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1.
Cell Rep ; 42(8): 113006, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37610870

RESUMO

Immune-suppressive effects of myeloid-derived suppressor cells (MDSCs) are well characterized during anti-tumor immunity. The complex mechanisms promoting MDSC development and their regulatory effects during autoimmune diseases are less understood. We demonstrate that the endogenous alarmin S100A8/A9 reprograms myeloid cells to a T cell suppressing phenotype during autoimmune arthritis. Treatment of myeloid precursors with S100-alarmins during differentiation induces MDSCs in a Toll-like receptor 4-dependent manner. Consequently, knockout of S100A8/A9 aggravates disease activity in collagen-induced arthritis due to a deficit of MDSCs in local lymph nodes, which could be corrected by adoptive transfer of S100-induced MDSCs. Blockade of MDSC function in vivo aggravates disease severity in arthritis. Therapeutic application of S100A8 induces MDSCs in vivo and suppresses the inflammatory phenotype of S100A9ko mice. Accordingly, the interplay of T cell-mediated autoimmunity with a defective innate immune regulation is crucial for autoimmune arthritis, which should be considered for future innovative therapeutic options.


Assuntos
Artrite , Calgranulina A , Calgranulina B , Células Supressoras Mieloides , Animais , Camundongos , Artrite/imunologia , Artrite/metabolismo , Artrite/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Modelos Animais de Doenças , Diferenciação Celular , Óxido Nítrico/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo
2.
FASEB J ; 37(4): e22883, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36934410

RESUMO

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína 1 com Domínio SAM e Domínio HD , Telomerase , Humanos , Nucleotídeos de Desoxiguanina , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo
3.
Adv Sci (Weinh) ; 9(36): e2201505, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36310133

RESUMO

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).


Assuntos
Monócitos , Receptor 4 Toll-Like , Humanos , Receptor 4 Toll-Like/metabolismo , Cálcio/metabolismo , Calgranulina B/metabolismo , Calgranulina A/química , Calgranulina A/metabolismo , Inflamação/metabolismo
4.
Histochem Cell Biol ; 156(1): 35-46, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33728539

RESUMO

The influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.


Assuntos
Tecido Adiposo/metabolismo , Grelina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Tecido Adiposo/efeitos dos fármacos , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Neurônios/efeitos dos fármacos
5.
J Allergy Clin Immunol ; 146(5): 1137-1151, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32199911

RESUMO

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células da Medula Óssea/metabolismo , Dermatite Alérgica de Contato/imunologia , Inflamação/imunologia , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Células da Medula Óssea/imunologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Imunomodulação , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/genética , Transcriptoma
6.
FASEB J ; 34(1): 631-647, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914608

RESUMO

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Fibroblastos/metabolismo , Mutação/genética , Malformações do Sistema Nervoso/genética , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Dano ao DNA/genética , Replicação do DNA/genética , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética
7.
Front Oncol ; 9: 987, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632918

RESUMO

Introduction: Adverse effects of radiotherapy (RT) significantly affect patient's quality of life (QOL). The possibility to identify patient-related factors that are associated with individual radiosensitivity would optimize adjuvant RT treatment, limiting the severity of normal tissue reactions, and improving patient's QOL. In this study, we analyzed the relationships between genetic features and toxicity grading manifested by RT patients looking for possible biomarkers of individual radiosensitivity. Methods: Early radiation toxicity was evaluated on 143 oncological patients according to the Common Terminology Criteria for Adverse Events (CTCAE). An individual radiosensitivity (IRS) index defining four classes of radiosensitivity (highly radiosensitive, radiosensitive, normal, and radioresistant) was determined by a G2-chromosomal assay on ex vivo irradiated, patient-derived blood samples. The expression level of 15 radioresponsive genes has been measured by quantitative real-time PCR at 24 h after the first RT fraction, in blood samples of a subset of 57 patients, representing the four IRS classes. Results: By applying univariate and multivariate statistical analyses, we found that fatigue was significantly associated with IRS index. Interestingly, associations were detected between clinical radiation toxicity and gene expression (ATM, CDKN1A, FDXR, SESN1, XPC, ZMAT3, and BCL2/BAX ratio) and between IRS index and gene expression (BBC3, FDXR, GADD45A, and BCL2/BAX). Conclusions: In this prospective cohort study we found that associations exist between normal tissue reactions and genetic features in RT-treated patients. Overall, our findings can contribute to the identification of biological markers to predict RT toxicity in normal tissues.

8.
Mutat Res Rev Mutat Res ; 779: 126-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31097149

RESUMO

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.


