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Sulfur is essential for plant growth, and the uptake of sulfate by plant roots is the primary source of plant sulfur. Previous studies have shown that the OAS-TL gene is a key enzyme in the sulfur metabolic pathway and regulates cysteine (Cys) synthase production. However, the interaction mechanism of the glycine max OAS-TL3 Cys synthase (OAS-TL3) gene on soybean root morphology construction and seed protein accumulation is unclear. This study shows that mutant M18 has better root growth and development, higher seed protein content, and higher methionine (Met) content in sulfur-containing amino acids than wild-type JN18. By transcriptome sequencing, the differentially expressed OAS-TL3 gene was targeted in the mutant M18 root line. The relative expression of the OAS-TL3 gene in roots, stems, and leaves during the seedling, flowering, and bulking stages of the OAS-TL3 gene overexpression lines is higher than that of the recipient material. Compared to the recipient material JN74, the enzymatic activities, Cys, and GSH contents of OAS-TL are higher in the sulfur metabolic pathway of seedling roots. The receptor material JN74 is exogenously applied with different concentrations of reduced glutathione. The results demonstrate a positive correlation between reduced glutathione on total root length, projected area, surface area, root volume, total root tip number, total bifurcation number, and total crossing number. The Met and total protein contents of sulfur-containing amino acids in soybean seeds of the OAS-TL3 gene overexpression lines are higher than those of the recipient material JN74, while the gene-edited lines show the opposite results. In conclusion, the OAS-TL3 gene positively regulates soybean root growth, root activity, and the content of Met in the seeds through the OAS-TL-Cys-GSH pathway. It breaks the limitation of other amino acids and facilitates the increase of total seed protein content. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01348-y.
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In order to study the role of GmXTH1 gene in alleviating drought stress, soybean seeds with GmXTH1 gene were transferred by T4 treated with PEG6000 concentration of 0%, 5%, 10%, and 15% respectively. The germination potential, germination rate, germination index, and other indicators were measured. The results showed that the germination potential, germination rate, and germination index of OEA1 and OEA2 strains overexpressed in T4 generation were significantly higher than those of the control material M18. After 0-day, 7-day, and 15-day drought stress, the analysis of seedling phenotypes and root-shoot of different T4 generation transgenic soybean lines showed that under stress conditions, the growth of GmXTH1 overexpression material was generally better than that of the control material M18. The growth of GmXTH1 interference expression material was generally worse than that of the control material M18, with significant differences in plant phenotypes. The root system of GmXTH1 overexpressed material was significantly developed compared with that of the control material M18. The analysis of physiological and biochemical indexes showed that the relative water content and the activity of antioxidant enzymes (superoxide dismutase and peroxidase) of GmXTH1 transgenic soybean material were significantly higher than those of the control material M18, and the accumulation of malondialdehyde was lower under the same stress conditions at seedling stage. Fluorescence quantitative PCR assay showed that the relative expression of GmXTH1 gene in transgenic soybean was significantly increased after drought stress. The results showed that the overexpression of GmXTH1 could increase the total root length, surface area, total projection area, root volume, average diameter, total cross number, and total root tip number, thereby increasing the water intake and reducing the transpiration of water content in leaves, thus reducing the accumulation of MDA and producing more protective enzymes in a more effective and prompt way, reducing cell membrane damage to improve drought resistance of soybean.
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BACKGROUND: The ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. METHODS: We combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. RESULTS: We identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)-positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. CONCLUSIONS: Together, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.
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Complexo Nuclear Basolateral da Amígdala , Núcleo Central da Amígdala , Núcleos Septais , Camundongos , Animais , Neurônios/fisiologia , Medo/fisiologiaRESUMO
BACKGROUND: With the rapidly increasing prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM), neuronal complications associated with these diseases have resulted in significant burdens on healthcare systems. Meanwhile, effective therapies have remained insufficient. A novel fatty acid called S-9-PAHSA has been reported to provide metabolic benefits in T2DM by regulating glucose metabolism. However, whether S-9-PAHSA has a neuroprotective effect in mouse models of T2DM remains unclear. METHODS: This in vivo study in mice fed a high-fat diet (HFD) for 5 months used fasting blood glucose, glucose tolerance, and insulin tolerance tests to examine the effect of S-9-PAHSA on glucose metabolism. The Morris water maze test was also used to assess the impact of S-9-PAHSA on cognition in the mice, while the neuroprotective effect of S-9-PAHSA was evaluated by measuring the expression of proteins related to apoptosis and oxidative stress. In addition, an in vitro study in PC12 cells assessed apoptosis, oxidative stress, and mitochondrial membrane potential with or without CAIII knockdown to determine the role of CAIII in the neuroprotective effect of S-9-PAHSA. RESULTS: S-9-PAHSA reduced fasting blood glucose levels significantly, increased insulin sensitivity in the HFD mice and also suppressed apoptosis and oxidative stress in the cortex of the mice and PC12 cells in a diabetic setting. By suppressing oxidative stress and apoptosis, S-9-PAHSA protected both neuronal cells and microvascular endothelial cells in in vivo and in vitro diabetic environments. Interestingly, this protective effect of S-9-PAHSA was reduced significantly when CAIII was knocked down in the PC12 cells, suggesting that CAIII has a major role in the neuroprotective effect of S-9-PAHSA. However, overexpression of CAIII did not significantly enhance the protective effect of S-9-PAHSA. CONCLUSION: S-9-PAHSA mediated by CAIII has the potential to exert a neuroprotective effect by suppressing apoptosis and oxidative stress in neuronal cells exposed to diabetic conditions. Furthermore, S-9-PAHSA has the capability to reduce fasting blood glucose and LDL levels and enhance insulin sensitivity in mice fed with HFD.
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Diabetes Mellitus Tipo 2 , Resistência à Insulina , Fármacos Neuroprotetores , Ácido Palmítico , Ácidos Esteáricos , Animais , Camundongos , Ratos , Apoptose , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , Anidrase Carbônica III/efeitos dos fármacos , Anidrase Carbônica III/metabolismoRESUMO
Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.
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Neoplasias do Colo , Ácido Mevalônico , Humanos , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Dissulfeto de Glutationa/metabolismo , Dissulfeto de Glutationa/farmacologia , Ácido Mevalônico/farmacologia , NADP/metabolismo , NADP/farmacologia , Via de Pentose Fosfato/fisiologia , Estresse Oxidativo , Neoplasias do Colo/tratamento farmacológicoRESUMO
Licking behavior is important for water intake. The deep mesencephalic nucleus (DpMe) has been implicated in instinctive behaviors. However, whether the DpMe is involved in licking behavior and the precise neural circuit behind this behavior remains unknown. Here, we found that the activity of the DpMe decreased during water intake. Inhibition of vesicular glutamate transporter 2-positive (VGLUT2+) neurons in the DpMe resulted in increased water intake. Somatostatin-expressing (SST+), but not protein kinase C-δ-expressing (PKC-δ+), GABAergic neurons in the central amygdala (CeA) preferentially innervated DpMe VGLUT2+ neurons. The SST+ neurons in the CeA projecting to the DpMe were activated at the onset of licking behavior. Activation of these CeA SST+ GABAergic neurons, but not PKC-δ+ GABAergic neurons, projecting to the DpMe was sufficient to induce licking behavior and promote water intake. These findings redefine the roles of the DpMe and reveal a novel CeASST-DpMeVGLUT2 circuit that regulates licking behavior and promotes water intake.
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Núcleo Central da Amígdala , Animais , Comportamento Animal , Neurônios GABAérgicos/fisiologia , Mesencéfalo/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HTâ©vGluT3 neurons projecting to BA parvalbumin (DRN5-HTâ©vGluT3-BAPV) and pyramidal (DRN5-HTâ©vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HTâ©vGluT3âBAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HTâ©vGluT3âBA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.
