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Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P

Background: There has been growing interest in the development of highly potent and selective protein tyrosine phosphatase (PTP1B) inhibitors for the past 2-3 decades. Though most PTPs share a common active site motif, the interest in selective inhibitors, particularly against PTP1B is increasing to discover new chemical entities as antidiabetic agents. In the current paradigm to find potent and selective PTP1B inhibitors, which is currently considered as one of the best validated biological targets for non-insulin-dependent diabetic and obese individuals, resistance to insulin due to decreased sensitivity of the insulin receptor is a pathological factor and is also genetically linked, causing type II diabetes. Objectives: Insulin receptor sensitization is performed by a signal transduction mechanism via a selective protein tyrosine phosphatase (PTP1B). After the interaction of insulin with its receptor, autophosphorylation of the intracellular part of the receptor takes place, turning it into an active kinase (sensitization). PTP1B is involved in the desensitization of the receptor by dephosphorylation. PTP1b inhibitors delay the receptor desensitization, prolonging insulin effect and making PTP1B as a drug target for the treatment of diabetes II. Therefore, it has become a major target for the discovery of potent drugs for the treatment of type II diabetes and obesity. An attempt has been made in the present study to discuss the latest design and discovery of protein tyrosine phosphatase (PTP1B) inhibitors. Methods: Many PTP1B inhibitors such as diaminopyrroloquinazoline, triazines, pyrimido triazine derivatives, 2-(benzylamino)-1-phenylethanol, urea, acetamides and piperazinylpropanols, phenylsulphonamides and phenylcarboxamide, benzamido, arylcarboxylic acid derivatives, arylsupfonyl derivatives, thiazoles, isothiozolidiones and thiazolodinones have been discussed, citing the disease mechanisms. Results: The reader will gain an overview of the structure and biological activity of recently developed PTPs inhibitors. Conclusion: The co-crystallized ligands and the screened inhibitors could be used as a template for the further design of potent congeners.

The residue located at 15 positions before the most conserved residue in TM7 (7.35 of Ballesteros-Weinstein number) plays important roles in ligand binding and the receptor activity for class A GPCRs. Nevertheless, its regulation mechanism has not been clearly clarified in experiments, and some controversies also exist for its impact on μ-opioid receptors (μOR) bound by agonists. Thus, we chose the μ-opioid receptor (μOR) of class A GPCRs as a representative and utilized a microsecond accelerated molecular dynamics simulation (aMD) coupled with a protein structure network (PSN) to explore the effect of W318sup7.35/supon its functional activity induced by the agonist endomorphin2 mainly by a comparison of the wild system and its W7.35A mutant. When endomorphin2 binds to the wild-type μOR, TM6 in μOR moves outward to form an open intracellular conformation that is beneficial to accommodating the β-arrestin transducer, rather than the G-protein transducer due to the clash with the α5 helix of G-protein, thus acting as a β-arrestin biased agonist. However, the W318A mutation induces the intracellular part of μOR to form a closed state, which disfavors coupling with either G-protein or β-arrestin. The allosteric pathway analysis further reveals that the binding of endomorphin2 to the wild-type μOR transmits more activation signals to the β-arrestin binding site while the W318A mutation induces more structural signals to transmit to common binding residues of the G protein and β-arrestin. More interestingly, the residue at the 7.35 position regulates the shortest allosteric pathway in indirect ways by influencing the interactions between other ligand-binding residues and endomorphin2. W293sup6.48/supand F289sup6.44/supare important for regulating the different activities of μOR induced either by the agonist or by the mutation. Y336sup7.53/sup, F343sup8.50/sup, and D340sup8.47/supplay crucial roles in activating the β-arrestin biased signal induced by the agonist endomorphin2, while L158sup3.43/supand V286sup6.41/supdevote important contributions to the change in the activity of endomorphin2 from the β-arrestin biased agonist to the antagonist upon the W318A mutation.

Purpose: N6-methyladenosine (m6A) is among the most abundant mRNA modifications in eukaryote. The aim of the present study was to investigate function of m6A mRNA methylation in lung cancer and the underlying mechanism. Methods: Microarray analysis was performed to detect the differences in RNA expression between cancerous and adjacent non-cancerous tissue samples. The target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hierarchical clustering of RNAs was conducted to identify distinct m6A methylation or expression patterns between the samples. Results: In the present study, some differentially expressed genes (DEGs) of mRNAs were identified, including up-regulated secret phosphoprotein 1 (SPP1) and down-regulated pRB. Functional enrichment analysis revealed that while differential hypermethylation was related to cell cycle, intracellular part and protein binding, the main pathway involved herpes simplex virus 1 infection related to down-regulated AKT, Araf1 and BCL2A1. In the meantime, sexual reproduction, cohesin complex and protein C-terminus binding was functionally linked to differential hypomethylation, while fluid shear stress and atherosclerosis were identified as the main pathways related to up-regulated GST and CNP. Conclusions: We showed that lung cancer development involved differential expression of SPP1 and pRB mRNA, as well as m6A mRNA methylation in AKT, APAF1, BCL2A1, GST and CNP genes.

Host genetic factors influencing the intracellular part of HIV live cycle and regulating of HIV-specific immune response are reviewed. Its include genes coding proteins which support viral replication and assembly of new virions, genes coding antiviral defense proteins, HLA genes and some others. Variants of these genes and its compositions affect individual succeptibility/resistance to HIV infection, influence the pathogenesis of the disease and also associate with efficacy of antiretroviral therapy.

Molecular switches in GPCRs enable passing the signal from the agonist binding site, usually located close to the extracellular surface, to the intracellular part of the receptor. The switches are usually associated with conserved structural motifs on transmembrane helices (TMs), and they are accompanied by adjacent residues which provide the signal to the central residue in the toggle switch. In case of locks being the molecular switches, they are breaking (permanently or temporarily) upon agonist binding. Cascade action of switches is correlated with influx of water molecules to form a pathway linking both sides of the receptor. The switches remove the hydrophobic barriers and facilitate water movement while water molecules help to rearrange the hydrogen bond network inside the receptor.

