scholarly journals Applications of the In Vitro Virus (IVV) Method for Various Protein Functional Analyses

10.5772/55483 ◽  
2013 ◽  
Author(s):  
Noriko Tabata ◽  
Kenichi Horisawa ◽  
Hiroshi Yanagaw
Keyword(s):  
Functional Analyses

scholarly journals Functional analyses of phosphatidylserine/PI(4)P exchangers with diverse lipid species and membrane contexts set unanticipated rules on lipid transfer

2021 ◽  
Author(s):  
Souade Ikhlef ◽  
Nicolas-Frédéric Lipp ◽  
Vanessa Delfosse ◽  
Nicolas Fuggetta ◽  
William Bourguet ◽  
...  
Keyword(s):  
Lipid Transfer ◽  
Oxysterol Binding Protein ◽  
Lipid Exchange ◽  
Exchange Processes ◽  
Lipid Species ◽  
Acyl Chains ◽  
Phosphatidylinositol 4 ◽  
Functional Analyses ◽  
Related Proteins

Several members of the oxysterol-binding protein-related proteins (ORPs)/oxysterol-binding homology (Osh) family exchange phosphatidylserine (PS) and phosphatidylinositol 4-phosphate (PI(4)P) at the endoplasmic reticulum/plasma membrane (PM) interface. It is unclear whether they preferentially exchange PS and PI(4)P with specific acyl chains to tune the features of the PM, whether they use phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) instead of PI(4)P for exchange processes and whether their activity is influenced by the association of PS with sterol in the PM. Here, we measured in vitro how the yeast Osh6p and human ORP8 transported diverse PS and PI(4)P subspecies, including major cellular species, between membranes. We established how their activity is impacted by the length and unsaturation degree of these lipids. Surprisingly, the speed at which they individually transfer these ligands inversely depends on their affinity for them. To be fast, the transfer of high-affinity ligands requires an exchange with a counterligand of equivalent affinity. Besides, we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for intracellular lipid exchange, as they have a low affinity for this lipid compared to PI(4)P, and do not transfer more PS into sterol-rich membranes. This study provides insights into PS/PI(4)P exchangers and sets unanticipated rules on how the activity of lipid transfer proteins relates to their affinity for ligands.

scholarly journals Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

2009 ◽  
Vol 106 (37) ◽  
pp. 15616-15621 ◽  
Author(s):  
Masataka Umitsu ◽  
Hiroshi Nishimasu ◽  
Akiko Noma ◽  
Tsutomu Suzuki ◽  
Ryuichiro Ishitani ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Binding Pocket ◽  
Structural Basis ◽  
Binding Modes ◽  
Substrate Binding Pocket ◽  
Methyl Group ◽  
Group Transfer ◽  
Canonical Class ◽  
Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

scholarly journals Allelic variation contributes to bacterial host specificity

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Min Yue ◽  
Xiangan Han ◽  
Leon De Masi ◽  
Chunhong Zhu ◽  
Xun Ma ◽  
...  
Keyword(s):  
Allelic Variation ◽  
Multinomial Logistic Regression ◽  
Gene Gain ◽  
Nucleotide Polymorphisms ◽  
Genome Wide ◽  
Serovar Typhimurium ◽  
Functional Analyses ◽  
High Degree

Abstract Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

scholarly journals The GATA3 X308_Splice breast cancer mutation is a hormone context-dependent oncogenic driver

Oncogene ◽  
2020 ◽  
Vol 39 (32) ◽  
pp. 5455-5467
Author(s):  
Natascha Hruschka ◽  
Mark Kalisz ◽  
Maria Subijana ◽  
Osvaldo Graña-Castro ◽  
Francisco Del Cano-Ochoa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Progesterone Receptors ◽  
Transcriptional Response ◽  
Molecular Data ◽  
Fine Tuning ◽  
Driver Mutations ◽  
Breast Cancer Patients ◽  
Oncogenic Driver ◽  
Functional Analyses

Abstract As the catalog of oncogenic driver mutations is expanding, it becomes clear that alterations in a given gene might have different functions and should not be lumped into one class. The transcription factor GATA3 is a paradigm of this. We investigated the functions of the most common GATA3 mutation (X308_Splice) and five additional mutations, which converge into a neoprotein that we called “neoGATA3,” associated with excellent prognosis in patients. Analysis of available molecular data from >3000 breast cancer patients revealed a dysregulation of the ER-dependent transcriptional response in tumors carrying neoGATA3-generating mutations. Mechanistic studies in vitro showed that neoGATA3 interferes with the transcriptional programs controlled by estrogen and progesterone receptors, without fully abrogating them. ChIP-Seq analysis indicated that ER binding is reduced in neoGATA3-expressing cells, especially at distal regions, suggesting that neoGATA3 interferes with the fine tuning of ER-dependent gene expression. This has opposite outputs in distinct hormonal context, having pro- or anti-proliferative effects, depending on the estrogen/progesterone ratio. Our data call for functional analyses of putative cancer drivers to guide clinical application.

scholarly journals Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia—A Retrospective Analysis in 337 Patients

2020 ◽  
Vol 21 (8) ◽  
pp. 3025 ◽  
Author(s):  
Klaus Geissler ◽  
Eva Jäger ◽  
Agnes Barna ◽  
Michael Gurbisz ◽  
Temeida Graf ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Treatment Strategies ◽  
Colony Growth ◽  
Chronic Myelomonocytic Leukemia ◽  
Peripheral Blood Mononuclear ◽  
Ras Pathway ◽  
Increased Risk ◽  
Functional Analyses ◽  
Myelomonocytic Leukemia

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

scholarly journals From the Field to the Pot: Phytochemical and Functional Analyses of Calendula officinalis L. Flower for Incorporation in an Organic Yogurt

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 559 ◽  
Author(s):  
Bragueto Escher ◽  
Cardoso Borges ◽  
Sousa Santos ◽  
Mendanha Cruz ◽  
Boscacci Marques ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Biological Activities ◽  
Total Phenolic ◽  
Antioxidant Compounds ◽  
Calendula Officinalis ◽  
Antioxidant Power ◽  
Ferric Reducing Antioxidant Power ◽  
Functional Analyses ◽  
Calendula Officinalis L

Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of water and ethyl alcohol, and the lyophilized extract with higher content of antioxidant compounds was incorporated into an organic yogurt. Results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolic content (TPC), flavonoids, and antioxidant activity (ferric reducing antioxidant power (FRAP), total reducing capacity (TRC), and Cu2+/Fe2+ chelating ability). Phenolic acids and flavonoids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract (LME) also inhibited 12% of Wistar rat’s brain lipid oxidation in vitro, inhibited α-amylase, and α-glucosidase activities, but showed no cytotoxicity towards cancerous cells (HCT8 and A549). However, marigold flower extract protected human erythrocytes against mechanical stress. When added into an organic yogurt model (0 to 1.5%), LME increased TPC and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and TRC), and the sensory analysis showed that the organic yogurt had an acceptance of 80.4%. Our results show that the use of LME may be a technological strategy to increase the content of bioactive compounds in yogurts.

scholarly journals Vascular Transdifferentiation in the CNS: A Focus on Neural and Glioblastoma Stem-Like Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Sophie Guelfi ◽  
Hugues Duffau ◽  
Luc Bauchet ◽  
Bernard Rothhut ◽  
Jean-Philippe Hugnot
Keyword(s):  
Stem Cells ◽  
Brain Tumors ◽  
Neural Stem Cells ◽  
Vascular Cells ◽  
Therapeutic Approaches ◽  
Tumorigenic Potential ◽  
New Directions ◽  
Functional Analyses

Glioblastomas are devastating and extensively vascularized brain tumors from which glioblastoma stem-like cells (GSCs) have been isolated by many groups. These cells have a high tumorigenic potential and the capacity to generate heterogeneous phenotypes. There is growing evidence to support the possibility that these cells are derived from the accumulation of mutations in adult neural stem cells (NSCs) as well as in oligodendrocyte progenitors. It was recently reported that GSCs could transdifferentiate into endothelial-like and pericyte-like cells bothin vitroandin vivo, notably under the influence of Notch and TGFβsignaling pathways. Vascular cells derived from GBM cells were also observed directly in patient samples. These results could lead to new directions for designing original therapeutic approaches against GBM neovascularization but this specific reprogramming requires further molecular investigations. Transdifferentiation of nontumoral neural stem cells into vascular cells has also been described and conversely vascular cells may generate neural stem cells. In this review, we present and discuss these recent data. As some of them appear controversial, further validation will be needed using new technical approaches such as high throughput profiling and functional analyses to avoid experimental pitfalls and misinterpretations.

scholarly journals An immunologically anomalous but considerably bioactive GH produced by a novel GH1 mutation (p.D116E)

2009 ◽  
Vol 161 (2) ◽  
pp. 301-306 ◽  
Author(s):  
Sumito Dateki ◽  
Kazuko Hizukuri ◽  
Toshiaki Tanaka ◽  
Noriyuki Katsumata ◽  
Paravee Katavetin ◽  
...  
Keyword(s):  
Rare Condition ◽  
Normal Range ◽  
Functional Studies ◽  
Provocation Tests ◽  
Old Japanese ◽  
Igf Binding ◽  
Functional Analyses ◽  
Igf Binding Protein

ContexAlthough GH values measured by an immunoassay usually reflect GH bioactivities, discrepancy exists between immunoactivity and bioactivity in a rare condition known as ‘bioinactive GH’.ObjectiveTo report an immunologically anomalous but considerably bioactive GH.MethodsWe performed mutational and functional analyses of GH1 in a 7-year-old Japanese boy with short stature (−3.0 s.d.) in whom serum GH values measured with a Tosoh immunoassay kit were all undetectable in three provocation tests, whereas urine GH value measured with a Hitachi immunoassay kit was within the normal range. Serum IGF-1 was at a low-normal range, and IGF-binding protein-3 was below the normal range.ResultsMutation analysis showed a missense GH produced by a novel GH1 mutation (p.D116E) of paternal origin and a frameshift mutation (p.Q68fsX106) of maternal origin. Genotype–phenotype correlations in this family and in vitro functional studies indicated that the p.D116E-GH was immeasurable with the Tosoh kit but was measurable, though maybe not precise, with a Daiichi kit, and had a reduced in vivo bioactivity. The p.Q68fsX106 yielded no GH protein.ConclusionsThe results suggest that the p.D116E affects the GH epitope primarily recognized by the Tosoh kit but not by the Hitachi or the Daiichi kits, thereby producing an immunologically anomalous but considerably bioactive GH. The presence of such a hormone discordant for immunoactivity and bioactivity should be kept in mind, to allow for an appropriate assessment of endocrine data.

scholarly journals Inhibition of β-Catenin Activity Abolishes LKB1 Loss-Driven Pancreatic Cystadenoma in Mice

2021 ◽  
Vol 22 (9) ◽  
pp. 4649
Author(s):  
Mei-Jen Hsieh ◽  
Ching-Chieh Weng ◽  
Yu-Chun Lin ◽  
Chia-Chen Wu ◽  
Li-Tzong Chen ◽  
...  
Keyword(s):  
Normal Development ◽  
Genetic Alterations ◽  
Epithelial Polarity ◽  
The Past ◽  
Upstream Kinase ◽  
Pancreatic Cystadenoma ◽  
Peutz Jeghers Syndrome ◽  
Functional Analyses

Pancreatic cancer (PC) is the seventh leading cause of cancer death worldwide, and remains one of our most recalcitrant and dismal diseases. In contrast to many other malignancies, there has not been a significant improvement in patient survival over the past decade. Despite advances in our understanding of the genetic alterations associated with this disease, an incomplete understanding of the underlying biology and lack of suitable animal models have hampered efforts to develop more effective therapies. LKB1 is a tumor suppressor that functions as a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), which is an important mediator in the regulation of cell growth and epithelial polarity pathways. LKB1 is mutated in a significant number of Peutz–Jeghers syndrome (PJS) patients and in a small proportion of sporadic cancers, including PC; however, little is known about how LKB1 loss contributes to PC development. Here, we report that a reduction in Wnt/β-catenin activity is associated with LKB1 tumor-suppressive properties in PC. Remarkably, in vivo functional analyses of β-catenin in the Pdx-1-Cre LKB1L/L β-cateninL/L mouse model compared to LKB1 loss-driven cystadenoma demonstrate that the loss of β-catenin impairs cystadenoma development in the pancreas of Pdx-1Cre LKB1L/L mice and dramatically restores the normal development and functions of the pancreas. This study further determined the in vivo and in vitro therapeutic efficacy of the β-catenin inhibitor FH535 in suppressing LKB1 loss-driven cystadenoma and reducing PC progression that delineates the potential roles of Wnt/β-catenin signaling in PC harboring LKB1 deficiency.

Effect of Pax3 Overexpression on the Engraftment of Mesenchymal Stem Cells in Dystrophic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1924-1924
Author(s):  
Eun Ji Gang ◽  
Radbod Darabi ◽  
Darko Bosnakovski ◽  
Zhaohui Xu ◽  
Rita Perlingeiro
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Myogenic Differentiation ◽  
Control Mscs ◽  
Poor Differentiation ◽  
Functional Analyses ◽  
Expansion Capacity ◽  
Myogenic Program ◽  
Dystrophic Mice

Abstract Mesenchymal stem cells (MSCs) residing within the bone marrow (BM) not only support hematopoiesis, but also differentiate into multiple lineages, including fat, bone, and cartilage. Because MSCs are multipotent and have a great expansion capacity in vitro, these cells are attractive candidates for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, treatment of muscle degenerative diseases with MSCs has been hampered by the poor differentiation of MSCs into the muscle lineage. To date most in vitro methods require the presence of strong non-physiological agents, such as azacytidine. Accordingly, limited engraftment has been observed following the transplantation of BM into dystrophic mice (less than 4%). In the present study we explored the potential of Pax3, the master regulator of the embryonic myogenic program, to promote myogenic differentiation from human BM-derived MSCs. Upon genetic modification with lentiviral vectors encoding Pax3-GFP or GFP only (vector control), MSCs were evaluated for their potential to differentiate towards muscle in vitro. Real time PCR analyses revealed up-regulation of several myogenic regulatory factors in Pax3-transduced MSC cultures in various differentiation conditions, whereas control GFP-MSCs showed undetectable levels. When transplanted into cardiotoxin-injured tibialis anterior (TA) muscles of Rag2−/− γc−/− immunodeficient as well as dystrophic mice, superior engraftment was observed with Pax3-transduced MSCs (31% vs. 12% and 12% vs. 6.5%, respectively). To assess whether such levels of engraftment are therapeutically relevant, functional analyses of transplanted muscles are required. We are currently undertaking these experiments.

Sign in / Sign up

Export Citation Format

Share Document