RESUMO
Hair loss is one of the most common skin problems experienced by more than half of the world's population. In East Asia, medicinal herbs have been used widely in clinical practice to treat hair loss. Recent studies, including systematic literature reviews, indicate that medicinal herbs may demonstrate potential effects for hair loss treatment. In a previous study, we identified medical herbs used frequently for alopecia treatment. Herein, we explored the potential novel therapeutic mechanisms of 20 vital medicinal herbs for alopecia treatment that could distinguish them from known mechanisms of conventional drugs using network pharmacology analysis methods. We determined the herb-ingredient-target protein networks and ingredient-associated protein (gene)-associated pathway networks and calculated the weighted degree centrality to define the strength of the connections. Data showed that 20 vital medicinal herbs could exert therapeutic effects on alopecia mainly mediated via regulation of various target genes and proteins, including acetylcholinesterase (AChE), phospholipase A2 (PLA2) subtypes, ecto-5-nucleotidase (NTE5), folate receptor (FR), nicotinamide N-methyltransferase (NNMT), and quinolinate phosphoribosyltransferase (QPRT). Findings regarding target genes/proteins and pathways of medicinal herbs associated with alopecia treatment offer insights for further research to better understand the pathogenesis and therapeutic mechanism of medicinal herbs for alopecia treatment with traditional herbal medicine.
Assuntos
Alopecia/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Farmacologia em Rede , Plantas Medicinais , Acetilcolinesterase/genética , Alopecia/genética , Alopecia/prevenção & controle , Ásia Oriental , Receptor 1 de Folato/genética , Humanos , Medicina Tradicional Chinesa , Nicotinamida N-Metiltransferase/genética , Nucleotidases/genética , Pentosiltransferases/genética , Fosfolipases A2/genética , Fitoterapia , Preparações de Plantas/química , Preparações de Plantas/uso terapêuticoRESUMO
Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Glicosídeo Hidrolases/metabolismo , Niacinamida/análogos & derivados , Compostos de Piridínio/farmacocinética , Células A549 , ADP-Ribosil Ciclase/genética , Administração Oral , Envelhecimento/efeitos dos fármacos , Animais , Antígenos CD/genética , Suplementos Nutricionais , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Microbioma Gastrointestinal , Glicosídeo Hidrolases/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Camundongos , Camundongos Knockout , Niacina/metabolismo , Niacinamida/administração & dosagem , Niacinamida/metabolismo , Niacinamida/farmacocinética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Compostos de Piridínio/administração & dosagemRESUMO
Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.
Assuntos
Antineoplásicos/uso terapêutico , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Pró-Fármacos/administração & dosagem , Animais , Antineoplásicos/farmacologia , Engenharia Celular/métodos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Citosina Desaminase/genética , Exossomos/genética , Flucitosina/administração & dosagem , Flucitosina/metabolismo , Fluoruracila/metabolismo , Proteínas Fúngicas/genética , Vetores Genéticos/genética , Humanos , Camundongos , Pentosiltransferases/genética , Pró-Fármacos/metabolismo , Proteínas Recombinantes de Fusão/genética , Retroviridae/genética , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Distroglicanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Distroglicanas/genética , Edição de Genes , Marcação de Genes , Loci Gênicos , Glicosilação/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem Molecular , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/etiologia , Distrofias Musculares/metabolismo , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismoRESUMO
Exosomes derived from human mesenchymal stem cells (MSCs) engineered to express the suicide gene yeast cytosine deaminase::uracil phosphoribosyl transferase (yCD::UPRT) represent a new therapeutic approach for tumor-targeted innovative therapy. The yCD::UPRT-MSC-exosomes carry mRNA of the suicide gene in their cargo. Upon internalization by tumor cells, the exosomes inhibit the growth of broad types of cancer cells in vitro, in the presence of a prodrug. Here we describe the method leading to the production and testing of these therapeutic exosomes. The described steps include the preparation of replication-deficient retrovirus possessing the yCD::UPRT suicide gene, and the preparation and selection of MSCs transduced with yCD::UPRT suicide gene. We present procedures to obtain exosomes possessing the ability to induce the death of tumor cells. In addition, we highlight methods for the evaluation of the suicide gene activity of yCD::UPRT-MSC-exosomes.
