Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca\u3csup\u3e2+\u3c/sup\u3e regulation

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOXcatalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/ lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation

Aspectos Moleculares da Regeneração de Fígado de Rato. I - Atividade da Aspartato Transcarbamilase em Função do Tempo de Regeneração em Fígado de Ratos Submetidos a Hepatectomia Parcial

A Aspartato Transcarbamilase foi estudada em fígado de rato em regeneração. Sua atividade sofre um aumento em função do tempo de regeneração, com aumento de até 47% sobre sua atividade basal na décima segunda hora após a hepatectomia

Reliability and suitability of physiological exercise response and recovery markers

There is currently insufficient evidence about the reliable quantification of exercise load and athlete’s recovery management for monitoring training processes. Therefore, this test–retest study investigated the reliability of various subjective, muscle force, and blood-based parameters in order to evaluate their suitability for monitoring exercise and recovery cycles. 62 subjects completed two identical 60-min continuous endurance exercise bouts intermitted by a four-week recovery period. Before, immediately after, three, and 24 h after each exercise bout, analysis of parameters were performed. Significant changes over time were found for rating of perceived exertion (RPE), multidimensional mood state questionnaire (MDMQ), maximum voluntary contraction parameters (MVCs), and blood-based biomarkers (p 0.90). A good reliability was found for thiobarbituric acid reactive substances (TBARS) (ICC = 0.79) and haematological markers (ICC = 0.75–0.86). For RPE, MDMQ, interleukin (IL-) 1RA, IL-6, IL-8, IL-15, cortisol, lactate dehydrogenase (LDH), creatine kinase (CK) only moderate reliability was found (ICC < 0.75). Significant associations for IL1-RA and CK to MVC were found. The excellent to moderate reliability of TBARS, LDH, IL-1RA, six measured haematological markers, MVCs and MDMQ implicate their suitability as physiological exercise response and recovery markers for monitoring athletes’ load management. © 2020, The Author(s)

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.National Institutes of Health (U.S.) (Grant R01 GM52339)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

Reduction of peritoneal carcinomatosis by intraperitoneal administration of phospholipids in rats

<p>Abstract</p> <p>Background</p> <p>Intraperitoneal tumor cell attachment after resection of gastrointestinal cancer may lead to a developing of peritoneal carcinosis. Intraabdominal application of phospholipids shows a significant decrease of adhesion formation even in case of rising tumor cell concentration.</p> <p>Methods</p> <p>In experiment A 2*10⁶colonic tumor cells (DHD/K12/Trb) were injected intraperitonely in female BD-IX-rats. A total of 30 rats were divided into three groups with treatments of phospholipids at 6% or 9% and the control group. In experiment B a total of 100 rats were divided into ten groups with treatments of phospholipids at 9% and the control group. A rising concentration of tumor cells (10,000, 50,000, 100,000, 250,000 and 500,000) were injected intraperitonely in female BD-IX-rats of the different groups. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of attachment and the Peritoneal Cancer Index (PCI).</p> <p>Results</p> <p>In experiment A, we found a significant reduction (control group: tumor volume: 12.0 ± 4.9 ml; area of tumor adhesion: 2434.4 ± 766 mm²; PCI 28.5 ± 10.0) of peritoneal dissemination according to all evaluation methods after treatment with phospholipids 6% (tumor volume: 5.2 ± 2.2 ml; area of tumor adhesion: 1106.8 ± 689 mm²; PCI 19.0 ± 5.0) and phospholipids 9% (tumor volume: 4.0 ± 3.5 ml; area of tumor adhesion: 362.7 ± 339 mm²; PCI 13.8 ± 5.1). In experiment B we found a significant reduction of tumor volume in all different groups of rising tumor cell concentration compared to the control. As detected by the area of attachment we found a significant reduction in the subgroups 1*10⁴, 25*10⁴and 50*10⁴. The reduction in the other subgroups shows no significance. The PCI could be reduced significantly in all subgroups apart from 5*10⁴.</p> <p>Conclusion</p> <p>In this animal study intraperitoneal application of phospholipids resulted in reduction of the extent of peritoneal carcinomatosis after intraperitoneal administration of free tumor cells. This effect was exceptionally noticed when the amount of intraperitoneal tumor cells was limited. Consequently, intraperitoneal administration of phospholipids might be effective in reducing peritoneal carcinomatosis after surgery of gastrointestinal tumors in humans.</p

A comprehensive analysis of coherent rainfall patterns in China and potential drivers. Part II: intraseasonal variability

The causes of subseasonal precipitation variability in China are investigated using observations and reanalysis data for extended winter (November–April) and summer (May–October) seasons from 1982 to 2007. For each season, the three dominant regions of coherent intraseasonal variability are identified with Empirical Orthogonal Teleconnection (EOT) analysis. While previous studies have focused on particular causes for precipitation variability or on specific regions, here a comprehensive analysis is carried out with an objective method. Furthermore, the associated rainfall anomaly timeseries are tied to specific locations in China, which facilitates their interpretation. To understand the underlying processes associated with spatially coherent patterns of rainfall variability, fields from observations and reanalysis are regressed onto EOT timeseries. The three dominant patterns in winter together explain 43% of the total space–time variance and have their origins in midlatitude disturbances that appear two pentads in advance. Winter precipitation variability along the Yangtze River is associated with wave trains originating over the Atlantic and northern Europe, while precipitation variability in southeast China is connected to the Mediterranean storm track. In summer, all patterns have a strong relationship with the Boreal Summer Intraseasonal Oscillation and are modulated by the seasonal cycle of the East Asian summer monsoon. The wet and dry phases of the regional patterns can substantially modulate the frequency of daily rainfall across China. The discovered links between weather patterns, precursors, and effects on local and remote precipitation may provide a valuable basis for hydrological risk assessments and the evaluation of numerical weather prediction models

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain