NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells

Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 μM) were detected by (1)H and (31)P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of (1)H-NMR spectroscopy demonstrated that the CH(2)/CH(3 )and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH(3 )and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. (31)P-NMR spectroscopy indicated levels of phosphomonoesters (PME) and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from (1)H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells

Modeling of Performance Creative Evaluation Driven by Multimodal Affective Data

Performance creative evaluation can be achieved through affective data, and the use of affective featuresto evaluate performance creative is a new research trend. This paper proposes a “Performance Creative—Multimodal Affective (PC-MulAff)” model based on the multimodal affective features for performance creative evaluation. The multimedia data acquisition equipment is used to collect the physiological data of the audience, including the multimodal affective data such as the facial expression, heart rate and eye movement. Calculate affective features of multimodal data combined with director annotation, and defined “Performance Creative—Affective Acceptance (PC-Acc)” based on multimodal affective features to evaluate the quality of performance creative. This paper verifies the PC-MulAff model on different performance data sets. The experimental results show that the PC-MulAff model shows high evaluation quality in different performance forms. In the creative evaluation of dance performance, the accuracy of the model is 7.44% and 13.95% higher than that of the single textual and single video evaluation

Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location - dimer interface, interlobar helices, protein surface, or within other secondary structural elements.The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Multiple roles of PPAR alpha in brown adipose tissue under constitutive and cold conditions

Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a member of the nuclear receptor family, regulating fatty acid degradation in many organs. Two-dimensional SDS-PAGE of brown adipose tissue (BAT) from PPAR alpha-null mice produced a higher-density spot. Proteomic analysis indicated that the protein was pyruvate dehydrogenase beta (PDH beta). To observe PDH beta regulation in BAT, the organ was stimulated by long-term cold exposure, and the activities of associated enzymes were investigated. Histological and biochemical analyses of BAT showed a significant decrease in the triglyceride content in wild-type mice and some degree of decrease in PPAR alpha-null mice on cold exposure. Analyses of molecules related to glucose metabolism showed that the expression of PDH beta is under PPAR alpha-specific regulation, and that glucose degradation ability may decrease on cold exposure. In contrast, analyses of molecules related to fatty acid metabolism showed that numerous PPAR alpha/gamma target molecules are induced on cold exposure, and that fatty acid degradation ability in wild-type mice is markedly enhanced and also increases to same degree in PPAR alpha-null mice on cold exposure. Thus, this study proposes novel and multiple roles of PPAR alpha in BAT.ArticleGENES TO CELLS. 15(2):91-100 (2010)journal articl

Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form “a carboxylate clamp” with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins

Early tissue and healing responses after maxillary sinus augmentation using horizontal platelet rich fibrin bone blocks.

BACKGROUND The effects of horizontal platelet-rich fibrin (H-PRF) bone block on the healing and immune response during sinus augmentation have not been fully investigated histologically at early time points. METHODS Eighteenth male New Zealand white rabbits underwent bilateral sinus augmentation and were divided into two groups: deproteinized bovine bone mineral (DBBM) alone and H-PRF + DBBM (H-PRF bone block) group. Maxilla samples were collected at 3, 7 and 14 days post sinus augmentation procedures and analyzed using histological staining for the number of inflammatory cells, new blood vessels and evidence for early osteoclast bone turnover/remodeling. Furthermore, the effects of H-PRF bone blocks on the migration of osteoblasts and THP-1 macrophages were evaluated using a Transwell assay in vitro. RESULTS A higher number of immune cells were found in the H-PRF bone block group at 3 and 7 days post-surgery when compared to the DBBM alone group,most notably in the regions close to the mucosal lining and bone plates. Furthermore, a significantly greater number of new blood vessel formations and early signs of osteoclast development were found in the H-PRF bone block group at 14 days. The in vitro transwell assay further confirmed that culture medium from H-PRF bone block markedly promote the migration of osteoblasts and THP-1 macrophages. CONCLUSIONS The findings from this study have shown that H-PRF bone block is capable of increasing early immune cell infiltration leading to the acceleration of neovascularization and speeding the process of bone metabolism in vivo following maxillary sinus grafting with DBBM