Assuntos
Exossomos , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Retroviridae/genética , Animais , Linhagem Celular , Citosina Desaminase/genética , Portadores de Fármacos , Proteínas Fúngicas/genética , Vetores Genéticos , Humanos , Células-Tronco Mesenquimais , Camundongos , Pentosiltransferases/genética , Leveduras/enzimologiaRESUMO
The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.
Assuntos
Exossomos/metabolismo , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Células-Tronco Mesenquimais/metabolismo , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Exossomos/genética , Flucitosina/metabolismo , Fluoruracila/metabolismo , Fluoruracila/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Pró-Fármacos/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
The intestinal mucus layer plays an important role in the management of inflammatory bowel disease. The aim of this study was to investigate the effects of oxyresveratrol (OXY), an antioxidant, on the stimulation of mucin production in human LS 174T goblet cells and the underlying mechanism thereof. OXY increased MUC2 expression at both the mRNA and protein levels. By performing two-dimensional gel electrophoresis, we found that the expression of nicotinic acid phosphoribosyltransferase1 (NaPRT1) in OXY-treated LS 174T cells was greatly increased compared with that in negative control cells. In addition, the NAD+/NADH ratio was increased in proportion to OXY in LS 174T cells. The expression of NAD+-synthesis enzymes, NaPRT1, nicotinamide riboside kinase1 (NRK1) and nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) was significantly increased at both the mRNA and protein levels in OXY-treated LS 174T cells. The inhibition of NaPRT1 and NRK1 did not decrease MUC2 expression after inhibiting by small interfering RNA (siRNA)-NaPRT1 and siRNA-NRK1, respectively; however, inhibition of Nmnat by an Nmnat inhibitor decreased MUC2 expression in a dose-dependent manner. In conclusion, OXY increases NAD+ levels, resulting in the stimulation of MUC2 expression in LS 174T cells. These findings present a novel role for NAD+ in stimulation of MUC2 expression.
Assuntos
Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucinas/biossíntese , NAD/metabolismo , Extratos Vegetais/farmacologia , Estilbenos/farmacologia , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Mucosa Intestinal/citologia , Mucina-2/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Magnetic hyperthermia, or the heating of tissues using magnetic materials, is a promising approach for treating cancer. We found that human mesenchymal stem cells (MSCs) isolated from various tissues and MSCs expressing the yeast cytosine deaminaseâ·uracil phosphoribosyl transferase suicide fusion gene (yCDâ·UPRT) can be labeled with Venofer, an iron oxide carbohydrate nanoparticle. Venofer labeling did not affect cell proliferation or the ability to home to tumors. All Venofer-labeled MSCs released exosomes that contained iron oxide. Furthermore, these exosomes were efficiently endocytosed by tumor cells. Exosomes from Venofer-labeled MSCs expressing the yCDâ·UPRT gene in the presence of the prodrug 5-fluorocytosine inhibited tumor growth in a dose-dependent fashion. The treated tumor cells were also effectively ablated following induction of hyperthermia using an external alternating magnetic field. Cumulatively, we found that magnetic nanoparticles packaged into MSC exosomes are efficiently endocytosed by tumor cells, facilitating targeted tumor cell ablation via magnetically induced hyperthermia.
Assuntos
Exossomos/química , Compostos Férricos/química , Ácido Glucárico/química , Hipertermia Induzida/métodos , Células-Tronco Mesenquimais/química , Linhagem Celular Tumoral , Proliferação de Células , Citosina Desaminase/genética , Compostos Férricos/farmacocinética , Óxido de Ferro Sacarado , Células HeLa , Humanos , Campos Magnéticos , Masculino , Nanopartículas/química , Pentosiltransferases/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas Recombinantes/genéticaRESUMO
Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of ß(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the ß(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking ß(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.
