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1.
J Clin Invest ; 131(6)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33555272

RESUMO

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animais , Linhagem Celular Tumoral , Quimiotaxia de Leucócito , Criança , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Progressão da Doença , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Biogênese de Organelas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais
2.
BMC Plant Biol ; 13: 220, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24359620

RESUMO

BACKGROUND: The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level. RESULTS: By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells. CONCLUSIONS: Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.


Assuntos
Artemisia annua/citologia , Tricomas/citologia , Perfilação da Expressão Gênica
3.
J Neuroimmune Pharmacol ; 8(4): 934-43, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23640520

RESUMO

Serotonin reuptake inhibitors (SRIs) are widely used drugs in the treatment of depression and anxiety disorders. Although SRIs are generally regarded as safe drugs with relatively few side effects, literature suggests that high concentrations of SRIs may alter immune function. We investigated whether high-dose treatment with fluoxetine was able to suppress acute graft-versus-host disease (GvHD) in a MHC-matched, minor histocompatibility antigen mismatched murine bone marrow transplantation model. We found that high doses fluoxetine induce a significant reduction of clinical symptoms and increase survival of these animals. The amelioration of clinical GvHD was accompanied by a reduced expansion of alloreactive T cells. We further analyzed the direct in vitro effect of six SRIs on the viability and proliferation of human T cells and found an anti-proliferative and pro-apoptotic effect that was significantly larger in activated than in resting T cells. We discuss these results in the light of potential future exploration of SRIs as a novel class of T cell immunosuppressive drugs.


Assuntos
Transplante de Medula Óssea , Fluoxetina/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Imunossupressores/uso terapêutico , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Linfócitos T/efeitos dos fármacos , Animais , Transplante de Medula Óssea/efeitos adversos , Feminino , Fluoxetina/farmacologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunossupressores/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C3H , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Linfócitos T/imunologia
4.
New Phytol ; 189(1): 176-89, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20874804

RESUMO

• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.


Assuntos
Antimaláricos/metabolismo , Artemisia annua/metabolismo , Artemisininas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/fisiologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antimaláricos/química , Artemisia annua/crescimento & desenvolvimento , Artemisininas/química , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Citocininas/metabolismo , Citocininas/farmacologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Giberelinas/metabolismo , Giberelinas/farmacologia , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Sesquiterpenos/metabolismo
5.
Am J Med Genet B Neuropsychiatr Genet ; 153B(1): 167-76, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19521967

RESUMO

Obsessive-Compulsive Disorder (OCD) is characterized by the presence of obsessions and compulsions, and shows considerable phenotypic variability. Family and twin studies have indicated a genetic component in the etiology of OCD, and the catechol-O-methyl transferase (COMT) gene is an important candidate gene for OCD. This study investigates the influence of the functional COMT Val158Met polymorphism on the phenotypic expression of OCD, using an item-level factor-analytic approach in a large sample. The COMT Val158Met variant was genotyped in 373 patients and 462 controls. It was tested whether there was an association between the COMT Val158Met polymorphism and OCD or dimensional phenotypes such as YBOCS severity score, age of onset of obsessive-compulsive symptoms and six symptom dimensions recently found in a large item-level factor-analytic study [Katerberg et al., submitted]. We further investigated possible sex-specific associations between the COMT Val158Met polymorphism and OCD or dimensional phenotypes. There was a trend for an association of the COMT 158Met allele with OCD in males, and an interaction between the COMT Val158Met genotype and sex on the somatic and sensory phenomena symptom dimension, with females showing lower scores. In conclusion, a dimensional approach seems fruitful in detecting genes of importance for OCD.


Assuntos
Catecol O-Metiltransferase/genética , Metionina/genética , Transtorno Obsessivo-Compulsivo/genética , Polimorfismo Genético , Valina/genética , Estudos de Casos e Controles , Análise Fatorial , Feminino , Genótipo , Humanos , Masculino , Fenótipo
6.
Int J Psychiatry Clin Pract ; 13(1): 345-348, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20174590

RESUMO

The cornerstone of pharmacotherapy for OCD is serotonin reuptake inhibition, either with clomipramine or with selective serotonin reuptake inhibitors (SSRIs). In spite of the success of serotonin reuptake inhibiting drugs, nearly half of OCD patients do not respond to treatment. Treatment response may be affected by genetic polymorphisms of the P450 metabolic system. The four most common enzyme-activity reducing polymorphisms of the P450 CYP2D6 enzyme were determined in 91 outpatients with primary OCD according to DSM-IV criteria, receiving dosages titrated upward to 300 mg/day of venlafaxine or 60 mg/day of paroxetine, using a fixed dosing schedule. Our results show that the investigated CYP2D6 polymorphisms are not a decisive factor in the response to paroxetine and venlafaxine treatment in OCD in spite of their highly significant effect on the blood levels of these medicines.

7.
Methods Mol Biol ; 384: 401-14, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18392576

RESUMO

This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N', N' id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be detected by a difference in mass.


Assuntos
Eletroforese Capilar/métodos , Oligonucleotídeos/análise , Oligonucleotídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sistemas On-Line
8.
J Chromatogr A ; 1118(2): 180-7, 2006 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-16650427

RESUMO

This study reports the development and validation of a rapid, sensitive and selective assay for the quantitation of artemisinin, arteannuin B, artemisitene and artemisinic acid in Artemisia annua L. by reversed phase high performance liquid chromatography (HPLC) electrospray (ESI) quadrupole time of flight (Q-TOF) tandem mass spectrometry (MS/MS). A recovery of >97% for all analytes was achieved by immersing one gram of fresh plant material in chloroform for 1 min. This result supports the hypothesis that artemisinin and some of its structural analogs present in the leaves A. annua L. are localized entirely in the subcuticular space of the glands on the surface of the leaves. We validated the use of this chloroform extract, without additional sample preparation steps, for quantitative Q-TOF MS/MS. No ion suppression (matrix effect) resulting from interference with other compounds was detected. For every concentration within the range of the standard curve (0.1 to 3.00 microg/ml), accuracy was between 85% and 115%. Within- and between-day variations for the analysis of A. annua L. samples were <20%.


Assuntos
Artemisia/metabolismo , Artemisininas/análise , Cromatografia Líquida de Alta Pressão/métodos , Sesquiterpenos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Artemisininas/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sesquiterpenos/metabolismo
9.
Electrophoresis ; 26(7-8): 1412-23, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15765471

RESUMO

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.


Assuntos
Eletroforese Capilar/métodos , Oligonucleotídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Soluções Tampão , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Oligonucleotídeos/isolamento & purificação , Controle de Qualidade
10.
Electrophoresis ; 26(7-8): 1221-53, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15759298

RESUMO

This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.


Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Ácidos Nucleicos/análise
11.
J Chromatogr A ; 1052(1-2): 93-101, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15527125

RESUMO

A fast quality control method is developed allowing the desalting and characterization of oligonucleotides by capillary liquid chromatography and on-line nano-electrospray ionization quadrupole time-of-flight mass spectrometry using column switching. The influence of addition of ammonium acetate, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, formic acid or acetic acid to the sample, addition of ammonium acetate to the trapping solvent and variation of the trapping time on the further reduction of cation adduction was studied. Final conditions were the addition of 0.1 M ammonium acetate to the sample, the use of a trapping solvent consisting of 0.4 M aqueous 1,1,1,3,3,3-hexafluoro-2-propanol (HFLP) adjusted to pH 7.0 with triethylamine plus 10 mM ammonium acetate during 8 min and the elution of the oligonucleotides with 0.4 M HFIP in 50% methanol. The potential of the optimized procedure is demonstrated for different synthetic oligonucleotides.


Assuntos
Cromatografia Líquida/métodos , Oligonucleotídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Bases , Primers do DNA , Nanotecnologia
12.
Electrophoresis ; 23(24): 4092-103, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12481265

RESUMO

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/análise , Adutos de DNA/análise , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , Adutos de DNA/química , Eletroforese Capilar/métodos , Conformação Molecular , Espectrometria de Massas por Ionização por Electrospray/métodos
13.
Artigo em Inglês | MEDLINE | ID: mdl-12361746

RESUMO

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.


Assuntos
Anfetaminas/análise , Cromatografia Líquida/métodos , Cocaína/análogos & derivados , Cocaína/análise , Entorpecentes/análise , Saliva/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Humanos , Padrões de Referência , Reprodutibilidade dos Testes
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