology-directed repair, requires long sequence homology between donor and acceptor DNA to complete the recombination step by recruiting a distinct set of repair proteins such as RAD54, RAD52, and RAD51 to the break sites (14, 15).Various studies on AID mutations in the N-terminal or C-terminal regions (4, 8, 9, 16–19) have shown that N-terminal AID mutants are compromised for CSR and are defective in SHM, indicating that the N-terminal region of AID is required for DNA cleavage (9, 16, 19). On the other hand, the C-terminal region of AID, which contains a nuclear-export signal and is responsible for AID’s shuttling activity between the nucleus and cytoplasm, is required for CSR-specific activity but not for DNA cleavage activity and SHM (8, 16). Among the series of AID C-terminal mutants examined, two mutants show characteristic features: P20, which has an insertion of 34 amino acids at residue 182 and normal nuclear-cytoplasmic shuttling activity, and JP8Bdel, which has a 16-amino acid truncation at residue 183, accumulates in the nucleus, and shows higher DNA break activity at the donor switch (S) region (16, 17). Although several reports suggest that the C-terminal region of AID is involved in protein stability (20, 21), C-terminal mutants of AID stabilized by fusing the hormone-binding domain of estrogen receptor (ER) also show similar CSR-defective phenotypes (8). Taken together, these data suggest that the DNA cleavage activity and CSR-specific activity depend on different regions of AID (8, 19). In addition, the C-terminal region of AID is essential for the interaction of AID with poly (A)⁺ RNA via a specific cofactor (22). Because CSR requires de novo protein synthesis, we proposed that after DNA cleavage the C-terminal region of AID may be involved in the regulation of the recombination step through generation of a new protein (8, 16, 22).DSBs induced by AID during CSR ultimately are joined by the efficient DNA repair pathway that requires C-NHEJ factors such as Ku70/80 (12, 23). However, in the absence of C-NHEJ, the A-EJ pathway that relies on microhomology can join the broken DNA ends, although this pathway is associated with chromosomal translocations (11, 24). Previously, we reported that JP8Bdel enhances aberrant c-myc/IgH translocations and that it fails to carry out the efficient recombination between donor and acceptor S regions in the IgH locus (8). Therefore, it is important to examine whether the AID C-terminal mutants affect the S–S joining in CSR.In the current work we examined whether the C-terminal region of AID is involved in DNA synapse formation and recombination during CSR in CH12F3-2 and spleen B cells. We also examined the effect of AID C-terminal mutations on GC in chicken DT40 cells, which depends on HR between pseudo V genes and the downstream IgVλ region. Using these CSR- and GC-monitoring systems, we demonstrate that efficient CSR and GC require the C-terminal region of AID for the formation of DSB from SSB and subsequent end synapse. Considering these findings together, we conclude that, in addition to DNA cleavage, AID has a unique function in the generation of DSBs, which is required for S–S synapse formation and joining in CSR and recombination in GC.