Assuntos
Terapia Biológica , Fucose/metabolismo , Glicoproteínas/metabolismo , Nicotiana/genética , Xilose/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Edição de Genes , Vetores Genéticos , Glicoproteínas/genética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polissacarídeos , Proteínas Recombinantes , Nicotiana/metabolismo , UDP Xilose-Proteína XilosiltransferaseRESUMO
Ethambutol (EMB) is an essential first-line drug for tuberculosis (TB) treatment. Nucleotide substitutions at embB codon 306 (embB306) have been proposed to be a potential marker for EMB resistance and a predictor of broad drug resistance in clinical Mycobacterium tuberculosis isolates. However, discordant findings about the association between embB306 mutations and EMB resistance were reported. Hebei Province is located in the Beijing-Tianjin-Hebei integration region in China; however, little information about the genetic diversity of the embB locus in this area is available. In this study, we sequenced the region surrounding embB306 (codons 207 to 445) in 62 ethambutol-resistant (EMBr) isolates, 214 ethambutol-susceptible isolates resistant to other first-line drugs (EMBs isolates), and 100 pan-sensitive isolates. Our data indicated that none of the pan-sensitive isolates showed mutations at embB306 and 63 drug-resistant isolates harbored embB306 substitutions, with these substitutions being found in 56.5% (35/62) of EMBr isolates and 13.1% (28/214) of EMBs isolates. A significant association between the embB306 mutation and resistance to isoniazid, rifampin, EMB, and multiple drugs was observed, and the rate of mutation of embB306 increased with increasing numbers of first-line drugs to which the isolates were resistant. The embB306 mutation is not the sole causative factor for EMB resistance, and the poor sensitivity limits its utility as a marker for drug-resistant TB. However, it may be a potential marker for broad drug resistance, especially for multidrug resistance. The mycobacterial interspersed repetitive unit-variable-number tandem-repeat profiles may serve as markers for predicting the embB306 substitutions that may occur in drug-resistant M. tuberculosis isolates under antimicrobial selection pressure.
Assuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Etambutol/uso terapêutico , Mycobacterium tuberculosis/genética , Pentosiltransferases/genética , Tuberculose Pulmonar/tratamento farmacológico , Sequência de Bases , China , DNA Bacteriano/genética , Humanos , Isoniazida/uso terapêutico , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/uso terapêutico , Análise de Sequência de DNA , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-ß-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.
Assuntos
Ganoderma/metabolismo , Glicosídeos/metabolismo , Trametes/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ganoderma/genética , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Humanos , Metabolômica , Interações Microbianas , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Filogenia , Trametes/genética , UDP Xilose-Proteína XilosiltransferaseRESUMO
All cells of terrestrial plants are fortified by walls composed of crystalline cellulose microfibrils and a variety of matrix polymers. Xylans are the second most abundant type of polysaccharides on Earth. Previous studies of Arabidopsis (Arabidopsis thaliana) irregular xylem (irx) mutants, with collapsed xylem vessels and dwarfed stature, highlighted the importance of this cell wall component and revealed multiple players required for its synthesis. Nevertheless, xylan elongation and substitution are complex processes that remain poorly understood. Recently, seed coat epidermal cells were shown to provide an excellent system for deciphering hemicellulose production. Using a coexpression and sequence-based strategy, we predicted several MUCILAGE-RELATED (MUCI) genes that encode glycosyltransferases (GTs) involved in the production of xylan. We now show that MUCI21, a member of an uncharacterized clade of the GT61 family, and IRX14 (GT43 protein) are essential for the synthesis of highly branched xylan in seed coat epidermal cells. Our results reveal that xylan is the most abundant xylose-rich component in Arabidopsis seed mucilage and is required to maintain its architecture. Characterization of muci21 and irx14 single and double mutants indicates that MUCI21 is a Golgi-localized protein that likely facilitates the addition of xylose residues directly to the xylan backbone. These unique branches seem to be necessary for pectin attachment to the seed surface, while the xylan backbone maintains cellulose distribution. Evaluation of muci21 and irx14 alongside mutants that disrupt other wall components suggests that mucilage adherence is maintained by complex interactions between several polymers: cellulose, xylans, pectins, and glycoproteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicosiltransferases/metabolismo , Pentosiltransferases/metabolismo , Mucilagem Vegetal/metabolismo , Sementes/metabolismo , Xilanos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Celulose/metabolismo , Genes Reporter , Glicosiltransferases/genética , Microfibrilas/química , Microfibrilas/metabolismo , Mutação , Pectinas/metabolismo , Pentosiltransferases/genética , Filogenia , Mucilagem Vegetal/química , Polímeros/química , Polímeros/metabolismo , Polissacarídeos/metabolismo , Sementes/genética , Análise de Sequência de DNA , Xilanos/química , Xilema/genética , Xilema/metabolismoRESUMO
Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.
