RESUMO
Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Humanos , Endotoxinas/genética , Edição de Genes/métodos , Proteínas RepressorasRESUMO
The human leukocyte antigen (HLA) system comprises cell-surface proteins responsible for the presentation of peptide antigens. HLAs play an essential role in the regulation of the human immune system, and their studies have been crucial to its understanding. To create a sustainable model for the investigation of HLAs, we successfully generated the human iPSC line MURAi003-A derived from the peripheral blood mononuclear cells of a donor with homozygous Class I and Class II HLAs (A*11:01, B*46:01; C*01:02; DRB1*09:01; DQB1*03:03) using non-integrative reprogramming episomes. MURAi003-A exhibited pluripotent stem cell characteristics with consistent demonstrations of pluripotency and expression of stem cell-associated markers.
Assuntos
Células-Tronco Pluripotentes Induzidas , Leucócitos Mononucleares , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/citologia , Linhagem Celular , Homozigoto , Diferenciação Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/genéticaRESUMO
Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes. TET1 missense mutations are previously reported in many types of leukemia. Here, the human induced pluripotent stem cell line MURAi001-A was generated from skin fibroblasts derived from a 56-year-old female patient carrying the TET1 gene mutation c.4404A > G (p.I1468M), who had a history of ovarian germ cell tumor. The MURAi001-A cell line demonstrated embryonic-like characteristics as it expressed specific stemness markers, differentiated into the three germ layers, and retained normal karyotyping.
Assuntos
Fibroblastos , Células-Tronco Pluripotentes Induzidas , Oxigenases de Função Mista , Proteínas Proto-Oncogênicas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fibroblastos/metabolismo , Feminino , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutação , Linhagem Celular , Pele/citologia , Pele/patologia , Diferenciação CelularRESUMO
BACKGROUND: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS. RESULTS: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS. CONCLUSIONS: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.
Assuntos
Enterocytozoon/patogenicidade , Penaeidae/microbiologia , Animais , Sistema Digestório/microbiologia , Sistema Digestório/patologia , Enterocytozoon/genética , Enterocytozoon/fisiologia , Hibridização In Situ/veterinária , Penaeidae/anatomia & histologia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico/genética , Alinhamento de Sequência/veterináriaRESUMO
OBJECTIVE: The aim of this study was to compare semen parameters and sperm DNA fragmentation (SDF) and explore the relationship between semen parameters and SDF between 2 and 7 days of abstinence and a short abstinence period (within 4 hours) in oligozoospermic infertile patients. METHODS: Two semen samples were collected from infertile oligozoospermic men (n=34) after an abstinence period of 2 to 7 days and within 4 hours, respectively. Sperm parameters were compared between the two abstinence duration groups, including semen volume, sperm concentration, total sperm count, sperm motility, total motile sperm count (TMSC), morphology, and SDF. RESULTS: The semen volume, concentration, and total sperm count were significantly decreased after 4 hours of abstinence than after 2 to 7 days of abstinence, with median differences of 1.2 mL (p<0.001), 2×106/mL (p=0.011), and 9.6×106/ejaculation (p<0.001), respectively. TMSC was significantly lower after a short abstinence, with a median difference of 4.24×106/ejaculate (p<0.001). However, there were no significance differences in the percentage of motility, the SDF, and the percentage of sperm with normal morphology. Interestingly, volume, concentration, total sperm count, sperm motility, and SDF, but not TMSC, exhibited significant linear correlations between the two abstinence groups in univariate regression analysis, except for TMSC. CONCLUSION: In oligozoospermic men, the volume, concentration, and total sperm count were significantly lower after a short abstinence period, but without adverse effects on sperm motility and SDF.
RESUMO
Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P<0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.
