Um novo olhar sobre a agricultura brasileira.

bitstream/item/133378/1/Um-novo-olhar-sobre-a-agricultura-brasileira.pd

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

Background: The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of gene

Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2–7 complex

Mcm2–7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2–7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2–4–6–7 in purified systems, rather modulates Mcm2–7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2–7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication

Investigation of the Acetylation Mechanism by GCN5 Histone Acetyltransferase

The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes

Efficacy and Safety of bimekizumab in elderly patients: real-world multicenter retrospective study - IL PSO (Italian Landscape Psoriasis)

Purpose of the article: The aim of this multicenter observational study is to report data from real world on the use of bimekizumab in patients aged ≥ 65 years with moderate-to-severe plaque psoriasis. Elderly patients are poorly represented in clinical trials on bimekizumab for plaque psoriasis, and real-world studies are important to guide clinical choices. Materials and methods: A retrospective multicenter study was conducted in 33 dermatological outpatient clinics in Italy. Patients aged ≥ 65 years, with moderate-to-severe plaque psoriasis and treated with bimekizumab were enrolled. No exclusion criteria were applied. Bimekizumab was administered following the Italian Guidelines for the management of plaque psoriasis and according to the summary of product characteristics, in adult patients who were candidates for systemic treatments. Overall, 98 subjects were included, and received bimekizumab up to week 36. Clinical and demographic data were collected before the initiation of treatment with bimekizumab. At baseline and each dermatological examination (4, 16, and 36 weeks), clinical outcomes were measured by the following parameters: (1) PASI score; (2) site-specific (scalp, palmoplantar, genital, nail) Psoriasis Global Assessment (PGA). At each visit, the occurrence of any adverse events (AEs) was recorded, including serious AEs and AEs leading to bimekizumab discontinuation. Results: The mean PASI score was 16.6 ± 9.4 at baseline and significantly decreased to 4.3 ± 5.2 after 4 weeks (p < 0.001), and 1.1 ± 1.7 after 16 week (p < 0.001). This level of improvement was maintained after 36 weeks (p < 0.001). PASI ≤2 was recorded in 36 (36.7%) at week 4, 68% and 69.4% at week 16 and 36, respectively. By week 16, 86/98 (87.8%) patients reached PASI75, 71/98 (72.4%) obtained PASI90, and 52/98 (53.1%) PASI100. Binary logistic regression tests showed a significant association of PASI100 by week 4 with lower PASI at baseline. PASI 100 at 16 or 36 weeks was not associated with baseline PASI, obesity, age, gender, previously naïve state, and presence of psoriatic arthritis. Patients naïve to biologics at baseline had similar response to bimekizumab as non-naïve subjects. Conclusions: Bimekizumab is a suitable option for elder patients as it is effective, tolerated and has a convenient schedule

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption