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1.
EMBO J ; 37(12)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29764980

RESUMO

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.


Assuntos
Comunicação Celular , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Transativadores/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adipogenia , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Proteínas com Homeodomínio LIM/biossíntese , Camundongos , Camundongos SCID , Miócitos Cardíacos/citologia , Transativadores/genética , Fatores de Transcrição/biossíntese , Proteínas WT1/biossíntese , Proteína rhoA de Ligação ao GTP/genética
2.
J Org Chem ; 87(13): 8437-8444, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35679839

RESUMO

An organocatalyzed, formal (3+3) cycloaddition reaction is described for the practical synthesis of substituted pyridines. Starting from readily available enamines and enal/ynal/enone substrates, the protocol affords tri- or tetrasubstituted pyridine scaffolds bearing various functional groups. This method was demonstrated on a 50 g scale, enabling the synthesis of 2-isopropyl-4-methylpyridin-3-amine, a raw material used for the manufacture of sotorasib. Mechanistic analysis using two-dimensional nuclear magnetic resonance (NMR) spectrometry revealed the transformation proceeds through the reversible formation of a stable reaction off-cycle species that precedes pyridine formation. In situ reaction progress kinetic analysis and control NMR studies were employed to better understand the role of FeCl3 and pyrrolidine hydrochloride in promoting the reaction.


Assuntos
Aldeídos , Cetonas , Aldeídos/química , Catálise , Reação de Cicloadição , Cetonas/química , Cinética , Piridinas/química
3.
EMBO J ; 35(6): 618-34, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26903601

RESUMO

Transcription factor Stat3 directs self-renewal of pluripotent mouse embryonic stem (ES) cells downstream of the cytokine leukemia inhibitory factor (LIF). Stat3 upregulates pivotal transcription factors in the ES cell gene regulatory network to sustain naïve identity. Stat3 also contributes to the rapid proliferation of ES cells. Here, we show that Stat3 increases the expression of mitochondrial-encoded transcripts and enhances oxidative metabolism. Chromatin immunoprecipitation reveals that Stat3 binds to the mitochondrial genome, consistent with direct transcriptional regulation. An engineered form of Stat3 that localizes predominantly to mitochondria is sufficient to support enhanced proliferation of ES cells, but not to maintain their undifferentiated phenotype. Furthermore, during reprogramming from primed to naïve states of pluripotency, Stat3 similarly upregulates mitochondrial transcripts and facilitates metabolic resetting. These findings suggest that the potent stimulation of naïve pluripotency by LIF/Stat3 is attributable to parallel and synergistic induction of both mitochondrial respiration and nuclear transcription factors.


Assuntos
Respiração Celular , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/fisiologia , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Fator Inibidor de Leucemia/metabolismo , Camundongos , Mitocôndrias/genética
4.
J Org Chem ; 84(8): 4615-4628, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30900880

RESUMO

In recent years, there has been a growing interest in therapeutic peptides within the pharmaceutical industry with more than 50 peptide drugs on the market, approximately 170 in clinical trials, and >200 in preclinical development. However, the current state of the art in peptide synthesis involves primarily legacy technologies with use of large amounts of highly hazardous reagents and solvents and little focus on green chemistry and engineering. In 2016, the ACS Green Chemistry Institute Pharmaceutical Roundtable identified development of greener processes for peptide API as a critical unmet need, and as a result, a new Roundtable team formed to address this important area. The initial focus of this new team is to highlight best practices in peptide synthesis and encourage much needed innovations. In this Perspective, we aim to summarize the current challenges of peptide synthesis and purification in terms of sustainability, highlight possible solutions, and encourage synergies between academia, the pharmaceutical industry, and contract research organizations/contract manufacturing organizations.


Assuntos
Desenvolvimento de Medicamentos , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Humanos , Estrutura Molecular , Peptídeos/química
5.
J Org Chem ; 84(8): 4763-4779, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30557503

RESUMO

A robust process to manufacture AMG 232 was developed to deliver drug substance of high purity. Highlights of the commercial process development efforts include the following: (i) use of a novel bench-stable Vilsmeier reagent, methoxymethylene- N, N-dimethyliminium methyl sulfate, for selective in situ activation of a primary alcohol intermediate; (ii) use of a new crystalline and stable isopropyl calcium sulfinate reagent ensuring robust preparation of a sulfone intermediate; (iii) development of a safe ozonolysis process conducted in an aqueous solvent mixture in either batch or continuous manufacturing mode; and (iv) control of the drug substance purity by crystallization of a salt rejecting impurities effectively. The new process was demonstrated to afford the drug substance (99.9 LC area %) in 49.8% overall yield from starting material DLAC (1).


Assuntos
Acetatos/síntese química , Ozônio/química , Piperidonas/síntese química , Acetatos/química , Acetatos/isolamento & purificação , Estrutura Molecular , Piperidonas/química , Piperidonas/isolamento & purificação
6.
Stem Cell Reports ; 18(9): 1744-1752, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37703820

RESUMO

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.


Assuntos
Pesquisa com Células-Tronco , Humanos , Reprodutibilidade dos Testes
7.
J Am Chem Soc ; 134(44): 18241-4, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23098215

RESUMO

A highly asymmetric vinylogous addition of acyclic silyl enol ethers to siloxyvinyldiazoacetate is described. The reaction features a diastereoselective 1,4-siloxy group migration event. Products are obtained in up to 97% ee. When more sterically crowded silyl enol ethers are employed, an enantioselective formal [3+2] cycloaddition becomes the dominant reaction pathway. Control experiments reveal the (Z)-olefin geometry to be critical for high levels of enantiocontrol.


Assuntos
Acetatos/química , Alcenos/química , Éteres/química , Ródio/química , Compostos Azo/química , Catálise , Ciclização , Estereoisomerismo
8.
J Am Chem Soc ; 133(46): 18618-21, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22026505

RESUMO

The coupling of carbonyl electrophiles at the donor position of donor-acceptor cyclopropanes is described, representing an inversion of polarity with respect to conventional reactivity modes displayed by these reagents. Specifically, upon exposure of donor-acceptor cyclopropanes to alcohols in the presence of a cyclometalated iridium catalyst modified by (S)-BINAP, catalytic C-C coupling occurs, providing enantiomerically enriched products of carbonyl allylation. Identical products are obtained upon isopropanol-mediated transfer hydrogenation of donor-acceptor cyclopropanes in the presence of aldehydes. The reaction products are directly transformed to cis-4,5-disubstituted δ-lactones.


Assuntos
Ciclopropanos/química , Irídio/química , Lactonas/química , Álcoois/química , Catálise , Estrutura Molecular , Oxirredução , Estereoisomerismo
9.
FASEB J ; 24(3): 700-11, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19850773

RESUMO

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.


Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Multipotentes/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Citometria de Fluxo , Heterozigoto , Humanos , Imuno-Histoquímica , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase , Fatores de Transcrição
10.
J Am Chem Soc ; 132(28): 9688-92, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20572661

RESUMO

An enantioselective preparation of 2,5-cis-disubstituted pyrrolidines has been achieved via a dynamic kinetic asymmetric transformation (DyKAT) of racemic donor-acceptor cyclopropanes and (E)-aldimines. Mechanistic studies suggest that isomerization of the aldimine or resultant iminium to the Z geometry is not a pathway that furnishes the observed 2,5-cis-disubstituted products.


Assuntos
Ciclopropanos/química , Pirrolidinas/síntese química , Cinética , Espectroscopia de Ressonância Magnética , Pirrolidinas/química , Estereoisomerismo
11.
Biochem Soc Trans ; 38(4): 1027-32, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20658998

RESUMO

Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.


Assuntos
Células-Tronco Pluripotentes/fisiologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator 4 de Crescimento de Fibroblastos/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Humanos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/fisiologia
12.
Biochem Soc Trans ; 38(4): 1067-71, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20659005

RESUMO

The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.


Assuntos
Diagnóstico por Imagem/métodos , Glioma/patologia , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Células-Tronco/fisiologia , Glioma/diagnóstico , Humanos , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/efeitos dos fármacos
13.
PLoS Biol ; 4(5): e121, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16594731

RESUMO

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.


Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Receptores Notch/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Neurônios/metabolismo , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais
14.
Mol Cell Biol ; 22(5): 1526-36, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11839818

RESUMO

Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Humanos , Camundongos , Mutação , Fator 3 de Transcrição de Octâmero , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Fatores de Transcrição/genética
15.
Nat Biotechnol ; 21(4): 443-7, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12627172

RESUMO

The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.


Assuntos
Alelos , Cromossomos Artificiais Bacterianos/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Genoma , Animais , Células Cultivadas , Escherichia coli/genética , Camundongos , Mutagênese Sítio-Dirigida , Recombinação Genética , Células-Tronco/fisiologia
16.
Bone ; 36(5): 758-69, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15794925

RESUMO

Reliable in vitro conversion of pluripotent embryonic stem (ES) cells into bone and cartilage-forming cells would expand opportunities for experimental investigations of skeletogenesis and could also provide new cellular sources for pharmaceutical screening and for cell therapy applications. Here, we evaluate the generation of mesenchymal cell lineages from mouse ES cells following treatment of embryoid bodies with retinoic acid, previously reported to induce development of adipocyte precursors. We find that retinoic acid reduces mesodermal differentiation but enhances expression of markers of neural crest, an alternative origin of mesenchymal elements. Runx1 and Ptprv appear to provide early markers of mesenchymal potential. Subsequently, different mesenchymal fates are generated in response to particular growth factors. Substitution of the adipogenic factors insulin and triiodothyronine with bone morphogenetic protein (BMP-4) results in suppression of adipogenesis and development of a mature osteogenic phenotype. In contrast, treatment with transforming growth factor-beta (TGF-beta3) promotes chondrogenic differentiation. Thus, the use of appropriate growth factors and culture milieu steers differentiation of ES cell-derived precursors into distinct mesenchymal compartments.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrócitos/citologia , Embrião de Mamíferos/citologia , Osteoblastos/citologia , Células-Tronco/citologia , Fosfatase Alcalina/biossíntese , Animais , Sequência de Bases , Biomarcadores , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/fisiologia , Linhagem da Célula , Meios de Cultura , Primers do DNA , Indução Enzimática , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Methods Enzymol ; 365: 327-41, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14696356

RESUMO

The efficiency of monolayer differentiation establishes that commitment of ES cells to a neural fate needs neither multicellular aggregation nor extrinsic inducers. The entire process by which pluripotent ES cells acquire neural specification can be visualized and recorded at the level of individual colonies. Furthermore this simple culture system is amenable to cellular and molecular dissection, promising to yield new insights into the mechanism underlying neural determination in mammals and perhaps to deliver the goal of "directed" homogeneous differentiation of ES cells.


Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Animais , Morte Celular , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas HMGB , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Contraste de Fase , Proteínas Nucleares/metabolismo , Fatores de Transcrição SOXB1 , Fatores de Transcrição
18.
Thromb Haemost ; 88(6): 967-74, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12529747

RESUMO

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.


Assuntos
Deficiência do Fator XIII/sangue , Deficiência do Fator XIII/genética , Fator XIII/genética , Animais , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , DNA/genética , Modelos Animais de Doenças , Éxons , Fator XIII/administração & dosagem , Deficiência do Fator XIII/tratamento farmacológico , Feminino , Marcação de Genes , Hemostasia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Fenótipo
19.
Biol Open ; 1(1): 58-65, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213369

RESUMO

The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.

20.
Org Lett ; 13(8): 1996-9, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21395241

RESUMO

A diastereoselective synthesis of pentasubstituted tetrahydrofurans via a Lewis acid catalyzed (3 + 2)-annulation of quaternary donor site cyclopropanes and aldehydes is described. The reaction is catalyzed by Sn(OTf)(2), SnCl(4), or Hf(OTf)(4) in yields up to 95% and diastereomeric ratios as high as 99:1.


Assuntos
Aldeídos/química , Ciclopropanos/química , Furanos/química , Catálise , Estrutura Molecular , Estereoisomerismo
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