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1.
Front Cell Dev Biol ; 12: 1329218, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38529405

RESUMO

Introduction: Mesenchymal stromal cells (MSCs) are activated upon inflammation and/or tissue damage and migrate to suppress inflammation and repair tissues. Migration is the first important step for MSCs to become functional; however, the migration potency of umbilical cord-derived MSCs (UC-MSCs) remains poorly understood. Thus, we aimed to assess the migration potency of UC-MSCs in comparison with those of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) and investigate the influence of chemotactic factors on the migration of these cells. Methods: We compared the migration potencies of UC-, BM-, and AD-MSCs toward allogeneic stimulated mononuclear cells (MNCs) in mixed lymphocyte reaction (MLR). The number of MSCs in the upper chamber that migrated toward the MLR in the lower chamber was counted using transwell migration assay. Results and discussion: UC-MSCs showed significantly faster and higher proliferation potencies and higher migration potency toward unstimulated MNCs and MLR than BM- and AD-MSCs, although the migration potencies of the three types of MSCs were comparable when cultured in the presence of fetal bovine serum. The amounts of CCL2, CCL7, and CXCL2 in the supernatants were significantly higher in UC-MSCs co-cultured with MLR than in MLR alone and in BM- and AD-MSCs co-cultured with MLR, although they did not induce the autologous migration of UC-MSCs. The amount of CCL8 was higher in BM- and AD-MSCs than in UC-MSCs, and the amount of IP-10 was higher in AD-MSCs co-cultured with MLR than in UC- and BM-MSCs. The migration of UC-MSCs toward the MLR was partially attenuated by platelet-derived growth factor, insulin-like growth factor 1, and matrix metalloproteinase inhibitors in a dose-dependent manner. Conclusion: UC-MSCs showed faster proliferation and higher migration potency toward activated or non-activated lymphocytes than BM- and AD-MSCs. The functional chemotactic factors may vary among MSCs derived from different tissue sources, although the roles of specific chemokines in the different sources of MSCs remain to be resolved.

2.
Org Lett ; 25(40): 7390-7394, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37782042

RESUMO

An intramolecular Pauson-Khand reaction with enantioenriched N-C axially chiral N-allyl-N-(2-alkynylphenyl)sulfonamide derivatives proceeded with complete chirality transfer from axial chirality (P configuration) to central chirality (R configuration), affording chiral nitrogen-containing tricyclic compounds (tetrahydrocyclopentaquinolin-2-one derivatives).

3.
Molecules ; 27(22)2022 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-36431920

RESUMO

Recently, catalytic enantioselective syntheses of N-C axially chiral compounds have been reported by many groups. Most N-C axially chiral compounds prepared through a catalytic asymmetric reaction possess carboxamide or nitrogen-containing aromatic heterocycle skeletons. On the other hand, although N-C axially chiral sulfonamide derivatives are known, their catalytic enantioselective synthesis is relatively underexplored. We found that the reaction (Tsuji-Trost allylation) of allyl acetate with secondary sulfonamides bearing a 2-arylethynyl-6-methylphenyl group on the nitrogen atom proceeds with good enantioselectivity (up to 92% ee) in the presence of (S,S)-Trost ligand-(allyl-PdCl)2 catalyst, affording rotationally stable N-C axially chiral N-allylated sulfonamides. Furthermore, the absolute stereochemistry of the major enantiomer was determined by X-ray single crystal structural analysis and the origin of the enantioselectivity was considered.


Assuntos
Paládio , Sulfonamidas , Estereoisomerismo , Paládio/química , Catálise , Sulfanilamida , Nitrogênio
4.
Int J Hematol ; 116(5): 754-769, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35908021

RESUMO

This study investigated the safety, efficacy, and immunological influence of allogeneic umbilical cord-derived mesenchymal stromal cells (IMSUT-CORD) processed in serum-free medium and cryoprotectant, for treating steroid-resistant acute graft-versus-host disease (aGVHD). In a phase I dose-escalation trial, IMSUT-CORD were infused intravenously twice weekly over two cycles with up to two additional cycles. Four patients received a dose of 1 × 106 cells/kg, while three received 2 × 106/kg. Of 76 total adverse events, fourteen associated or possibly associated adverse events included 2 cases of a hot flash, headache, and peripheral neuropathy, 1 each of upper abdominal pain, hypoxia, increased γ-GTP, somnolence, peripheral vascular pain at the injection site, thrombocytopenia, hypertension, and decreased fibrinogen. At 16 weeks after the initial IMSUT-CORD infusion, three patients showed complete response (CR), two partial response (PR), one mixed response, and one no response. The overall response rate was 71.4%, and the continuous CR/PR rate was 100% for over 28 days after CR/PR. NK cell count significantly increased and correlated with treatment response, whereas IL-12, IL-17, and IL-33 levels decreased, but did not correlate with treatment response. CCL2 and CCL11 levels increased during IMSUT-CORD therapy. IMSUT-CORD are usable in patients with steroid-resistant aGVHD (UMIN000032819: https://www.umin.ac.jp/ctr ).


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Humanos , Fibrinogênio/uso terapêutico , Doença Enxerto-Hospedeiro/terapia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Guanosina Trifosfato/uso terapêutico , Interleucina-12/uso terapêutico , Interleucina-17/uso terapêutico , Interleucina-33/uso terapêutico , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Esteroides/uso terapêutico , Cordão Umbilical
5.
Molecules ; 27(7)2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35408762

RESUMO

The halogen bond has been widely used as an important supramolecular tool in various research areas. However, there are relatively few studies on halogen bonding related to molecular chirality. 3-(2-Halophenyl)quinazoline-4-thione derivatives have stable atropisomeric structures due to the rotational restriction around an N-C single bond. In X-ray single crystal structures of the racemic and optically pure N-C axially chiral quinazoline-4-thiones, we found that different types of intermolecular halogen bonds (C=S⋯X) are formed. That is, in the racemic crystals, the intermolecular halogen bond between the ortho-halogen atom and sulfur atom was found to be oriented in a periplanar conformation toward the thiocarbonyl plane, leading to a syndiotactic zig-zag array. On the other hand, the halogen bond in the enantiomerically pure crystals was oriented orthogonally toward the thiocarbonyl plane, resulting in the formation of a homochiral dimer. These results indicate that the corresponding racemic and optically pure forms in chiral molecules are expected to display different halogen bonding properties, respectively, and should be separately studied as different chemical entities.


Assuntos
Halogênios , Tionas , Halogênios/química , Modelos Moleculares , Conformação Molecular , Quinazolinas
6.
Front Immunol ; 12: 686356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484183

RESUMO

Mesenchymal stromal cells (MSCs) are known to have immunosuppressive ability and have been used in clinical treatment of acute graft-versus-host disease, one of severe complications of the hematopoietic stem cell transplantation. However, MSCs are activated to suppress the immune system only after encountering an inflammatory stimulation. Thus, it will be ideal if MSCs are primed to be activated and ready to suppress the immune reaction before being administered. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb-Tripterygium wilfordii Hook.f. It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aimed to use TPL to prime umbilical cord-derived MSCs (TPL-primed UC-MSCs) to enter a stronger immunosuppressive status. UC-MSCs were primed with TPL, which was washed out thoroughly, and the TPL-primed UC-MSCs were resuspended in fresh medium. Although TPL inhibited the proliferation of UC-MSCs, 0.01 µM TPL for 24 h was tolerable. The surface markers of TPL-primed UC-MSCs were identical to those of non-primed UC-MSCs. TPL-primed UC-MSCs exhibited stronger anti-proliferative effect for activated CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction assay than the non-primed UC-MSCs. TPL-primed UC-MSCs promoted the expression of IDO-1 in the presence of IFN-γ, but TPL alone was not sufficient. Furthermore, TPL-primed UC-MSCs showed increased expression of PD-L1. Conclusively, upregulation of IDO-1 in the presence of IFN-γ and induction of PD-L1 enhances the immunosuppressive potency of TPL-primed UC-MSCs on the proliferation of activated T cells. Thus, TPL- primed MSCs may provide a novel immunosuppressive cell therapy.


Assuntos
Diterpenos/farmacologia , Células-Tronco Mesenquimais/fisiologia , Fenantrenos/farmacologia , Linfócitos T/citologia , Cordão Umbilical/citologia , Antígeno B7-H1/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Compostos de Epóxi/farmacologia , Humanos , Imunomodulação , Terapia de Imunossupressão , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Linfócitos T/imunologia
7.
Biotechnol J ; 16(6): e2000558, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33545746

RESUMO

Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.


Assuntos
Células-Tronco Mesenquimais , Animais , Reatores Biológicos , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Camundongos , Cordão Umbilical
8.
Acta Crystallogr E Crystallogr Commun ; 76(Pt 6): 820-825, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32523747

RESUMO

The title complex, Cu(L)2 or [Cu(C15HF10O2)2], comprised of one copper ion and two fully fluorinated ligands (L -), was crystallized with 3,4-ethyl-ene-dioxy-thio-phene (EDOT, C6H6O2S) as a guest mol-ecule to give in a di-chloro-methane solution a unique co-crystal, Cu(L)2·3C6H6O2S. In the crystal, the oxygen of one guest mol-ecule, EDOT-1, is coordinated to the metal to give an alternate linear arrangement, and the π-planes of the others, EDOT-2 and EDOT-3, inter-act weakly with the penta-fluoro-phenyl groups of the complex through arene-perfluoro-arene inter-actions. Head-to-tail columnar and head-to-head dimeric arrangements are observed for EDOT-2 and EDOT-3, respectively, in the crystal. The Hirshfeld surface analysis indicated that the most important contributions for the crystal packing are from the F⋯F (20.4%), F⋯H/H⋯F (24.5%) and F⋯C/C⋯F (9.6%) inter-actions. The density functional theory (DFT) optimized structure at the ωB97X-D 6-31G* level was compared with the experimentally determined mol-ecular structure in the solid state.

9.
Inorg Chem ; 59(10): 6709-6716, 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32186382

RESUMO

Metal halide perovskites are promising materials for light absorbers in solar cell applications. Use of the Br/I system enables us to control band gap energy and improves the efficiency of solar cells. Precise knowledge of lattice parameters and band gap energies as functions of compositions are crucially important for developing the devices using those materials. In this study, we have determined lattice parameters and band gap energies of CH3NH3Pb(I1-xBrx)3, one of the most intensively studied mix-halide perovskites, as functions of Br content x. We measured accurate Br contents and lattice parameters of CH3NH3Pb(I1-xBrx)3 (0 ≤ x ≤ 1) using single-crystalline samples by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) measurements, respectively. The CH3NH3Pb(I1-xBrx)3 crystal system is tetragonal for x ≤ 0.06 and cubic for x ≥ 0.08 at 300 K. Lattice parameters of CH3NH3Pb(I1-xBrx)3 strictly follow Vegard's law; i.e., they are linearly dependent on x. We give linear expressions of x of lattice parameters for the tetragonal and cubic phases of CH3NH3Pb(I1-xBrx)3 at 300 K. We have shown that these expressions can be used for determining the Br contents of CH3NH3Pb(I1-xBrx)3 polycrystalline thin-film samples based on XRD measurements and, in addition, demonstrated that XPS measurements on polycrystalline samples may be erroneous because of impure ingredients in the samples. Furthermore, we determined band gap energies of CH3NH3Pb(I1-xBrx)3 (0 ≤ x ≤ 1) at room temperature using absorption spectra of polycrystalline thin films taking account of excitonic effects.

10.
Chemistry ; 26(22): 5051-5060, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32026510

RESUMO

The flexible host framework of a perfluorinated mononuclear copper complex, [Cu(L1 )2 ] (1, HL1 =3-hydroxy-1,3-bis(pentafluorophenyl)-2-propen-1-one), with a CuO4 core reversibly encapsulated several organic guest molecules through electrostatic interactions in its crystals. Hence, the corresponding dinuclear complex, [Cu2 (L2 )2 ] (2, H2 L2 =1,5-dihydroxy-1,5-bis(pentafluorophenyl)-1,4-pentadien-3-one), was prepared to enhance guest recognition and the ability to separate molecular mixtures. Complex 2 comprises a Cu2 O6 core and four pentafluorophenyl groups. In crystal 2, cavities are formed on the axial sites of the metal core that are surrounded by pentafluorophenyl groups. The crystal of 2 encapsulates various guest molecules, that is, benzene (3), toluene (4), xylene (5), mesitylene (6), durene (7), and anisole (8). X-ray crystallographic and thermogravimetric (TG) studies show that three guest molecules are present in the crystal cavities. The number of guest molecules found in complex 2 was higher than that in complex 1, for example, (2)3 ⋅(6)10 >1⋅(6)2 , (2)2 ⋅(7)7 >1⋅7, or 2⋅(8)3 >1⋅(8)2 . Naphthalene (9), was encapsulated in 2 to give 2⋅(9)3 , but not in 1. In the crystal of complex 2, heteromolecular guest encapsulation was confirmed, designated as 2⋅(3)2 ⋅9. TG analysis indicates that the thermal stability of the guest-included crystals of 2 is higher than that of 1.

11.
Sci Rep ; 9(1): 15911, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685837

RESUMO

Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.


Assuntos
Antígeno AC133/metabolismo , Cálcio/metabolismo , Extensões da Superfície Celular/metabolismo , Cloretos/metabolismo , Quinases Associadas a rho/metabolismo , Antígeno AC133/química , Antígeno AC133/genética , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Colesterol/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Transdução de Sinais
12.
Langmuir ; 35(50): 16576-16582, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31763843

RESUMO

Protein-encapsulated nanoclusters (NCs) are emerging as a versatile platform for in-vivo imaging and other biomedical applications due to their ultrasmall size and excitation in the near-infrared region. Encapsulation may however affect protein structure, size, charge, and its interaction with lipid membranes. In this study, bulk characterization methods along with surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy were employed to study the secondary structure of bovine serum albumin (BSA) with blue-emitting Au8NCs at the air/water and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) lipid/water interfaces. With this approach, the difference in the adsorption behavior between native BSA and BSA with an increasing number of blue-emitting NCs was investigated under different pH conditions. At pH 7, at which both BSA and the lipid are negatively charged, protein molecules are found to associate with the DPPG monolayer via hydrophobic interactions with no preferential orientation across the lipid monolayer. At pH 3, adsorption of BSA at the DPPG monolayer occurs mainly due to electrostatic interactions between the negatively charged lipid headgroups and the positively charged protein, resulting in a uniform orientation of the protein across the lipid monolayer. Complimentary bulk studies by circular dichroism and particle size measurements show that the encapsulation of Au8NCs is associated with the loss of BSA helicity, which makes BSA-encapsulated Au8NCs prone to oligomerization, especially at a high content of Au8NCs at one BSA protein. The results indicate that the hydrodynamic diameter of BSA with Au8NCs strongly depends on the molar fraction of gold, the pH, and the storage time. A prolonged storage of Au8NCs@BSA at pH 7 increases the rate of protein oligomerization.


Assuntos
Ar , Ouro/química , Lipídeos/química , Soroalbumina Bovina/química , Água/química , Adsorção , Animais , Bovinos , Cor , Modelos Moleculares , Conformação Proteica
13.
Development ; 146(17)2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31444216

RESUMO

Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity in the neural progenitor cells changes dynamically during differentiation, but the mechanisms regulating this dynamicity are not fully understood. Here, we show that temporal change of intracellular cAMP levels confers the temporal Shh signal, and the purinergic G-protein-coupled receptor GPR17 plays an essential role in this regulation. GPR17 is highly expressed in the ventral progenitor regions of the neural tube and acts as a negative regulator of the Shh signal in chick embryos. Although the activation of the GPR17-related signal inhibits ventral identity, perturbation of Gpr17 expression leads to aberrant expansion of ventral neural domains. Notably, perturbation of Gpr17 expression partially inhibits the negative feedback of Gli activity. Moreover, GPR17 increases cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulates Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas Hedgehog/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/embriologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Padronização Corporal/fisiologia , Diferenciação Celular/genética , Embrião de Galinha , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Transfecção , Proteína Gli3 com Dedos de Zinco/metabolismo
14.
Dalton Trans ; 48(25): 9062-9066, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31161178

RESUMO

Two different coordination types of fully fluorinated dinuclear metal complexes, [Fe2L4(OMe)2] and [M2L4(OH2)2] (M = Co2+, Ni2+ and HL = bis(pentafluorobenzoyl)methane), were obtained. All of the complexes form non-porous crystals, which act as hosts for the adsorption of various benzene derivatives, e.g., benzene, toluene, p-xylene, anisole, and small gas molecules, e.g., CO2, O2, and NO. The complex of iron selectively adsorbs NO in high amounts and the complex of cobalt is found to store adsorbed O2.

15.
Acta Crystallogr C Struct Chem ; 75(Pt 3): 265-270, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30833520

RESUMO

Calix[4]arene and oxacalix[4]arene derivatives have eight possible conformations in the up and down directions of their four aromatic rings from the mean plane of a bridged central ring, the conformations of which determine the functionality of the host frameworks. Despite being a known compound for five decades, oxacalix[4]arene, C24H16O4, has not been characterized previously by crystallographic methods. It crystallizes from hexane/CH2Cl2 solution to give two polymorphs, i.e. prismatic and block-shaped crystals as twisted 1,3-alternate structures with S4 and C2 symmetry, respectively. These were previously suggested as the prefered stable conformations by density functional theory (DFT) calculations.

16.
Cell Mol Life Sci ; 74(2): 213-229, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27484406

RESUMO

Centriolar satellites comprise cytoplasmic granules that are located around the centrosome. Their molecular identification was first reported more than a quarter of a century ago. These particles are not static in the cell but instead constantly move around the centrosome. Over the last decade, significant advances in their molecular compositions and biological functions have been achieved due to comprehensive proteomics and genomics, super-resolution microscopy analyses and elegant genetic manipulations. Centriolar satellites play pivotal roles in centrosome assembly and primary cilium formation through the delivery of centriolar/centrosomal components from the cytoplasm to the centrosome. Their importance is further underscored by the fact that mutations in genes encoding satellite components and regulators lead to various human disorders such as ciliopathies. Moreover, the most recent findings highlight dynamic structural remodelling in response to internal and external cues and unexpected positive feedback control that is exerted from the centrosome for centriolar satellite integrity.


Assuntos
Centríolos/fisiologia , Grânulos Citoplasmáticos/fisiologia , Animais , Ciclo Celular , Cílios/metabolismo , Humanos , Microtúbulos/metabolismo , Estresse Fisiológico
17.
Exp Anim ; 65(3): 231-44, 2016 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26923756

RESUMO

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.


Assuntos
Técnicas Genéticas , Integrases/genética , Camundongos Transgênicos/genética , Recombinação Genética , Tamoxifeno , Transgenes , Animais , Antígenos Transformantes de Poliomavirus/genética , Fusão Gênica , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Oncogenes/genética , Fenótipo , Ativação Transcricional
18.
EMBO Rep ; 17(3): 326-37, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26755742

RESUMO

Centrioles are the major constituents of the animal centrosome, in which Plk4 kinase serves as a master regulator of the duplication cycle. Many eukaryotes also contain numerous peripheral particles known as centriolar satellites. While centriolar satellites aid centriole assembly and primary cilium formation, it is unknown whether Plk4 plays any regulatory roles in centriolar satellite integrity. Here we show that Plk4 is a critical determinant of centriolar satellite organisation. Plk4 depletion leads to the dispersion of centriolar satellites and perturbed ciliogenesis. Plk4 interacts with the satellite component PCM1, and its kinase activity is required for phosphorylation of the conserved S372. The nonphosphorylatable PCM1 mutant recapitulates phenotypes of Plk4 depletion, while the phosphomimetic mutant partially rescues the dispersed centriolar satellite patterns and ciliogenesis in cells depleted of PCM1. We show that S372 phosphorylation occurs during the G1 phase of the cell cycle and is important for PCM1 dimerisation and interaction with other satellite components. Our findings reveal that Plk4 is required for centriolar satellite function, which may underlie the ciliogenesis defects caused by Plk4 dysfunction.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Cílios/metabolismo , Fase G1 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética
19.
Int J Oral Maxillofac Implants ; 31(1): 232-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26800183

RESUMO

PURPOSE: To optimize methods for seeding cells on granular-type beta-tricalcium phosphate (ß-TCP). MATERIALS AND METHODS: Bone marrow stromal cells were obtained from rat long bones and cultured in flasks with Minimum Essential Medium, Alpha Modification (αMEM) supplemented with 10% fetal bovine serum (FBS), dexamethasone, ascorbic acid, ß -glycerophosphate, and antibiotics. The influence of differential cell seeding densities and dynamic cell seeding conditions (rotation) was investigated using different sizes of ß -TCP granules and a subcutaneous implantation model. RESULTS: Higher cell seeding densities contributed to efficient in vivo bone formation. The rotational seeding did not affect the efficiency but contributed to the uniformity. Although the granule size did not affect the efficiency under the conditions used in this study, large granules showed more uniform distribution of bone regeneration, while small granules showed nonuniform but dense bone formation. Mixtures of relatively large and small granules may be beneficial for both uniform and efficient bone regeneration. CONCLUSION: These findings may contribute to stable bone tissue engineering with bone marrow stromal cells and ß -TCP granules as a scaffold.


Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , Células-Tronco Mesenquimais/fisiologia , Alicerces Teciduais/química , Animais , Regeneração Óssea/fisiologia , Adesão Celular/fisiologia , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteogênese/fisiologia , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Rotação , Tela Subcutânea/cirurgia , Engenharia Tecidual/métodos
20.
Biochem Biophys Res Commun ; 468(1-2): 39-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26545777

RESUMO

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, as one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation.


Assuntos
Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Centrossomo/ultraestrutura , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas/análise , Proteínas/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura
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