Assuntos
Antineoplásicos/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Hematopoese/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Niacina/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Trombocitopenia/induzido quimicamente , Animais , Antineoplásicos/química , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Suplementos Nutricionais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Interações Alimento-Droga , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macaca fascicularis , Masculino , Megacariócitos/citologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Estrutura Molecular , Niacina/uso terapêutico , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Ratos Sprague-Dawley , Trombocitopenia/metabolismo , Trombocitopenia/prevenção & controleRESUMO
Desbuquois dysplasia (DBQD) is a severe condition characterized by short stature, joint laxity, and advanced carpal ossification. Based on the presence of additional hand anomalies, we have previously distinguished DBQD type 1 and identified CANT1 (calcium activated nucleotidase 1) mutations as responsible for DBQD type 1. We report here the identification of five distinct homozygous xylosyltransferase 1 (XYLT1) mutations in seven DBQD type 2 subjects from six consanguineous families. Among the five mutations, four were expected to result in loss of function and a drastic reduction of XYLT1 cDNA level was demonstrated in two cultured individual fibroblasts. Because xylosyltransferase 1 (XT-I) catalyzes the very first step in proteoglycan (PG) biosynthesis, we further demonstrated in the two individual fibroblasts a significant reduction of cellular PG content. Our findings of XYLT1 mutations in DBQD type 2 further support a common physiological basis involving PG synthesis in the multiple dislocation group of disorders. This observation sheds light on the key role of the XT-I during the ossification process.
Assuntos
Anormalidades Craniofaciais/genética , Nanismo/genética , Instabilidade Articular/genética , Mutação , Ossificação Heterotópica/genética , Pentosiltransferases/genética , Polidactilia/genética , Adolescente , Adulto , Osso e Ossos/metabolismo , Criança , Consanguinidade , DNA Complementar/metabolismo , Exoma , Feminino , Fibroblastos/metabolismo , Predisposição Genética para Doença , Homozigoto , Humanos , Masculino , Repetições de Microssatélites/genética , Pentosiltransferases/metabolismo , Proteoglicanas/metabolismo , Análise de Sequência de DNA , UDP Xilose-Proteína XilosiltransferaseRESUMO
Plant cell walls are complex, multi-macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell-wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell-wall matrix glycan sub-populations and inter-connections. The method combines chromatographic separations with use of glycan-directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell-wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub-populations and their modulation in A. thaliana organs.