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Transtornos de Ansiedade , Complexo Nuclear Basolateral da Amígdala , Serotonina , Animais , Camundongos , Tonsila do Cerebelo , Ansiedade , Receptores de GABA-BRESUMO
Heteroatom doping is a promising strategy to optimize the electronic structure of transition metal phosphides so enhancing the hydrogen evolution reaction (HER). However, complex and harsh experimental design is often required to achieve homogeneous doping of corresponding elements while achieving the best regulating effect. Herein, a facile ion-exchange (IE) strategy is applied to dope Mo/V species evenly into Ni-Co phosphides under mild conditions while maintaining the nanoneedle morphology. The electrochemical characterization verifies Mo dopants have a better electronic regulation effect on NiCoP crystal than V dopants, corresponding to the better hydrogen evolution performance of Mo-NiCoP/NF. Notably, due to the highly dispersed nanoneedle morphology, the synergistic effect of Ni-Co phosphides, and the optimized electronic structure, Mo-NiCoP/NF demonstrates a higher activity than that of the noble metal Pt/C at the high current density (>99 mA cm-2). The present work is supposed to open new sights for the development of high-performance catalysts by ion-exchange strategy.
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The amygdala, which is involved in various behaviors and emotions, is reported to connect with the whole brain. However, the long-range inputs of distinct cell types have not yet been defined. Here, we used a retrograde trans-synaptic rabies virus to generate a whole-brain map of inputs to the main cell types in the mouse amygdala. We identified 37 individual regions that projected to neurons expressing vesicular glutamate transporter 2, 78 regions to parvalbumin-expressing neurons, 104 regions to neurons expressing protein kinase C-δ, and 89 regions to somatostatin-expressing neurons. The amygdala received massive projections from the isocortex and striatum. Several nuclei, such as the caudate-putamen and the CA1 field of the hippocampus, exhibited input preferences to different cell types in the amygdala. Notably, we identified several novel input areas, including the substantia innominata and zona incerta. These findings provide anatomical evidence to help understand the precise connections and diverse functions of the amygdala.
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Tonsila do Cerebelo , Mapeamento Encefálico , Tonsila do Cerebelo/fisiologia , Animais , Região CA1 Hipocampal/fisiologia , Masculino , Camundongos , Vias Neurais/fisiologia , Parvalbuminas/fisiologia , Proteínas Vesiculares de Transporte de Glutamato/fisiologiaRESUMO
INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a representative rare disease characterised by progressive, fatal motor neuron degeneration. Due to the unknown aetiology and variability of the phenotypes, there are no accurate reports concerning the epidemiology or clinical characteristics of the disease. The low prevalence, as previously reported, makes it difficult to carry out studies with large samples. The aim of this study was to explore the natural history and clinical features of ALS in mainland China through a multicentre, prospective cohort study. The findings will both offer a better understanding of ALS and also support the development of a model to study other rare diseases. METHODS AND ANALYSIS: Patients from 88 representative hospitals in different parts of mainland China will be recruited through a specially designed online data system (http://www.chalsr.net/). We aim to recruit 4752 ALS patients over a 3-year period. Baseline data will be recorded, and follow-up data will be collected every 3 months. The primary outcome is effective survival. Overall survival and indices of disease progression will be measured as the secondary outcomes. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the ethics committee of Peking University Third Hospital (M2019388). Informed written consent will be obtained from each participant. Dissemination of the study protocol and data will take place primarily through a specially designed online data system (http://www.chalsr.net/). The collective results of the study will be published in peer-reviewed journals and shared in scientific presentations. TRIAL REGISTRATION NUMBER: NCT04328675.
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Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/epidemiologia , China/epidemiologia , Progressão da Doença , Humanos , Estudos Multicêntricos como Assunto , Estudos Prospectivos , Sistema de RegistrosRESUMO
Methyl-CpG-binding protein 2 (MeCP2) encoded by the MECP2 gene is a transcriptional regulator whose mutations cause Rett syndrome (RTT). Mecp2-deficient mice show fear regulation impairment; however, the cellular and molecular mechanisms underlying this abnormal behavior are largely uncharacterized. Here, we showed that Mecp2 gene deficiency in cholinergic interneurons of the nucleus accumbens (NAc) dramatically impaired fear learning. We further found that spontaneous activity of cholinergic interneurons in Mecp2-deficient mice decreased, mediated by enhanced inhibitory transmission via α2-containing GABAA receptors. With MeCP2 restoration, opto- and chemo-genetic activation, and RNA interference in ChAT-expressing interneurons of the NAc, impaired fear retrieval was rescued. Taken together, these results reveal a previously unknown role of MeCP2 in NAc cholinergic interneurons in fear regulation, suggesting that modulation of neurons in the NAc may ameliorate fear-related disorders.
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Neurônios Colinérgicos/metabolismo , Medo/fisiologia , Interneurônios/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Receptores de GABA-A/metabolismo , Animais , Modelos Animais de Doenças , Aprendizagem/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleo Accumbens/metabolismo , Interferência de RNARESUMO
In the version of this article originally published, there were several errors in Fig. 4. In Fig. 4a, the title read '3D repeated optical inhibition after CSDS.' It should have read '3-day repeated optical inhibition after CSDS.' In Fig. 4c, two labels that should have been aligned with the time axis appeared in the wrong place in the figure. The ticks labeled 'SI' and 'Fiber implant' should have also been labeled with '10' and '14,' respectively. Additionally, in Fig. 4j, a label that should have been aligned with the time axis appeared in the wrong place in the figure. The tick labeled 'Fiber implant' should have also been labeled with '14.' The errors have been corrected in the print, PDF and HTML versions of the manuscript.
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Major depressive disorder is a devastating psychiatric disease that afflicts up to 17% of the world's population. Postmortem brain analyses and imaging studies of patients with depression have implicated basal lateral amygdala (BLA) dysfunction in the pathophysiology of depression. However, the circuit and molecular mechanisms through which BLA neurons modulate depressive behavior are largely uncharacterized. Here, in mice, we identified that BLA cholecystokinin (CCK) glutamatergic neurons mediated negative reinforcement via D2 medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and that chronic social defeat selectively potentiated excitatory transmission of the CCKBLA-D2NAc circuit in susceptible mice via reduction of presynaptic cannabinoid type-1 receptor (CB1R). Knockdown of CB1R in the CCKBLA-D2NAc circuit elevated synaptic activity and promoted stress susceptibility. Notably, selective inhibition of the CCKBLA-D2NAc circuit or administration of synthetic cannabinoids in the NAc was sufficient to produce antidepressant-like effects. Overall, our studies reveal the circuit and molecular mechanisms of depression.
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Tonsila do Cerebelo/metabolismo , Comportamento Animal , Colecistocinina/metabolismo , Depressão/metabolismo , Ácido Glutâmico/metabolismo , Neurônios Aferentes/metabolismo , Núcleo Accumbens/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Regulação para Baixo , Masculino , Camundongos Endogâmicos C57BL , Optogenética , Estresse Psicológico/metabolismo , Sinapses/metabolismoRESUMO
To clarify whether expression of the programmed cell death 5 (PDCD5) gene in leukemic cells is abnormal, real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) was used to examine its expression in marrow cells from leukemia patients. We found lower PDCD5 in both AML and CML marrow cells than in normal donor marrow cells. A negative correlation was found between relative levels of PDCD5 and BCR/ABL expression in all CML patients and in CML patients in the advanced phase. Treatment with the ABL tyrosine kinase inhibitor Imatinib mesylate increased PDCD5 expression in K562 and MEG-01 cells. These findings suggest that abnormal expression of PDCD5 in leukemia may be involved in the pathomechanism of AML and CML.
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Proteínas Reguladoras de Apoptose/biossíntese , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biossíntese , Adulto , Proteínas Reguladoras de Apoptose/genética , Benzamidas , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologiaRESUMO
OBJECTIVE: To evaluate ABL tyrosine kinase point mutations in imatinib treated chronic myelogenous leukemia (CML) patients. METHODS: A total of 45 bone marrow samples from 30 CML patients were included in this work. The patients were in accelerated/blast phase (AP/BP) or late-chronic phase (CP) at the start of imatinib and usually showed resistance to imatinib. ABL kinase domain of BCR-ABL allele was amplified by nested reverse transcriptase-polymerase chain reaction technique, followed by direct sequencing and sequence homologous analyzing. RESULTS: The ABL point mutation was detected in 13 of 30 patients, 12 of them had progressed to advanced phase, The other patient who was in late chronic phase showed point mutation when she was at 45th months of imatinib treatment, but she was still in complete cytogenetic remission at 50th months and is doing well. 4 patients had Glu255Lys mutation and 4 had Gly250Glu, the other types of mutation were Phe359Cys, Glu355Gly, Met244Val, Tyr253His and Asp276Gly, each was tested in one patient. 11/12 patients who progressed to advanced disease and showed point mutation were collected samples in advanced stage, 8 patients showed homozygote mutation, and 3 patients had a mixture of wild and mutant type. In advanced stage patients, mutations were detected in a median of 5 months (ranged 0.5-30 months), it appeared much earlier than that in late CP patients (25.5 months, ranged 11-45 months, P < 0.05). In 4/7 followed up patients, the intensity of point mutation increased gradually within 7-15 months before disease progression. 6 patients did not showed ABL point mutation while their disease were in progression. CONCLUSIONS: Abl kinase point mutation is one of the main mechanisms of CML secondary resistance to imatinib. Long term regular monitoring of ABL kinase point mutation is necessary during imatinib treatment.
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Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Piperazinas/uso terapêutico , Mutação Puntual , Proteínas Tirosina Quinases/genética , Pirimidinas/uso terapêutico , Adolescente , Adulto , Idoso , Antineoplásicos/uso terapêutico , Sequência de Bases , Benzamidas , Análise Mutacional de DNA , Feminino , Seguimentos , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
To further elucidate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF(121)cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The growth of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow endothelial cells (BMECs), and examined the synergic effects of antisense VEGF(121)cDNA and IFNalpha or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF(121)cDNA showed growth retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF(121)cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNalpha. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.
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Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Apoptose , Comunicação Celular , Divisão Celular , DNA Antissenso/genética , DNA Antissenso/farmacologia , DNA Complementar/genética , Humanos , Interferon-alfa/farmacologia , Células K562/transplante , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: To analyze the immunophenotype and leukemia associated immunophenotype (LAIP) of leukemia cells from patients with acute myeloid leukemia (AML) in minimal residual disease (MRD) detection. METHODS: Four-color multiparametric flow cytometry (FCM) with CD45/SSC gating was used to determine the expression of the following antibodies of CD7, CD117, CD33, CD34, CD10, CD19, CD56, CD38, CD13, CD14, CD64, CD9, CD16, CD2, CD5, CD11b, CD123, HLA-DR in 610 AML patients and 20 normal bone marrow (NBM) samples. RESULTS: The mean percentages of CD34+ and CD117+ side scatter low (SSC(low)) cells in NBM mononuclear cells (BMMNCs) were (0.35 +/- 0.15)% and (0.76 +/- 0.31)%, respectively. The majority (84% -95%) of CD34+ SSC(low) cells co-expressed CD13, CD33, CD38, CD117 and HLA-DR. 33% and 20% of CD34+ SSC(low) cells were CD15+ and CD9, respectively. Only a small proportion (< 10%) of CD34 SSC(low) cell co-expressed CD11b, CD56, CD19 and CD64, while co-expressed CD7 was 12%. In CD117 SSC(low) cells, the relative proportions of CD19+, CD11b+, CD56+ and CD7+ were less than 10%, while CD9+ was 14%. CD38+ and HLA-DR+ were 87% and 91%, respectively. The expressions of CD15, CD34, CD13 and CD33 on CD117 SSC(low) cells were between 30% and 50%. In AML patients, most cases were CD117+ (95.08%), CD38+ (94.74%), CD9+ (84.93%), CD33+ (84.60%), HLA-DR+ (77.23%) and CD13 (75.25%). The proportions of CD64+ and CD34+ were 64.41% and 59.51%, and that of CD15 and CD11b+ were 43.06% and 22.07%, respectively. 86.39% of AML patients were found to have at least one LAIP, the highest incidence being in AML-M1 and M3 subtypes and the lowest in AML-M4Eo subtype. The cross-lineage antigen and asynchronous antigen expression were the most frequent aberrant phenotypes. CD7, CD19 and CD56 expressing on CD34+ cells were major cross-lineage antigen. For asynchronous antigen expression, CD34+ CD64+, CD117+ CD11b+ , CD34+ CD38(-/dim) and CD34+ HLA-DR(-/dim) were seldom expressed on normal BMMNCs (about 0.01%), and the logarithm difference between AML and NBM was larger than 3.0, being the more sensitive LAIP. CONCLUSION: MRD detection by multiparameter flow cytometry can be applied to more than 80% of AML patients.
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Imunofenotipagem , Leucemia Mieloide Aguda/imunologia , Neoplasia Residual/diagnóstico , Adulto , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The objective of this study was to estimate a novel apoptosis-promoting molecule PDCD5 expression in the bone marrow cells from adult acute myeloid leukemia (AML) for investigation of its significance in the pathogenesis of AML. Flow cytometry assay was used for detection of PDCD5 expression in the different groups of cells from bone marrow of AML patients and normal controls by using 21 monoclonal antibodies with different fluorescent markers. The PDCD5 expressions in bone marrow cells from some AML patients and normal controls were also detected by Western blot. The results showed that the mean PDCD5 fluorescence intensity in bone marrow nucleated cells (MNC) from the bone marrow of 36 untreated AML patients was significantly lower than that from the bone marrow of 30 normal controls (3059 +/- 1392) vs (7432 +/- 1261) (P < 0.01). The mean PDCD5 fluorescence intensity was lower in the marrow granulocytes, monocytes, blast cells, and lymphocytes from untreated AML patients than that from normal (3939 +/- 2121) vs (8367 +/- 1045); (3156 +/- 1635) vs (5917 +/- 2329); (2824 +/- 1592) vs (3998 +/- 2106); (1474 +/- 816) vs (3355 +/- 2042) respectively, (all P < 0.01). Western blot analysis demonstrated that PDCD5 expression was significantly decreased in the AML cells, as compared with normal cells. It is concluded that PDCD5 expression in MNC in untreated AML patients is lower than that in the normal. PDCD5 expression in the marrow granulocytes, monocytes, blast cells, and lymphocytes of untreated AML patients is significantly lower than that in the normal. It suggests that the abnormally low expression of PDCD5 may be involved in the pathogenesis of AML.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Células da Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Humanos , Leucemia Mieloide Aguda/patologiaRESUMO
This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.
Assuntos
Adenoviridae/metabolismo , Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Proteínas de Neoplasias/genética , Transfecção , Adenoviridae/genética , Antineoplásicos Fitogênicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Células K562 , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: To evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL). METHODS: MRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available. RESULTS: Of 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000). CONCLUSION: Relapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.