Assuntos
Penaeidae/enzimologia , Pró-Proteína Convertase 2/química , Pró-Proteína Convertase 2/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Imuno-Histoquímica , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurossecreção/genética , Reação em Cadeia da Polimerase , Pró-Proteína Convertase 2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-HíbridoRESUMO
MUi028-A human induced pluripotent stem cell (hiPSC) line was generated from normal fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT4, SOX2, KLF4, L-MYC, and LIN28, and TP53 shRNA in three episomal vectors were delivered by electroporation. The MUi028-A cell line had embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of in vitro differentiation into the three germ layers. This iPSC line can be used as a control to study disease mechanisms.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Feto , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
The accumulation of unbound α-globin chains in red blood cells is a crucial pathophysiology of ß-thalassemia. IOX1 (5-carboxy-8-hydroxyquinoline) is a broad-spectrum 2-oxoglutarate (2OG)-dependent oxygenase inhibitor that can reduce α-globin mRNA expression in human cord blood erythroid progenitor cells. Therefore, IOX1 has been proposed as a potential compound for ß-thalassemia treatment through the decrease in α-globin chain synthesis. However, there is no empirical evidence regarding the consequences of IOX1 in ß-thalassemia. In this study, the therapeutic effects of IOX1 were investigated in ß0-thalassemia/hemoglobin E (HbE) erythroid progenitor cells during in vitro erythropoiesis. The results indicated that IOX1 had no impact on α-globin gene expression, but it led instead to significant decreases in γ-globin and fetal hemoglobin (HbF, α2γ2) production without affecting well-known globin regulators: KLF1, BCL11A, LRF, and GATA1. In addition, differential mRNA expression of several genes in the hypoxia response pathway revealed the induction of EGLN1, the PHD2-encoding gene, as a result of IOX1 treatment. These findings suggested that IOX1 fails to lower α-globin gene expression; on the contrary, it mediates γ-globin and HbF silencing in ß0-thalassemia/HbE erythroid progenitor cells. Because of the negative correlation of EGLN1 and γ-globin gene expression after IOX1 treatment, repurposing IOX1 to study the hypoxia response pathway and γ-globin regulation may provide beneficial information for ß-thalassemia.
Assuntos
Hemoglobina E , Talassemia , Talassemia beta , Adulto , Proteínas de Transporte/metabolismo , Células Eritroides/metabolismo , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal , Hemoglobina E/genética , Hemoglobina E/metabolismo , Humanos , Hipóxia/metabolismo , RNA Mensageiro/genética , Talassemia/metabolismo , alfa-Globinas/metabolismo , Talassemia beta/terapia , gama-Globinas/genéticaRESUMO
Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of ß-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in ß0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells.
Assuntos
Talassemia , Talassemia beta , Adulto , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Humanos , Fatores de Transcrição/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
Hemoglobin Constant Spring (HbCS) is unstable hemoglobin resulting from a nucleotide substitution at the termination codon of the HBA2 gene (c.427 T > C). The homozygous state for HbCS is non-transfusion dependent in adults. Nevertheless, severe anemia is often observed in fetuses. Here, human induced pluripotent stem cell line MUi034-A was generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) derived from a 14-year-old female with homozygous HbCS who had a history of severe anemia and hydrops during fetal period. The MUi034-A cell line represented embryonic-like characteristics as they expressed specific pluripotency markers, differentiated into the three germ layers, and retained normal karyotyping.
Assuntos
Anemia , Células-Tronco Pluripotentes Induzidas , Humanos , AdolescenteRESUMO
The Krüppel-like factor (KLF) family dominates highly conserved three zinc finger DNA binding domains at the C-terminus and variable transactivation domains at the N-terminus. Humans possess 18 KLF genes that are differentially expressed in various tissues. Several KLFs recognize a specific CACCC DNA motif that is commonly found within hematopoietic-specific promoters. To investigate those KLFs that are involved in human hemoglobin (Hb) switching, the present study analyzed a previous microarray data set from fetal and adult erythroid cells and validated the mRNA expression levels of 18 KLFs by reverse transcription-quantitative PCR (RT-qPCR). KLF with a decreased expression level in the fetuses was selected for a functional study in human erythroid progenitor cells using lentiviral-based short hairpin RNA knockdown. The fetuses demonstrated a lower level of KLF4 mRNA expression when compared with the adults. Downregulation of KLF4 in erythroid progenitor cells from healthy individuals and individuals with ß0-thalassemia/HbE evidenced the increasing embryonic and fetal globin mRNA expression with neither significant cytotoxicity nor gene expression alteration of the examined globin regulators, KLF1, B-cell lymphoma/leukemia 11A and lymphoma/leukemia-related factor. These findings demonstrate that the downregulation of KLF4 is associated with increased embryonic and fetal globin gene expression in human erythroid progenitor cells. Moreover, identifying putative compounds or molecular approaches that effectively downregulate KLF4 and further induce embryonic globin expression may provide an alternative therapeutic strategy for α-globin substitution in severe α-thalassemia.
RESUMO
The 13q deletion syndrome is a rare chromosomal disorder caused by loss of the long arm of chromosome 13, and usually entails developmental delay, intellectual disability, behavioral problems and distinctive facial features. In this study, we successfully generated a human iPSC line (MUi015-A) from skin fibroblasts of a patient who had large deletion of chromosome 13, del(13)(q14q22). The MUi015-A line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into three germ layers. The cell line provides a good tool in studying pathophysiology of the tumors, drug testing and gene therapy.
Assuntos
Transtornos Cromossômicos , Células-Tronco Pluripotentes Induzidas , Neoplasias da Retina , Retinoblastoma , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 13/genética , Fibroblastos , Humanos , Retinoblastoma/genéticaRESUMO
Mutations in MYH9 gene is one of the major causes of inherited thrombocytopenia resulted from nonfunctional myosin-9 protein. We have generated a human induced pluripotent stem cell line MUi010-A from skin fibroblasts of a patient who had a point mutation c.2104C>T (p.R702C) in the exon 16 of MYH9 gene using a non-integrative reprogramming method. The MUi010-A exhibited embryonic stem cell-like characteristics with consistent pluripotent markers expression, was capable of all three embryonic germ layers differentiation, and had a normal karyotype.
Assuntos
Linhagem Celular , Células-Tronco Pluripotentes Induzidas , Cadeias Pesadas de Miosina/genética , Animais , Impressões Digitais de DNA , Fibroblastos , Humanos , Cariótipo , Masculino , Camundongos Endogâmicos BALB C , Mutação Puntual , Pele , Trombocitopenia/genéticaRESUMO
BACKGROUND: A key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease and ß-thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain. METHODS: We obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+ cells were cultured in vitro in a fetal bovine serum-based culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset. RESULTS: FL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by meta-analysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. The in vivo relevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogen-activated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways. DISCUSSION: The genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression.
RESUMO
The MUi019-A human induced pluripotent stem cell line was generated from peripheral blood CD34+ hematopoietic progenitors of a healthy woman using a non-integrative reprogramming method. Episomal vectors carrying reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 and EBNA-1 were delivered using electroporation. The iPSC line can be used as a control in studying disease mechanisms. Furthermore, gene editing approaches can be used to introduce specific mutations into the MUi019-A to model disease while the cell type affected by the disease is inaccessible.
Assuntos
Reprogramação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Adulto , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Fator 4 Semelhante a Kruppel , Microscopia de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The thalassemias are a group of genetic disorders characterized by a deficiency in the synthesis of globin chains. In this study the MUi009-A human induced pluripotent stem cell line was successfully generated from peripheral blood CD34+ haematopoietic progenitors of a 32year old male who had coinherited a homozygous ß°-thalassemia mutation at codon 41/42 (-TCTT) and a heterozygous α-thalassemia 4.2 deletion. The MUi009-A cell line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into the three germ layers. The cell line may provide a tool for drug testing and gene therapy studies.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Talassemia alfa/patologia , Adulto , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Deleção de Genes , Genótipo , Heterozigoto , Humanos , Cariótipo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Microscopia de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Talassemia alfa/genética , Talassemia alfa/metabolismoRESUMO
Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.
Assuntos
Reprogramação Celular , Hemoglobinas Anormais/genética , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Talassemia alfa/genética , Talassemia alfa/metabolismo , Talassemia alfa/patologiaRESUMO
Intracellular fatty acid-binding proteins (FABPs) are small members of the superfamily of lipid-binding proteins, which occur in invertebrates and vertebrates. Included in this superfamily are the cellular retinoic acid-binding proteins and retinol-binding proteins, which seem to be restricted to vertebrates. Here, we report the cDNA cloning and characterization of two FABPs from hemocytes of the freshwater crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon. In both these proteins, the binding triad residues involved in interaction with ligand carboxylate groups are present. From the sequence and homology modeling, the proteins are probably FABPs and not retinoic acid-binding proteins. The crayfish transcript (plFABP) was detected at high level in hemocytes, hepatopancreas, intestine and ovary and at low level in hematopoietic tissue and testis. Its expression in hematopoietic cells varied depending on the state of the crayfish from which it was isolated. Expression was 10-15 times higher in cultures isolated from crayfish with red colored plasma, in which hemocyte synthesis was high, if retinoic acid was added to the culture medium. In normal colored crayfish, with normal levels of hemocytes, no increase in expression of p1FABP was detected. Two other putative plFABP ligands, stearic acid and oleic acid, did not have any effect on plFABP expression in hematopoietic cells. These results suggest that retinoic acid-dependent signaling may be present in crustaceans.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Hemócitos/metabolismo , Sequência de Aminoácidos , Animais , Astacoidea , Sequência de Bases , Etiquetas de Sequências Expressas , Modelos Moleculares , Dados de Sequência Molecular , Ácido Oleico/metabolismo , Penaeidae , Homologia de Sequência de Aminoácidos , Ácidos Esteáricos/química , Distribuição Tecidual , Tretinoína/metabolismoRESUMO
Human iPSC line MU011.A-hiPS was generated from homozygous α-thalassemia (-(SEA)/-(SEA)) fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 contained in three episomal vectors were delivered using electroporation.