Assuntos
Mutagênicos/efeitos adversos , Aneugênicos/efeitos adversos , Aneuploidia , Animais , Transformação Celular Neoplásica/induzido quimicamente , Aberrações Cromossômicas , Europa (Continente) , Células Germinativas/efeitos dos fármacos , Humanos , Risco
9.
Genes Chromosomes Cancer ; 58(5): 260-269, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30387295

RESUMO

Mechanisms and events related to common fragile site (CFS) instability are well known in cancer cells. Here, we argue that normal cells remain an important experimental model to address questions related to CFS instability in the absence of alterations in cell cycle and DNA damage repair pathways, which are common features acquired in cancer. Furthermore, a major gap of knowledge concerns the stability of CFSs during gametogenesis. CFS instability in meiotic or postmeiotic stages of the germ cell line could generate chromosome deletions or large rearrangements. This in turn can lead to the functional loss of the several CFS-associated genes with tumor suppressor function. Our hypothesis is that such mutations can potentially result in genetic predisposition to develop cancer. Indirect evidence for CFS instability in human germ cells has been provided by genomic investigations in family pedigrees associated with genetic disease. The issue of CFS instability in the germ cell line should represent one of the future efforts, and may take advantage of the existence of sequence and functional conservation of CFSs between rodents and humans.


Assuntos
Instabilidade Cromossômica , Sítios Frágeis do Cromossomo , Células Germinativas/metabolismo , Animais , Gametogênese , Técnicas de Genotipagem/métodos , Células Germinativas/citologia , Humanos , Sequenciamento Completo do Genoma/métodos
10.
Front Immunol ; 9: 893, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867927

RESUMO

Although glucocorticoids (GC) represent the most frequently used immunosuppressive drugs, their effects are still not well understood. In our previous studies, we have shown that treatment of monocytes with GC does not cause a global suppression of monocytic effector functions, but rather induces differentiation of a specific anti-inflammatory phenotype. The anti-inflammatory role of peroxisome proliferator-activated receptor (PPAR)-γ has been extensively studied during recent years. However, a relationship between GC treatment and PPAR-γ expression in macrophages has not been investigated so far. Studies using PPAR-γ-deficient mice have frequently provided controversial results. A potential reason is the use of primary cells, which commonly represent inhomogeneous populations burdened with side effects and influenced by bystander cells. To overcome this constraint, we established ER-Hoxb8-immortalized bone marrow-derived macrophages from Ppargfl/fl and LysM-Cre Ppargfl/fl mice in this study. In contrast to primary macrophages, the ER-Hoxb8 system allows the generation of a homogeneous and well-defined population of resting macrophages. We could show that the loss of PPAR-γ resulted in delayed kinetic of differentiation of monocytes into macrophages as assessed by reduced F4/80, but increased Ly6C expression in early phases of differentiation. As expected, PPAR-γ-deficient macrophages displayed an increased pro-inflammatory phenotype upon long-term LPS stimulation characterized by an elevated production of pro-inflammatory cytokines TNF-α, IL1-ß, IL-6, IL-12 and a reduced production of anti-inflammatory cytokine IL-10 compared to PPAR-γ WT cells. Moreover, PPAR-γ-deficient macrophages showed impaired phagocytosis. GC treatment of macrophages led to the upregulation of PPAR-γ expression. However, there were no differences in GC-induced suppression of cytokines between both cell types, implicating a PPAR-γ-independent mechanism. Intriguingly, GC treatment resulted in an increased in vitro migration only in PPAR-γ-deficient macrophages. Performing a newly developed in vivo cell-tracking experiment, we could confirm that GC induces an increased recruitment of PPAR-γ KO, but not PPAR-γ WT macrophages to the site of inflammation. Our findings suggest a specific effect of PPAR-γ on GC-induced migration in macrophages. In conclusion, we could demonstrate that PPAR-γ exerts anti-inflammatory activities and shapes macrophage functions. Moreover, we identified a molecular link between GC and PPAR-γ and could show for the first time that PPAR-γ modulates GC-induced migration in macrophages.


Assuntos
Glucocorticoides/farmacologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , PPAR gama/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Modelos Animais de Doenças , Granuloma/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , PPAR gama/genética , Dermatopatias/imunologia
11.
Nucleic Acids Res ; 46(1): 267-278, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29165708

RESUMO

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis.


Assuntos
Replicação do DNA , DNA/química , Instabilidade Genômica , Conformação de Ácido Nucleico , Separase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , DNA/genética , DNA/metabolismo , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Genéticos , Ligação Proteica , Interferência de RNA , Separase/genética , Coesinas
12.
Dis Markers ; 2017: 9126560, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29180834

RESUMO

Peritonsillar abscess (PTA) is a very frequent reason for urgent outpatient consultation and otolaryngological hospital admission. Early, correct diagnosis and therapy of peritonsillar abscess are important to prevent possible life-threatening complications. Based on physical examinations, a reliable differentiation between peritonsillar cellulitis and peritonsillar abscess is restricted. A heterodimeric complex called calprotectin consists of the S100 proteins A8 and A9 (S100A8/A9) and is predominantly expressed not only in monocytes and neutrophils but also in epithelial cells. Due to its release by activated phagocytes at local sites of inflammation, we assumed S100A8/A9 to be a potential biomarker for peritonsillar abscess. We examined serum and saliva of patients with peritonsillitis, acute tonsillitis, peritonsillar abscess, and healthy controls and found significantly increased levels of S100A8/A9 in patients with PTA. Furthermore, we could identify halitosis, trismus, uvula edema, and unilateral swelling of the arched palate to be characteristic symptoms for PTA. Using a combination of these characteristic symptoms and S100A8/A9 levels, we developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and peritonsillar abscess with a sensitivity of 92% and specificity of 93%.


Assuntos
Calgranulina A/sangue , Calgranulina B/sangue , Abscesso Peritonsilar/sangue , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Abscesso Peritonsilar/metabolismo , Abscesso Peritonsilar/patologia , Saliva/metabolismo
13.
Materials (Basel) ; 10(8)2017 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-28773208

RESUMO

Polyurethane-resin doming is currently one of the fastest growing markets in the field of industrial graphics and product identification. Semi-rigid bio-based polyurethanes were prepared deriving from soybean oil as a valuable alternative to fossil materials for digital doming and applied to digital mosaic technology. Bio-resins produced can favorably compete with the analogous fossil polymers, giving high-quality surface coatings (ascertained by SEM analyses). In addition, polyurethane synthesis was accomplished by using a mercury- and tin-free catalyst (the commercially available zinc derivative K22) bringing significant benefits in terms of cost efficiency and eco-sustainability.

14.
Molecules ; 22(2)2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28230803

RESUMO

An innovative and eco-friendly one-pot synthesis of bio-based polyurethanes is proposed via the epoxy-ring opening of epoxidized soybean oil (ESO) with methanol, followed by the reaction of methoxy bio-polyols intermediates with 2,6-tolyl-diisocyanate (TDI). Both synthetic steps, methanolysis and polyurethane linkage formation, are promoted by a unique catalyst, molybdenum(VI) dichloride dioxide (MoCl2O2), which makes this procedure an efficient, cost-effective, and environmentally safer method amenable to industrial scale-up.


Assuntos
Técnicas de Química Sintética , Poliuretanos/síntese química , Óleo de Soja/química , Catálise , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética
15.
Stem Cells Transl Med ; 5(8): 1098-105, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27151912

RESUMO

UNLABELLED: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. SIGNIFICANCE: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Doenças da Córnea/cirurgia , Transplante de Córnea/métodos , Displasia Ectodérmica/genética , Heterozigoto , Homozigoto , Mosaicismo , Mucosa Bucal/transplante , Medicina de Precisão/métodos , Transplante de Células-Tronco/métodos , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Células 3T3 , Adolescente , Animais , Estudos de Casos e Controles , Fenda Labial/complicações , Fenda Labial/diagnóstico , Fissura Palatina/complicações , Fissura Palatina/diagnóstico , Técnicas de Cocultura , Doenças da Córnea/diagnóstico , Doenças da Córnea/genética , Análise Mutacional de DNA , Displasia Ectodérmica/complicações , Displasia Ectodérmica/diagnóstico , Células Alimentadoras , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Mutação de Sentido Incorreto , Seleção de Pacientes , Fenótipo , Valor Preditivo dos Testes , Transfecção , Transplante Autólogo
16.
Cytogenet Genome Res ; 150(3-4): 176-184, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28192779

RESUMO

Genomic instability is a hallmark of cancer, and it is well-known that in several cancers the karyotype is unstable and rapidly evolving. Molecular cytogenetics has contributed to the description and interpretation of cancer karyotypes, in particular through multicolor FISH approaches which can define even complex chromosome rearrangements. The introduction of genome-wide methods has made available a powerful set of tools with higher resolution than cytogenetics, thus appropriate to comprehend the huge variability of cancer cells. This review focuses on novel findings deriving from the combination of cytogenetic and genomic approaches in cancer research.


Assuntos
Instabilidade Cromossômica , Citogenética , Genômica , Neoplasias/genética , Cromotripsia , Humanos , Cariotipagem
17.
Environ Mol Mutagen ; 56(7): 563-80, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25784636

RESUMO

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.


Assuntos
Cromossomos Humanos/genética , Instabilidade Genômica , Animais , Aberrações Cromossômicas , Dano ao DNA , Replicação do DNA , Células Germinativas/fisiologia , Humanos , Mitose , Neoplasias/genética , Neoplasias/patologia
18.
PLoS One ; 9(2): e88637, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24523925

RESUMO

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,ß (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, ß-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, ß downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression.


Assuntos
Cálcio/metabolismo , Células Dendríticas/citologia , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sequência de Bases , Células da Medula Óssea/citologia , Calbindina 1/metabolismo , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Potássio/metabolismo
19.
Cell Physiol Biochem ; 33(1): 222-36, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24496246

RESUMO

BACKGROUND/AIMS: The protein kinase Akt2/PKBß is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling. METHODS: DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. RESULTS: Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration. CONCLUSION: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.


Assuntos
Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CCL21/farmacologia , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Compostos Macrocíclicos/farmacologia , Camundongos , Modelos Biológicos , Oxazóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/deficiência
20.
Exp Cell Res ; 319(20): 3081-93, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24126019

RESUMO

Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.


Assuntos
Replicação do DNA/genética , Loci Gênicos/genética , Genoma Humano/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo
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