As multicellular organisms age, they undergo a reduction in tissue and organ function. Researchers have put forward a theory that stem cell aging is the main factor responsible for decreased tissue and organ function. The adult stem cells guarantee the maintenance and repair of adult tissues and organs. Among adult stem cells, mesenchymal stem cells (MSCs) are emerging as hopeful candidates for cell-based therapy of numerous diseases. In recent years, high-throughput sequencing technologies have evolved to identify circular RNAs (circRNAs) associated with an increasing number of diseases, such as cancer and age-related diseases. It has been reported that circRNAs can compete with microRNAs (miRNAs) to affect the stability or translation of target RNAs and further regulate gene expression at the transcriptional level. However, the role of circRNAs expressed in MSCs in aging mechanisms has not yet been deciphered. The aim of this study was to explore and analyze the expression profiles of age-related circRNAs in MSCs. In this study, bone marrow MSCs were extracted from aged and young rats and analyzed using high-throughput sequencing and bioinformatics. The reliability of high-throughput RNA sequencing was verified by quantitative real-time polymerase chain reaction. The most important circRNA functions and pathways were further selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) analysis. Age-related circRNAs were found in the circrNA–miRNA–mRNA interaction network. The results of high-throughput sequencing showed that 4,229 circRNAs were involved in age-related senescence of MSCs. Compared with the young group, there were 29 differentially expressed circRNAs in the aged group, of which four were upregulated and 25 were downregulated. GO analysis covered three domains: biological process (BP), cellular component (CC), and molecular function (MF). The terms assigned to the BP domain were cellular metabolic processes and cellular macromolecule metabolic processes. The identified CC terms were intracellular and intracellular part, and the identified MF terms were binding and protein binding. The top five KEGG pathways were mitophagy–animal–Rattus norvegicus, prostate cancer–Rattus norvegicus, pathways in cancer–Rattus norvegicus, lysosome–Rattus norvegicus, and autophagy–animal–Rattus norvegicus. Altogether, circRNAs may play a major role in age-related MSC senescence. This study provides new mechanistic insights into MSC senescence, possibly leading to novel therapeutic strategies for age-related diseases.

Activating killer cell immunoglobulin-like receptors (KIR) in macaques are thought to be derived by genetic recombination of the region encoding the transmembrane and intracellular part of KIR2DL4 and a KIR3D gene. As a result, all macaque activating KIR possess a positively charged arginine residue in the transmembrane region. As human KIR2DL4 associates with the FCER1G (also called Fc receptor-gamma, FcRγ) adaptor, we hypothesized that in contrast to human and great ape the activating KIRs of macaques associate with FcRγ instead of DAP12. By applying co-immunoprecipitation of transfected as well as primary cells, we demonstrate that rhesus macaque KIR3DS05 indeed associates with FcRγ and not with DAP12. This association with FcRγ results in increased and substantially stabilized surface expression of KIR3DS05. In addition, we demonstrate that binding of specific ligands of KIR3DS05, Mamu-A1*001 and A1*011, resulted in signal transduction in the presence of FcRγ in contrast to DAP12.

Cilia are involved in a plethora of motility and sensory-related functions. Ciliary defects cause several ciliopathies, some of which with late-onset, suggesting cilia are actively maintained. While much is known about cilia assembly, little is understood about the mechanisms of their maintenance. Given that intraflagellar transport (IFT) is essential for cilium assembly, we investigated the role of one of its main players, IFT88, in ciliary maintenance. We show that DmIFT88, the Drosophila melanogaster orthologue of IFT88, continues to move along fully formed sensory cilia, and that its acute knockdown in the ciliated neurons of the adult affects sensory behaviour. We further identify DmGucy2d, the Drosophila guanylyl cyclase 2d, as a DmIFT88 cargo, whose loss also leads to defects in sensory behaviour maintenance. DmIFT88 binds to the intracellular part of DmGucy2d, a highly, evolutionarily conserved and mutated in several degenerative retina diseases, taking the cyclase into the cilia. Our results offer a novel mechanism for the maintenance of sensory cilia function and its potential role in human diseases.

Background: IgA nephropathy (IgAN) is one of the most common forms of primary glomerulonephritis. Recent studies have indicated that small noncoding RNAs, such as tRNA-derived small RNAs (tsRNAs), might be novel biomarkers for glomerulonephritis. We therefore investigated the potential roles and possible functions of the tsRNAs in IgAN. Method: Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of the patients with IgAN and healthy control groups. The expression profiles of tsRNAs were assessed by small RNA sequencing (RNA-Seq) in PBMCs of the IgAN and control groups. Dysregulated tsRNAs were selected for validation by quantitative real-time polymerase chain reaction (qRT-PCR). Target gene prediction and enrichment were performed by bioinformatics analysis. Results: The results revealed that 143 significantly upregulated and 202 significantly downregulated tsRNAs were differentially altered in the IgAN group compared with the control group. Five upregulated tsRNAs (tRF-Val-AAC-007, tRF-Ala-AGC-063, tRF-Gln-CTG-010, tRF-Tyr-GTA-011 and tRF-Thr-AGT-007) and 3 downregulated tsRNAs (tiRNA-Val-TAC-004, tRF-Gly-CCC-005 and tRF-His-GTG-006) were selected for validation by qRT-PCR; the results were consistent with the sequencing data. Gene Ontology (GO) analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in “nucleic acid metabolic process," “intracellular part," and “ion binding," whereas the target genes predicted by downregulated tsRNAs were mostly enriched in “regulation of cellular component organization," “membrane-bound organelle," and “ion binding." Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in “herpes simplex virus 1 infection," whereas the target genes predicted by downregulated tsRNAs were mostly enriched in “circadian rhythm Conclusions: The present study confirmed the differential expression of tsRNAs in patients with IgAN, and these dysregulated tsRNAs might be novel potential targets for the diagnosis and treatment of IgAN.

Intracellular part of TLR4 signaling starts on the inner side of the cytoplasm and consists of two branches, namely, TLR4/TRIF/IRF3 and TLR4/MyD88/NF-κB. Through complex interactions including phosphorylation and ubiquitination, they lead to the activation of various transcription factors and are intrinsically regulated.

Colletotrichum camelliae is one of the most serious pathogens causing anthracnose in tea plants, but the interactive relationship between C. camelliae and tea plants has not been fully elucidated. This study investigated the gene expression changes in five different growth stages of C. camelliae based on transcriptome analysis to explain the lifestyle characteristics during the infection. On the basis of gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs) in comparisons of germ tube (GT)/conidium (Con), appressoria (App)/Con, and cellophane infectious hyphae (CIH)/Con groups, the cellular process in the biological process category and intracellular, intracellular part, cell, and cell part in the cellular component category were significantly enriched. Hydrolase activity, catalytic activity, and molecular_function in the molecular function category were particularly enriched in the infection leaves (IL)/Con group. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEGs were enriched in the genetic information processing pathway (ribosome) at the GT stage and the metabolism pathway (metabolic pathways and biosynthesis of secondary metabolism) in the rest of the stages. Interestingly, the genes associated with melanin biosynthesis and carbohydrate-active enzymes (CAZys), which are vital for penetration and cell wall degradation, were significantly upregulated at the App, CIH and IL stages. Subcellular localization results further showed that the selected non-annotated secreted proteins based on transcriptome data were majorly located in the cytoplasm and nucleus, predicted as new candidate effectors. The results of this study may establish a foundation and provide innovative ideas for subsequent research on C. camelliae.

The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 μs in total) to study conformational changes triggered by the inverse agonist R-(−)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY211962...

In this work, accelerated molecular dynamics (aMD) simulations were used to study different effects of G286F and R126 mutations on the activity of CCR5. Potential of Mean Force (PMF) results indicate that there are stable inactive-like states and active-like ones existing in the conformation space of the wild type (WT), confirming that CCR5 could possess to some extent constitutive activity. But the R126N mutation could constrain CCR5 in the inactive state through influencing the TXP motif and limiting the movements of TM5 and TM6. In contrast, the G286F mutation promotes the activity of the receptor by increasing the distance of TM2-TM6 and the flexibility of the intracellular part of TM5 and changing the H-bonding in the TXP motif. The observations from the cross correlation analysis further show that the R126N mutation dramatically reduces the motion correlations between TMs, which should partly contribute to the deactivation of CCR5. Compared with the WT system, TM6 and TM7 in the G286F mutant are loosely correlated with other regions, which should be conducive to drive the movement of TM6 and TM7 toward the active conformation. In addition, the result from the protein structure network (PSN) analysis reveals that the shortest pathways connecting the extracellular and the intracellular domains are highly conserved in the three systems despite the different mutations, in which the hydrogen bond plays a pivotal role. However, the G286F mutation shortens the lifetime of the pathway with respect to the R126N mutation, which may be associated with the different activities of the two mutants. The pathway connecting the ligand-binding site and the G-protein region reveals that the allosteric communication between TM6 and TM7 is enhanced by the R126N mutation while the G286F mutation induces the activation of the G-protein pocket by arousing more residues in the NPxxY region to participate in the pathway.

Nanocarrier mediated targeted delivery and biosensing in reproductive health care is a major exploratory domain. This work demonstrates the loading of silver nanoparticle (AgNP) inside the multiwalled carbon nanotube (MWCNT) and their targetability to the intracellular part of the sperm cell for its further application in biosensing based infertility diagnosis. Ocimum tenuiflorum (tulsi extract) mediated photosynthesized AgNP exhibited spherical shape, 5–40 nm size and surface plasmonic resonance at 430 nm. After loading of freshly prepared AgNP into emulsified MWCNT, the loading was confirmed with spectroscopic and microscopic methods. FTIR analysis displayed significant shifting at 3450 cm−1 (–OH stretching) and 1615 cm−1 (CNT back bone) which validated the binding of AgNP with MWCNT and interestingly heat flow analysis revealed that Ag loaded MWCNT has greater stability than AgNP. Moreover, AFM based surface profile height analysis clearly showed the loading of AgNP inside MWCNT as surface heig...

Contents Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.

Adaptations to low temperature play a critical role in restricting the geographical distribution of insects. Decreasing day lengths and temperatures trigger seasonal cold adaptations in insects. These adaptions include changes in expression at the miRNA, mRNA and protein levels. The rice water weevil (RWW), Lissorhoptrus oryzophilus, introduced from the Mississippi River, is a globally invasive pest of wetland rice that can survive at the northern border of China. To investigate the changes in expression at the protein level in overwintering female RWW adults, 6-plex tandem mass tags (TMTs) were used in overwintering and summer adults. By using a proteome database available for Curculionidae, 1077 proteins were quantified, 183 of which differed significantly between the overwintering and summer samples. To further understand these differentially expressed proteins (DEPs), bioinformatics analyses such as gene ontology (GO) enrichment analyses were performed. DEPs associated with the terms binding, structural molecule activity, catalytic activity, multicellular organismal process, extracellular region, chitin binding, metabolic process, intracellular part and organic cyclic compound binding were altered by selection during winter. The changes in the expression of these proteins suggest that the proteins are important for RWW survival in winter.

The inactive conformations of glucagon receptor (GCGR) are widely reported by crystal structures that support the precision structure for drug discovery of type 2 diabetes. The previous study shows that the intracellular part is open in the glucagon-bound GCGR (glu-GCGR) and closed in the apo-GCGR by accelerated molecular dynamics (aMD) simulations. However, the crystal structure of GCGR in complex with partial agonist shows that the intracellular part is closed in the inactive conformation. To understand the differences between the studies of aMD simulations and crystal structure, the 2,500 ns conventional molecular dynamics (cMD) simulations are performed on the simulated model of glu-GCGR. The result shows that the transmembrane helices (TMH) 6 of glu-GCGR is outward similar to 4 angstrom to drive the intracellular part of glu-GCGR open until similar to 390 ns cMD simulations. The (TMH) 6 of glu-GCGR becomes closed after similar to 490 ns cMD simulations, which are consistent with the crystal structure of GCGR in complex with the partial agonist. To further elucidate the activation mechanism of GCGR deeply, the simulated models of glu-GCGR, apo-GCGR, and antagonist-bound GCGR (ant-GCGR) are constructed to perform 10 of parallel 300 ns aMD simulations, respectively. The results show that both of glu-GCGR and apo-GCGR can generate the open conformations of the intracellular part. But the glu-GCGR has the higher percentage of open conformations than apo-GCGR. The ant-GCGR is restricted to generate the open conformations of the intracellular part by antagonist MK-0893. It indicates that the glu-GCGR, apo-GCGR, and ant-GCGR can be distinguished by the aMD simulated method. Free energy landscape shows that the open conformations of the intracellular part of GCGR are in intermediate state. Our results show that aMD simulations enhance the space samplings of open conformations of GCGR via adding extra boost potential. It indicates that the aMD simulations are an effective way for drug discovery of GCGR.

Seed longevity is one of the most essential characters of seed quality. Two Chromosome segment substitution lines (CSSL) I178 and X178 with significant difference on seed longevity were subjected to transcriptome sequencing before (0d-AA) and after five days of accelerated ageing (5d-AA) treatments. Compared to the non-accelerated ageing treatment (0d-AA), 286 and 220 differential expressed genes (DEGs) were identified in I178 and X178, respectively Among those, 98 DEGs were detected in both I178 and X178 after 5d-AA, Enriched GO terms included cellular components of cell part, intracellular part, organelle and membrane etc., including carbohydrate derivative catabolic process, carbohydrate synthesis, sugar isomerase (SIS) family protein etc. Transcriptome analysis of I178 and X178 showed that Alternative splicing (AS) occurs in 63.6% of the expressed genes in all samples. Only 381 genes specifically occurred AS in I178 and X178 after 5d-AA, mostly enriched in nucleotide and nucleoside binding. Combined with the reported QTL mapping result, the DEG and the AS information, 13 DEGs in the mapping intervals and 7 AS-DEGs were potential candidates may directly or indirectly associated to seed ageing.

Seed longevity is one of the most essential characteristics of seed quality. Two chromosome segment substitution lines, I178 and X178, which show significant differences in seed longevity, were subjected to transcriptome sequencing before and after five days of accelerated aging (AA) treatments. Compared to the non-aging treatment, 286 and 220 differentially expressed genes (DEGs) were identified after 5 days of aging treatment in I178 and X178, respectively. Of these DEGs, 98 were detected in both I178 and X178, which were enriched in Gene Ontology (GO) terms of the cellular component of the nuclear part, intracellular part, organelle and membrane. Only 86 commonly downregulated genes were enriched in GO terms of the carbohydrate derivative catabolic process. Additionally, transcriptome analysis of alternative splicing (AS) events in I178 and X178 showed that 63.6% of transcript isoforms occurred AS in all samples, and only 1.6% of transcript isoforms contained 169 genes that exhibited aging-specific AS arising after aging treatment. Combined with the reported QTL mapping result, 7 DEGs exhibited AS after aging treatment, and 13 DEGs in mapping interval were potential candidates that were directly or indirectly related to seed longevity.

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical-to-mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi-open and open states. We observed spontaneous structural transition from closed to open state in the ADP-bound system and partial closure of MalK dimer from the semi-open state in the ATP-bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP- and ADP-bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. This article is protected by copyright. All rights reserved.

Milk fat globule membrane (MFGM) proteins have important biological functions, such as anticancer properties, preventing Helicobacter pylori infection, and immune functions. However, the full range of colostrum MFGM proteins has not completely identified. In this study, extracted colostrum MFGM proteins were identified and quantified using the isobaric tags for relative and absolute quantitation proteomic method. A total of 411 human and bovine colostrum MFGM proteins were identified and quantified. These MFGM proteins were classified according to gene ontology annotation. The major biological processes involved were response to stimulus, representing 19 % of total proteins; establishment of localization (18 %); and cellular component organization (17 %). The most prevalent cellular components were intracellular representing 19 %, and intracellular part represented 18 %. The most prevalent molecular function was protein binding (46 %). Hierarchical clustering was used for the quantitative analysis of colostrum MFGM levels in humans and bovines. Furthermore, one-factor analysis of variance identified 26 differentially expressed proteins. The differentially expressed proteins were involved in three major pathways: calcium signaling pathway, phagosome, and FcγR-mediated phagocytosis. This study examined the expression changes and functional differences of colostrum MFGM proteins between humans and bovines to provide useful information and potential research directions for the dairy industry.

Muraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure–activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field.

Acquired mutations in the intracellular part of CSF3R (colony stimulating factor 3 receptor, granulocyte) have been detected with a frequency of more than 30% in severe congenital neutropenia (CN) patients. CN is a preleukemic syndrome with a risk of approximately 20% to develop leukemia. More than 80% of CN patients who develop acute myeloid leukemia or myelodysplastic syndrome reveal CSF3R mutations, suggesting that they are involved in leukemogenesis. Using deep-sequencing technology, we were able to analyze large cohorts of CN patients for the entire CSF3R sequence as well as to identify cell clones carrying mutations in the intracellular part of CSF3R with very high sensitivity. Acquisition of CSF3R mutations is a CN-specific phenomenon and is associated with inherited mutations causing CN or cyclic neutropenia, such as ELANE mutations. In the group of CN patients negative for known germ-line mutations, biallelic CSF3R mutations were identified. In addition, CSF3R mutant clones are highly dynamic and may disappear and reappear during continuous granulocyte colony-stimulating factor (G-CSF) therapy. The time between the first detection of CSF3R mutations and overt leukemia is highly variable.

Despite the biological significance of insulin signaling, the molecular mechanisms of activation of the insulin receptor (IR) and other proteins from its family remain elusive. Current hypothesis on signal transduction suggests ligand-triggered structural changes in the extracellular domain followed by transmembrane (TM) domains closure and dimerization leading to trans-autophosphorylation and kinase activity in intracellular segments of the receptor. Using NMR spectroscopy, we detected dimerization of isolated TM segments of IR in different membrane-mimicking environments and observed multiple signals of NH groups of protein backbone possibly corresponding to several dimer conformations. Taking available experimental data as constraints, several atomistic models of dimeric TM domains of IR and insulin-like growth factor 1 (IGF-1R) receptors were elaborated. Molecular dynamics simulations of IR ectodomain revealed noticeable collective movements potentially responsible for closure of the C-termini of FnIII-3 domains and spatial approaching of TM helices upon insulin-induced receptor activation. In addition, we demonstrated that the intracellular part of the receptor does not impose restrictions on the positioning of TM helices in the membrane. Finally, we used two independent structure prediction methods to generate a series of dimer conformations followed by their cluster analysis and dimerization free energy estimation to select the best dimer models. Biological relevance of the later was further tested via comparison of the hydrophobic organization of TM helices for both wild-type receptors and their mutants. Based on these data, the ability of several segments from other proteins to functionally replace IR and/or IGF-1R TM domains was explained.

Protein and peptide hormones cannot pass through the cell membrane. Therefore, in order to be able to affect cellular processes, there should be a way to transfer their message through the cell membrane. This is generally performed by transmembrane receptors, which have an extracellular part able to recognize the hormone, and an intracellular part which transfers the activity to intracellular signal transduction pathways. Binding of the hormone to members of the G-protein coupled receptor (GPCR) family leads to dissociation of GDP from G-proteins, enabling association of GTP with a G-protein a-subunit leading to the production of second messengers. GPCRs are involved in signal transduction of a large number of hormones, such as biogenic amines, and peptide and protein hormones. Ligand binding to kinase-linked receptors usually leads to multimerization of receptors, followed by activation of the receptors by mutual phosphorylation at serine/threonine or tyrosine residues and subsequent phosphorylation of intracellular proteins which can transfer the hormonal message to the nucleus or other intracellular targets. Examples of this group are receptors for insulin, growth hormone (GH) and members of the TGFΒ-family of growth factors. After activation of the receptor at the cell membrane, subsequent receptor internalization can enhance the action of the hormone by continuation of receptor action after incorporation in endosomes, or alternatively terminate signaling by lysosomal degradation of the hormone-receptor complex. Finally, activity of the receptors can be affected by the action of synthetic or endogenous agonists and antagonists.

Thyroid-stimulating hormone receptor (TSHR) is a typical membrane receptor with 7-transmembrane helix domain (7TMR), coupled to the G protein. The mature receptor, present in the cell membrane, is composed of the A subunit comprising a large extracellular domain, and the B subunit, which consists of a short extracellular fragment anchored in the cell membrane and an intracellular part. The TSH receptor is subject to numerous post-translational modifications that determine its final structure and significantly affect its activity. One of them is glycosylation. TSHR is abundantly N-glycosylated, due to the presence of six N-glycosylation sites in the extracellular domain (Asn77, Asn99, Asn113, Asn177, Asn198, Asn302), mostly evolutionarily conserved. N-glycans constitute 30-40% of the receptor molecular weight. The glycans are necessary for the receptor trafficking to the plasma membrane and binding of TSH to the receptor. Fucosylated and sialylated N-oligosaccharides were found on TSHR molecules. The increased sialylation of TSHR glycans correlates positively with the receptor binding ability and prolongs the time of receptor incorporation into the cell membrane. TSHR is the main autoantigen in Graves' disease (GD), one of the thyroid autoimmune diseases. One hypothesis assumes that the higher N-glycosylation of THSR in human compared to animals influences the breaking of autotolerance and GD development. N-oligosaccharides are the important part of THSR molecule, necessary for the proper functioning of receptors and probably involved in thyroid autoimmunity in GD.

Background: Diabetes Mellitus (DM), is a metabolic disorder characterized by high blood glucose levels. The main types of diabetes mellitus are Diabetes mellitus type I, Diabetes mellitus type II, gestational diabetes and Diabetes of other etiology. Diabetes type II, the Non Insulin Dependent Type (NIDDM) is the most common type, characterized by the impairment in activation of the intracellular mechanism leading to the insertion and usage of glucose after interaction of insulin with its receptor, known as insulin resistance. Although, a number of drugs have been developed for the treatment of diabetes type II, their ability to reduce blood glucose levels is limited, while several side effects are also observed. Furthermore, none of the market drugs targets the enhancement of the action of the intracellular part of insulin receptor or recuperation of the glucose transport mechanism in GLUT4 dependent cells. The Protein Tyrosine Phosphatase (PTP1b) is the main enzyme involved in insulin receptor desensitization and has become a drug target for the treatment of Diabetes type II. Several PTP1b inhibitors have already been found, interacting with the binding site of the enzyme, surrounding the catalytic amino acid Cys215 and the neighboring area or with the allosteric site of the enzyme, placed at a distance of 20 Å from the active site, around Phe280. However, the research continues for finding more potent inhibitors with increased cell permeability and specificity. Objective: The aim of this review is to show the attempts made in developing of Protein Tyrosine Phosphatase (PTP1b) inhibitors with high potency, selectivity and bioavailability and to sum up the indications for favorable structural characteristics of effective PTP1b inhibitors. Methods: The methods used include a literature survey and the use of Protein Structure Databanks such as PuBMed Structure and RCSB and the tools they provide. Conclusion: The research for finding PTP1b inhibitors started with the design of molecules mimicking the Tyrosine substrate of the enzyme. The study revealed that an aromatic ring connected to a polar group, which preferably enables hydrogen bond formation, is the minimum requirement for small inhibitors binding to the active site surrounding Cys215. Molecules bearing two hydrogen bond donor/acceptor (Hb d/a) groups at a distance of 8.5-11.5 Å may form more stable complexes, interacting simultaneously with a secondary area A2. Longer molecules with two Hb d/a groups at a distance of 17 Å or 19 Å may enable additional interactions with secondary sites (B and C) that confer stability as well as specificity. An aromatic ring linked to polar or Hb d/a moieties is also required for allosteric inhibitors. A lower distance between Hb d/a moieties, around 7.5 Å may favor allosteric interaction. Permanent inhibition of the enzyme by oxidation of the catalytic Cys215 has also been referred. Moreover, covalent modification of Cys121, placed near but not inside the catalytic pocket has been associated with permanent inhibition of the enzyme.

Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1′241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies. Electronic supplementary material The online version of this article (10.1007/s00251-018-01099-1) contains supplementary material, which is available to authorized users.

Ethnopharmacological relevance Bai-Hu-Tang (BHT) has been traditionally used to clear heat and engender fluids. Aim of the study To reveal the alteration of differentially expressed genes (DEGs) between lipopolysaccharide (LPS) febrile syndrome in rabbits and treatment with BHT which is a classical anti-febrile formula in traditional Chinese medicine. Materials and methods Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT was gavaged to another group of febrile rabbits. After sacrifice of animals, total RNA of liver tissue was isolated, processed, and hybridized to rabbit cDNA microarrays obtained from Agilent Co. The data of DEGs were obtained by lazer scanning and analyzed with Cluster program 3.0. Then bioinformatic analysis of DEGs was conducted through gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, expression levels of four relative genes were detected by quantitative real time ployenzyme chain reaction (qRT-PCR) to validate the accuracy of microarrays. Result The results demonstrated that genes expression pattern could be clustered into three groups significantly, and there were 606 up-regulated genes and 859 down-regulated genes in the model group, and 106 up-regulated genes and 429 down-regulated genes in BHT treated group. There were 286 DEGs existed as the common in two experimental groups. Enrichment analysis of GO annotations indicated that DEGs in model and BHT treated animals mainly referred catalytic activity and oxidoreductase activity for metabolic processes located in the membrane system at intracellular part, and binding activities increased significantly in treatment with BHT. Enrichment of KEGG analysis showed that the pathways of phagosome and protein processing in endoplasmic reticulum contained the most altered genes in the LPS group, but the percentage of phagosome pathway almost doubled in BHT group. Most DEGs involved in the LPS signal recognition system was up-regulated in LPS group, but partly decreased in BHT group. RT-PCR results of eight relative genes were consistent with the results of microarrays. Conclusion DEGs of LPS febrile syndrome mainly involved oxidoreductase and catalytic activity of the metabolic processes, and pathways of processing protein for pyrotoxin recognition; BHT mostly regulated the DEGs in the phagosome pathway to clear LPS in the liver, and partly interfered with gene expression in LPS recognition system. The study provided an important pioneering result on gene expression profiling research, and will facilitate the clinical care or further studies of the formula.

The Δ-distance maps can detect local remodeling that is difficult to accurately determine using superimpositions. Transmembrane segments (TMSs) 11 in both LacY and XylE of the major facilitator superfamily uniquely contribute the greatest amount of mobile surface area in the outward-occluded state and undergo analogous movements. The intracellular part of TMS11 moves away from the C-terminal domain and into the substrate cavity during the conformational change from the outward-occluded to the inward-occluded state. A difference was noted between LacY and XylE when they assumed the inward open state after releasing a substrate to the inside in which TMS11 of LacY moved further into the substrate release space, whereas in XylE, TMS11 slightly retracted into the C-terminal domain. Independent movement of the N-terminal half of TMS11 suggests that it is flexible in the middle. Repeat-swapped homology modeling was used to discover that a loop connecting TMSs 10 and 11 in LacY probably moves during the transition between the unavailable outward-open state and the outward-occluded state. TMSs 11 and the other elements displaying a notable domain-independent movement colocalize with the interdomain linker, suggesting that these elements could drive the alternating access movement between the domain halves. Preliminary evidence indicates that analogous movements occur in other members of the major facilitator superfamily.

Cell adhesion molecules (CAMs) are glycoproteins uniformly present on the cell surface. They are responsible for cell-to-cell and cell-to-extracellular environment interactions such as adhesion, growth, migration. They are expressed on the surface of the immune cells and take part in various inflammatory events. CAMs are divided into four groups: immunoglobulin superfamily, selectins, cadherins and integrins. They all share the same structure: CAMs are composed of three fragments – extracellular domain responsible for receiving signals from the extracellular space, transmembrane fragment and intracellular part which interacts with cytoskeleton. CAMs are present on cell-membrane in their native original cell-bound forms which may be released to body fluids as circulating or soluble forms. The soluble forms likely have the same potential of binding to their natural counterparts as the native forms. It is generally admitted that increased concentration of the soluble form reflects its overexpression on the cell surface. We hypothesized that the soluble forms could be a sort of truncated false saturators for their natural counterpart which finally reduce the triggered inflammatory cascade. They should be then considered a self-limiting negative feed-back mechanism of the CAM-activated inflammatory pathway. We present current data on the possible involvement of CAMS in various human pathologies with special emphasis on the diseases of the nervous system.

© 2017 American Chemical Society. The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R2 > 0.97, p < 0.03) between the surface hydrophilicity index and α-helix percentages in the secondary structure indicated that the water affinity relied more on the spatial distribution of hydrophilic functional groups rather than the content. Destructing the secondary structure represented by the α-helix and stretching the polypeptide aggregation in the water phase through disulfide bond removal might be the key to eliminating the inhibitory effects of extracellular proteins on the interstitial water removal from WAS.

The production of biomass by micro-organisms is of great interest in more and more industrial fields. Their culture in fedbatch bioreactors is largely used and the optimization of the influent flow rate is a major challenge to set the microorganisms in the highest performance conditions. Usually, sets of experiments are achieved to compute macroscopic models expressing the observed growth as a function of the concentrations of glucose, cell density, and any kind of external parameters (pH, temperature, etc.). In contrast, this paper relies on an intracellular model of a bacteria, which is used to determine the optimal profile of influent flow rate in a fedbatch bioreactor to produce as much biomass as possible. Compared to macroscopic models, the additional elements of this model better describe biological phenomena. The intracellular part of the model also provides a clear interpretation of the obtained optimal influent profile.

It has been reported that circular RNA (circRNA) is associated with human cancer. However, few studies have been reported in active pulmonary tuberculosis (APTB). The global circRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of APTB patients (n=5) and health controls (HC) (n=5) by using high-throughput sequencing. According to the systematical bioinformatics analysis, the basic content of circRNAs and their fold changes in the two groups were calculated. We selected 6 significant differentially expressed circRNAs, hsa_circ_0005836, hsa_circ_0009128, hsa_circ_0003519, hsa_circ_0023956, hsa_circ_0078768, and hsa_circ_0088452 and validated the expression in PBMCs from APTB (n=10) and HC (n=10) by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Further, the verification of these specific circRNAs (hsa_circ_0005836 and hsa_circ_0009128) between APTB (n=34) and HC (n=30) in PBMCs was also conducted by qRT-PCRs. The RNA-seq data showed the significant differential expression of the 523 circRNAs between the APTB and HC groups (199 circRNAs were significantly up-regulated and 324 circRNAs were down-regulated). Hsa_circ_0005836 and hsa_circ_0009128 expression was significantly down-regulated in the PBMCs of APTB (P

Background: PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. Methods: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. Findings: We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. Interpretation: We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. Fund: CR UK, MRC and AstaZeneca. Keywords: Disease-linked variants, Phospholipase C gamma, Structure, Mechanism

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. VEGF receptor 2 (VEGFR 2) is the major receptor involved in vasculogenesis and angiogenesis and regulates endothelial cell survival migration and mitogenesis. Ligand mediated receptor dimerization instigates transmembrane signaling thereby promoting activation of the intracellular kinase domain. The intracellular part of the receptor comprises the juxtamembrane domain the catalytic kinase domain the kinase insert domain (KID) and the carboxy terminal domain (CD). Here we show that the CD inhibits VEGFR 2 activity in the absence of ligand whereas the KID particularly a tyrosine residue in this domain (Y951) is indispensable for downstream signaling by the activated kinase. Because of the lack of crystallographic data for the complete kinase domain we applied size exclusion chromatography multiangle laser scattering analytical ultracentrifugation and small angle X ray scattering to build and functionally validate structural models. Our data show substantial conformational changes of the kinase when it is switched from the inactive unphosphorylated state to the active phosphorylated state. Finally we structurally characterized recombinantly produced protein complexes between VEGFR 2 and T cell specific adapter protein a molecule involved in downstream signaling by VEGFR 2. Manni S. Kisko K. Schleier T. Missimer J. Ballmer Hofer K. Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation.

The structural behavior of the ligand-free form of adenosine receptor A2A in an explicit membrane-mimicking environment was investigated by molecular dynamics (MD) simulation. Principal components analysis was applied to the series of MD snapshots and to a collection of X-ray structures of the A2A receptor. The resulting charts revealed a correlation in the dynamic behavior of the receptor observed in the MD trajectories and in the experimental dataset. The most pronounced structural dynamics in the A2A receptor were observed in the intracellular part: TM 5 and 6 with the connecting loop, just as generally recognized in crystallographic studies and attributed to receptor activation. There are grounds for supposing that this pattern of intramolecular motions ensues directly from the spatial architecture (fold) of the A2A receptor.

The in vitro effects of oxo-bridged binuclear gold(III) complexes, i.e., [(bipy2Me)(2)Au-2(mu-O)(2)][PF6](2) (Auoxo6), Au-2[(bipydmb-H)(2)(mu-O)][PF6] (Au(2)bipyC) and [Au-2(phen(2Me))(2)(mu-O)(2)](PF6)(2) (Au(2)phen) on Na/K-ATPase, purified from the porcine cerebral cortex, were investigated. All three studied gold complexes inhibited the enzyme activity in a concentration-dependent manner achieving IC50 values in the low micromolar range. Kinetic analysis suggested an uncompetitive mode of inhibition for Auoxo6 and Au(2)bipyC, and a mixed type one for Au(2)phen. Docking studies indicated that the inhibitory actions of all tested complexes are related to E2-P enzyme conformation binding to ion channel and intracellular part between N and P sub-domain. In addition, Au(2)phen was able to inhibit the enzyme by interacting with its extracellular part as well. Toxic effects of the gold(III) complexes were evaluated in vitro by following lactate dehydrogenase activity in rat brain synaptosomes and incidence of micronuclei and cytokinesis-block proliferation index in cultivated human lymphocytes. All investigated complexes turned out to induce cytogenetic damage consisting of a significant decrease in cell proliferation and an increase in micronuclei in a dose-dependent manner. On the other hand, lactate dehydrogenase activity, an indicator of membrane integrity/viability, was not affected by Auoxo6 and Au(2)bipyC, while Au(2)phen slightly modified its activity.

Histamine H4 receptor (H4R), a member of histamine receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in histamine-induced chemotaxis in mast cells and eosinophils. Recently, the crystal structure of human histamine H1 receptor (H1R) was reported, which facilitates structure-based drug discovery of histamine receptor significantly. In the current work, the homology models of H4R and H3R are first constructed based on the crystal structure of H1R. Clobenpropit is then docked into the binding pocket of H4R and two different binding modes can be identified. In order to select a reasonable binding mode, several other ligands including agonists and antagonists are docked into H4R, and the results reveal that all ligands share one preferable binding mode: the protonated NH tightly interacts with Asp(3.32) and the imidazole NH interacts with Glu(5.46). By comparing H3R and H4R, we find that Glu(5.20) and Thr(6.55) in H4R involve in the selectivity of H4R. Then, we perform molecular dynamics (MD) simulations for H4R in complex with its compounds. MD results indicate that the preferable docking mode is more stable. Finally, we dock agonist histamine into H1R and H4R, and then perform 20ns MD simulations for the complexes. H1R or H4R bound with histamine show strong conformational changes from TM5, TM6 and TM7, outward movement of intracellular part of TM6, and conformational change of Tyr(7.53), which is consistent with the recent crystal structures of active GPCRs. Our results reveal the mechanism of selectivity and activation for H4R, which is important for developing selective antagonists and agonists for H4R.

In order to clarify the relation between ReKω and the electrical property of each intracellular part, we loaded temperature stress on the cells. In this experiment, the electrical property of the intracellular part means the cytoplasm electrical conductivity (σ3) and the cell membrane capacitance (Cm). ReKω of the cells was measured using dielectrophoresis (DEP) levitation, and the electrical property of each intracellular part was analyzed from the measurement result in applying a single shell model. In this experiment, mouse hybridoma 3-2H3 cells were used as the sample cells. Temperature stress was loaded on the sample cell at 5% CO2 in a CO2 incubator. The result of the frequency characteristic of ReKω using DEP levitation showed that both ReKω and the crossover frequency of the single cell in the high frequency region decreased with increasing temperature. Furthermore, both the cytoplasm electrical conductivity and the cell membrane capacitance decreased with increasing temperature stress.

International audience; Three major discoveries have recently profoundly modified our perception of the viral world: molecular ecologists have shown that viral particles are more abundant than cells in natural environments; structural biologists have shown that some viruses from the three domains of life, Bacteria, Eukarya and Archaea, are evolutionarily related, and microbiologists have discovered giant viruses that rival with cells in terms of size and gene content. I discuss here the scientific and philosophical impact of these discoveries on the debates over the definition, nature (living or not), and origin of viruses. I suggest that viruses have often been considered non-living, because they are traditionally assimilated to their virions. However, the term virus describes a biological process and should integrate all aspects of the viral reproduction cycle. It is especially important to focus on the intracellular part of this cycle, the virocell, when viral information is actively expressed and reproduced, allowing the emergence of new viral genes. The virocell concept theoretically removes roadblocks that prevent defining viruses as living organisms. However, defining a "living organism" remains challenging, as indicated by the case of organelles that evolved from intracellular bacteria. To bypass this problem, I suggest considering that all biological entities that actively participate in the process of life are living.

Exosomes are membranous vesicles found in biological fluids with important functions. However, milk-derived exosome proteins from humans and bovines have not been studied in detail. The advanced iTRAQ proteomic approach was used to analyze milk-derived exosomes in human and bovine colostrum and mature milk samples. A total of 920 milk exosome proteins were identified and quantified. Among these, 575 differentially expressed exosome proteins (P

In order to apply structure-based drug design techniques to G protein-coupled receptor complexes, it is essential to model their 3D structure and to identify regions that are suitable for selective drug binding. For this purpose, we have developed and tested a multi-component protocol to model the inactive conformation of the dopamine D2 receptor dimer, suitable for interaction with homobivalent antagonists. Our approach was based on protein–protein docking, applying the Rosetta software to obtain populations of dimers as present in membranes with all the main possible interfaces. Consensus scoring based on the values and frequencies of best interfaces regarding four scoring parameters, Rosetta interface score, interface area, free energy of binding and energy of hydrogen bond interactions indicated that the best scored dimer model possesses a TM4–TM5–TM7–TM1 interface, which is in agreement with experimental data. This model was used to study interactions of the previously published dopamine D2 receptor homobivalent antagonists based on clozapine,1,4-disubstituted aromatic piperidines/piperazines and arylamidoalkyl substituted phenylpiperazine pharmacophores. It was found that the homobivalent antagonists stabilize the receptor-inactive conformation by maintaining the ionic lock interaction, and change the dimer interface by disrupting a set of hydrogen bonds and maintaining water- and ligand-mediated hydrogen bonds in the extracellular and intracellular part of the interface. Graphical Abstract Structure of the final model of the dopamine D2 receptor homodimer, indicating the distancebetween Tyr37 and Tyr 5.42 in the apo form (left) and in the complex with the ligand (right). Electronic supplementary material The online version of this article (doi:10.1007/s00894-016-3065-2) contains supplementary material, which is available to authorized users.

International audience; Aims: Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism.Methods and results: We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146.Conclusions: These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Muraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure-activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field.

The recently discovered protein, leptin, is a 16 kD protein consisting of 146 amino acids which is synthesized primarily by adipose tissue and is secreted into the bloodstream after cleavage of the 21 amino acids signal peptide. Leptin impacts feed intake, the neuroendocrine-axis, metabolism and immunological processes. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. The hypothalamus appears to be the primary site of action, since leptin receptors are located within hypothalamic areas associated with control of appetite, reproduction and growth. Using herbs and essential oils depends on their antimicrobial activity. Most plants have favorable multifunctional properties, which are the specific content of bioactive components. Some authors characterize phytogenic substance such as natural substances plant origin, which leave no residues in animal products and is not necessary to keep the trade period before slaughter animals. Analyzes suggest that the structural function of the receptor exists as a dimer constructively in the plasma membrane. Each receptor dimer pair is reversibly bound to one molecule of leptin. When bound, signaling pathways are responsible for beginning the activation receptor associated Janus kinase 2 (JAK2) and tyrosine phosphorylation of two key residues in the intracellular part of receptor. The aim of our experiment was to optimize the methodology for monitoring the expression of the leptin receptor extracellular avian model. We used samples of internal organs and abdominal fat chickens that were fed spirit and also fat and organ samples from broiler chickens from the control group. In heard tissue, spleen, liver at a relatively high concentration of total cDNA in the sample length leptin receptor extracellular fragment located in the expected quantities.

D(3) receptor, a member of dopamine (DA) D(2)-like receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in neuropsychiatric disorders. Recently, the crystal structure of human dopamine D3 receptor was reported, which facilitates structure-based drug discovery of D3R significantly. We dock D3R-selective compounds into the crystal structure of D3R and homology structure of D2R. Then we perform 20 ns molecular dynamics (MD) of the receptor with selective compounds bound in explicit lipid and water. Our docking and MD results indicate the important residues related to the selectivity of D3R. Specifically, residue Thr(7.39) in D3R may contribute to the high selectivity of R-22 with D3R. Meanwhile, the 4-carbon linker and phenylpiperazine of R-22 improve the binding affinity and the selectivity with D3R. We also dock the agonists, including dopamine, into D3R and perform MD. Our molecular dynamics results of D3R with agonist bound show strong conformational changes from TM5, TM6, and TM7, outward movement of intracellular part of TM6, fluctuation of "ionic lock" motif and conformational change of Tyr(7.53), which is consistent with recent crystal structures of active GPCRs and illustrates the dynamical process during activation. Our results reveal the mechanism of selectivity and activation for D3R, which is important for developing high selective antagonists and agonists for D3R.