Assuntos
Parede Celular/química , Cromatografia/métodos , Epitopos/análise , Plantas/química , Polissacarídeos/análise , Anticorpos Monoclonais/imunologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Epitopos/imunologia , Glucanos/análise , Glucanos/química , Pectinas/análise , Pectinas/química , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/química , Brotos de Planta/genética , Brotos de Planta/metabolismo , Polissacarídeos/química , Polissacarídeos/imunologia , Reprodutibilidade dos Testes , Xilanos/análise , Xilanos/químicaRESUMO
Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⻹ xylose and produced 15.5 g L⻹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⻹ xylose, 2.1 g L⻹ glucose and 1.5 g L⻹ arabinose, it produced a final succinate concentration of 17.2 g L⻹ with a yield of 0.94 g g⻹ total sugars.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Escherichia coli K12/enzimologia , Microbiologia Industrial/métodos , Pentosiltransferases/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Succinatos/metabolismo , Anaerobiose , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Reatores Biológicos , Clonagem Molecular , Escherichia coli K12/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Fermentação , Genes Bacterianos , Genes Sintéticos , Hidrólise , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Extratos Vegetais/metabolismo , Brotos de Planta , Succinatos/isolamento & purificação , Resíduos , Xilose/metabolismo , Zea maysRESUMO
Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Parede Celular/química , Pectinas/metabolismo , Pentosiltransferases/metabolismo , Polissacarídeos/metabolismo , Anticorpos Monoclonais , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Fenômenos Biomecânicos , Epitopos , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inflorescência/química , Inflorescência/citologia , Inflorescência/genética , Inflorescência/fisiologia , Mutação , Especificidade de Órgãos , Pentosiltransferases/genética , Epiderme Vegetal/química , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Epiderme Vegetal/fisiologia , Caules de Planta/química , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/fisiologia , Plantas Geneticamente Modificadas , Polissacarídeos/imunologia , Plântula/química , Plântula/citologia , Plântula/genética , Plântula/fisiologia , Estresse MecânicoRESUMO
NAD(+) is synthesized from tryptophan either via the kynurenine (de novo) pathway or via the salvage pathway by reutilizing intermediates such as nicotinic acid or nicotinamide ribose. Quinolinic acid is an intermediate in the kynurenine pathway. We have discovered that the budding yeast Saccharomyces cerevisiae secretes quinolinic acid into the medium and also utilizes extracellular quinolinic acid as a novel NAD(+) precursor. We provide evidence that extracellular quinolinic acid enters the cell via Tna1, a high-affinity nicotinic acid permease, and thereby helps to increase the intracellular concentration of NAD(+). Transcription of genes involved in the kynurenine pathway and Tna1 was increased, responding to a low intracellular NAD(+) concentration, in cells bearing mutations of these genes; this transcriptional induction was suppressed by supplementation with quinolinic acid or nicotinic acid. Our data thus shed new light on the significance of quinolinic acid, which had previously been recognized only as an intermediate in the kynurenine pathway.
Assuntos
Vias Biossintéticas , Cinurenina/biossíntese , NAD/biossíntese , Ácido Quinolínico/metabolismo , Saccharomyces cerevisiae/fisiologia , Meios de Cultivo Condicionados , Expressão Gênica , Técnicas de Inativação de Genes , Genes Fúngicos , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Transcrição GênicaRESUMO
Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing uracil phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5-fluorouracil (5-FU), in immunodeficient, Ad-nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5-FU therapy and of its combination with CP for syngenic HaP-T1 pancreatic cancers in immunocompetent, Ad-permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5-FU in HaP-T1 cells in vitro. I.t. AxE1CAUP/5-FU treatment inhibited the growth of subcutaneous HaP-T1 allografts. The combination with high-dose CP inhibited serum Ad-neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5-FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically-approved, intermediate-dose CP also enhanced the efficacy of i.t. AxE1CAUP/5-FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad-permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV-mediated gene therapy.
Assuntos
Ciclofosfamida/uso terapêutico , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Pentosiltransferases/genética , Animais , Linhagem Celular Tumoral , Cricetinae , Feminino , Fluoruracila/uso terapêutico , Imunocompetência , Mesocricetus , Transdução GenéticaRESUMO
Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy.