Splicing function of mitotic regulators links R-loop–mediated DNA damage to tumor cell killing

Although studies suggest that perturbing mitotic progression leads to DNA damage and p53 activation, which in turn lead to either cell apoptosis or senescence, it remains unclear how mitotic defects trigger p53 activation. We show that BuGZ and Bub3, which are two mitotic regulators localized in the interphase nucleus, interact with the splicing machinery and are required for pre-mRNA splicing. Similar to inhibition of RNA splicing by pladienolide B, depletion of either BuGZ or Bub3 led to increased formation of RNA–DNA hybrids (R-loops), which led to DNA damage and p53 activation in both human tumor cells and primary cells. Thus, R-loop–mediated DNA damage and p53 activation offer a mechanistic explanation for apoptosis of cancer cells and senescence of primary cells upon disruption of the dual-function mitotic regulators. This demonstrates the importance of understanding the full range of functions of mitotic regulators to develop antitumor drugs.     

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ^−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.     

The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell Migration

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an “outside-in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

Choline Kinase β Mutant Mice Exhibit Reduced Phosphocholine,Elevated Osteoclast Activity,and Low Bone Mass

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase β (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase β mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5′-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.     

利用皇竹草处理城市污泥生产植物产品

利用大生物量植物——皇竹草(Pennisetum hydridum),处理城市污泥高效生产有用的植物产品。通过小型田间试验,采用根兜分株移栽和直接扦插方式种植皇竹草,比较土壤、新鲜污泥、土壤+新鲜污泥等体积混合、植物处理后的污泥4种介质的适应性,测定了皇竹草的成活率和生长状况,以及污泥自身性质的变化;通过盆栽试验及田间试验进一步探讨育苗后移栽在新鲜污泥上的可行性。结果表明,新鲜污泥上直接扦插的皇竹草无一存活,即使是根兜分株移栽其成活率也仅为16.67%,生长较差。而对于土壤+污泥混合物或植物处理后的污泥,采用直接扦插方式,皇竹草的成活率也分别达58.33%、75.00%,两个月干草产量达22.20、19.80 t/hm2,为土壤上的5.11、4.55倍。皇竹草吸收K较明显,3个有污泥的处理皇竹草K含量接近40 g/kg干重,N、P2O5、K2O的总含量大于70 g/kg,可作为有机K肥原料;皇竹草重金属Cu、Zn、Pb、Cd含量均符合国家饲料卫生标准(GB 13078—2001),作为饲料是安全的。盆栽试验及田间试验表明,采用育苗后移栽的方式,皇竹草在新鲜污泥上的成活率达66.67%以上。因此,城市污泥直接种植皇竹草可以实现资源化利用。     

不同水氮处理对玉米-大豆间作群体内作物光能截获、竞争和利用的影响

通过田间试验研究了不同水氮处理对玉米-大豆间作群体的光能截获、竞争与利用的影响。试验设置充分供水和水分亏缺两种水分处理以及施氮(亩施纯氮7.5 kg)和不施氮两种氮肥处理。结果表明,在生育中后期,同一氮肥处理条件下,充分供水处理间作作物的光能截获率显著高于水分亏缺处理;相同水分条件下,施氮处理间作大豆的光能截获率略高于不施氮处理,但未达到显著水平,而施氮处理间作玉米的光能截获率则显著高于不施氮处理。从播后第64天到成熟,同一氮肥处理条件下,充分供水提高了间作玉米的光能竞争比,但却降低了间作大豆的光能竞争比。从播后第73天到成熟,相同水分条件下,施氮处理间作玉米的光能竞争比显著高于不施氮处理,而大豆的光能竞争比在两个氮肥处理间则没有显著差异。充分供水条件下,施氮处理间作玉米的光能利用效率(LUE)为3.87 g/MJ,略高于不施氮处理(3.81 g/MJ);水分亏缺条件下,施氮处理间作玉米的LUE(3.86 g/MJ)比不施氮处理(3.72 g/MJ)高3.6%。充分供水条件下,施氮处理间作大豆的LUE(1.62 g/MJ)比不施氮处理(1.57 g/MJ)高3.2%;水分亏缺条件下,施氮处理间作大豆的LUE为1.55 g/MJ,与不施氮处理(1.54 g/MJ)基本相同,表明与氮肥处理相比,水分状况对大豆LUE的影响更为明显。