The Combination of Human Urinary Kallidinogenase and Mild Hypothermia Protects Adult Rats Against Hypoxic-Ischemic Encephalopathy-Induced Injury by Promoting Angiogenesis and Regeneration

Objectives: Human Urinary Kallidinogenase (HUK) is a tissue kallikrein that plays neuroprotective role in ischemic conditions via different mechanisms. Mild hypothermia (MH) is another robust neuroprotectant that reduces mortality but does not profoundly ameliorate the neurological outcome in hypoxic-ischemic encephalopathy (HIE) patients. However, whether the combination of HUK and MH can be used as a promising neuroprotective treatment in HIE is unknown.Methods: One-hundred and forty-four adult Wistar rats were randomly divided into five groups: Sham, HIE, HUK, MH and a combination of HUK and MH treatment. The HIE rat model was established by right carotid dissection followed by hypoxia aspiration. The survival curve was created within 7 days, and the neurological severity scores (NSS) were assessed at days 0, 1, 3, and 7. Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), immunofluorescent staining and western blotting were used to evaluate neuronal survival, apoptosis and necrosis, tight-junction proteins Claudin-1 and Zonula occludens-1 (ZO-1), vascular endothelial growth factor (VEGF), doublecortex (DCX), bradykinin receptor B1 (BDKRB1), BDKRB2 and Ki67 staining.Results: The combined treatment rescued all HIE rats from death and had a best survival curve compared to HIE. The Combination also reduced the NSS scores after HIE at days 7, better than HUK or MH alone. The combination of HUK and MH reserved more cells in Nissl staining and inhibited neuronal apoptosis and necrosis as well as significantly attenuated HIE-induced decreases in claudin-1, ZO-1, cyclin D1 and BDKRB1/B2 in comparison to HUK or MH treatment alone. Moreover, the combined treatment increased the expression of VEGF and DCX as well as the number of Ki67-labeled cells.Conclusions: This study demonstrates that both HUK and MH are neuroprotective after HIE insult; however, the combined therapy with HUK and MH enhanced the efficiency and efficacy of either therapy alone in the treatment of HIE, at least partially by promoting angiogenesis and regeneration and rescuing tight-junction loss. The combination of HUK and MH seems to be a feasible and promising clinical strategy to alleviate cerebral injury following HIE insult.Highlights: -The combination of HUK and MH distinctly reduces neurological dysfunction in HIE rats.-HUK enhances the neuroprotective effects of MH in HIE.-MH attenuates tight-junction disruption, upregulates the BDKR B1/2, DCX and cyclin D1.-The combination of MH and HUK enhances the expressions of MH/HUK mediated-BDKR B1/2, DCX, cyclin D1 and Ki67 positive cells

N-linked glycosylation enhances hemagglutinin stability in avian H5N6 influenza virus to promote adaptation in mammals

Clade 2.3.4.4 avian H5Ny viruses, namely H5N2, H5N6, and H5N8, have exhibited unprecedented intercontinental spread in poultry. Among them, only H5N6 viruses are frequently reported to infect mammals and cause serious human infections. In this study, the genetic and biological characteristics of surface hemagglutinin (HA) from clade 2.3.4.4 H5Ny avian influenza viruses (AIVs) were examined for adaptation in mammalian infection. Phylogenetic analysis identified an amino acid (AA) deletion at position 131 of HA as a distinctive feature of H5N6 virus isolated from human patients. This single AA deletion was found to enhance H5N6 virus replication and pathogenicity in vitro and in mammalian hosts (mice and ferrets) through HA protein acid and thermal stabilization that resulted in reduced pH threshold from pH 5.7 to 5.5 for viral-endosomal membrane fusion. Mass spectrometry and crystal structure revealed that the AA deletion in HA at position 131 introduced an N-linked glycosylation site at 129 which increases compactness between HA monomers thus stabilizes the trimeric structure. Our findings provide a molecular understanding of how HA protein stabilization promotes cross-species avian H5N6 virus infection to mammalian hosts

Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits.Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore