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1.
PLoS One ; 12(4): e0175894, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28437454

RESUMO

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals.


Assuntos
Envelhecimento/metabolismo , Abelhas/metabolismo , Proteínas de Insetos/metabolismo , Corpos Pedunculados/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/metabolismo
2.
Learn Mem ; 23(5): 195-207, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27084927

RESUMO

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees(Apis mellifera)we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.


Assuntos
Encéfalo/metabolismo , Proteína de Ligação a CREB/metabolismo , Condicionamento Clássico/fisiologia , Retenção Psicológica/fisiologia , Análise de Variância , Animais , Abelhas , Encéfalo/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Retenção Psicológica/efeitos dos fármacos , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos
3.
J Comp Neurol ; 524(6): 1165-80, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26355639

RESUMO

Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP-response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age-dependent division of labor. In honeybee brains, CREB-dependent genes are regulated in an age-dependent manner, indicating that there might be a role for neuronal honeybee CREB (Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB-dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB-dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age-dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age-dependent increase of pAmCREB. Our results demonstrate the IC to be candidate neurons involved in age-dependent division of labor. We hypothesize that the IC display a high level of CREB-dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior.


Assuntos
Envelhecimento/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Corpos Pedunculados/crescimento & desenvolvimento , Corpos Pedunculados/metabolismo , Animais , Animais Recém-Nascidos , Abelhas , Galinhas , Drosophila , Humanos , Corpos Pedunculados/citologia , Fosforilação/fisiologia
4.
Front Behav Neurosci ; 9: 91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25964749

RESUMO

In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli.

5.
Learn Mem ; 21(12): 676-85, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25403456

RESUMO

This study examines the role of stimulus duration in learning and memory formation of honeybees (Apis mellifera). In classical appetitive conditioning honeybees learn the association between an initially neutral, conditioned stimulus (CS) and the occurrence of a meaningful stimulus, the unconditioned stimulus (US). Thereby the CS becomes a predictor for the US eliciting a conditioned response (CR). Here we study the role of US duration in classical conditioning by examining honeybees conditioned with different US durations. We quantify the CR during acquisition, memory retention, and extinction of the early long-term memory (eLTM), and examine the molecular mechanisms of eLTM by interfering with protein synthesis. We find that the US duration affects neither the probability nor the strength of the CR during acquisition, eLTM retention, and extinction 24 h after conditioning. However, we find that the resistance to extinction 24 h after conditioning is susceptible to protein synthesis inhibition depending on the US duration. We conclude that the US duration does not affect the predictability of the US but modulates the protein synthesis underlying the eLTM's strength. Thus, the US duration differentially impacts learning, eLTM strength, and its underlying protein synthesis.


Assuntos
Condicionamento Clássico/fisiologia , Memória de Longo Prazo/fisiologia , Biossíntese de Proteínas , Animais , Comportamento Apetitivo/efeitos dos fármacos , Comportamento Apetitivo/fisiologia , Abelhas , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Condicionamento Clássico/efeitos dos fármacos , Sacarose Alimentar/administração & dosagem , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Líquidos/fisiologia , Emetina/farmacologia , Extinção Psicológica/efeitos dos fármacos , Extinção Psicológica/fisiologia , Memória de Longo Prazo/efeitos dos fármacos , Odorantes , Percepção Olfatória/efeitos dos fármacos , Percepção Olfatória/fisiologia , Estimulação Física , Probabilidade , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia
6.
Radiat Res ; 170(1): 41-8, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18582150

RESUMO

The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica/efeitos da radiação , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias/genética , Neoplasias/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade
7.
Ann Hematol ; 83(12): 745-50, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15340762

RESUMO

WT1 is a transcription factor involved in differentiation and proliferation of acute myeloid leukemia (AML) blasts and is expressed in 90% of cases, as determined by nested reverse transcription polymerase chain reaction (RT-PCR). It is proposed to be a key molecule in leukemia promotion. To assess the relevance of WT1 expression, we analyzed blood and bone marrow samples from 58 AML patients (37 at diagnosis, 8 in hematological remission, and 13 at relapse) for the level of WT1 expression, using quantitative real-time RT-PCR. In addition, 21 randomly chosen samples were also analyzed for the quantitative expression of the main WT1 splice variants. As expected, samples from patients at the time of diagnosis or relapse showed significantly higher WT1 expression compared to samples from patients in remission or control samples. No striking difference in expression levels was found between various French-American-British (FAB) subtypes. The level of WT1 expression observed in patients at the time of initial diagnosis was similarly high in patients at relapse. Expression of the four main isoforms (E5+/KTS+, E5-/KTS+, E5+/KTS-, and E5-/KTS-) was found in all samples with significantly higher expression levels of the E5+ variants. Together, these findings support the potential of WT1 as a target for novel treatment approaches in AML.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Splicing de RNA/genética , RNA Neoplásico/genética , Proteínas WT1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular/genética , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Neoplásico/sangue , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas WT1/metabolismo
8.
Clin Cancer Res ; 10(5): 1605-12, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15014010

RESUMO

PURPOSE: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells. EXPERIMENTAL DESIGN: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples. RESULTS: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/ microl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples). CONCLUSIONS: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.


Assuntos
Neoplasias Oculares/patologia , Melanoma/patologia , Antígenos de Neoplasias/genética , Sequência de Bases , Primers do DNA , Neoplasias Oculares/sangue , Neoplasias Oculares/genética , Humanos , Hidroximetilbilano Sintase/genética , Antígeno MART-1 , Melanoma/sangue , Melanoma/genética , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Monofenol Mono-Oxigenase/genética , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Plasmídeos/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , Antígeno gp100 de Melanoma
9.
Int J Cancer ; 108(2): 219-27, 2004 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-14639606

RESUMO

The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/sangue , Células Neoplásicas Circulantes/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Antígeno Carcinoembrionário/genética , Estudos de Casos e Controles , Colo/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Humanos , Proteínas de Filamentos Intermediários/genética , Calicreínas/genética , Queratina-20 , Prognóstico , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Sensibilidade e Especificidade , Células Tumorais Cultivadas
10.
Br J Haematol ; 123(2): 235-42, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14531904

RESUMO

Overexpression of the embryonic transcription factor, Wilms' tumour protein 1 (WT1), is common in acute myeloid leukaemias (AML). Mutations of Wilms' tumour gene 1 (WT1) in AML are rare and WT1 expression may be increased by other transcription factors. PAX2, PAX8 and GATA-1 are known physiological regulators of WT1. In the present study, we analysed either bone marrow or blood samples of 43 AML patients for the expression levels of WT1, PAX2, PAX8 and GATA-1 by real-time reverse transcription polymerase chain reaction (LightCycler). Bone marrow samples of patients without haematological malignancies and stem cell preparation samples from healthy donors and lymphoma patients served as controls. PAX2 expression was found in 11 of 43 AML samples, with a clear correlation of PAX2 with WT1 expression levels observed. PAX8 expression was found in two additional samples. GATA-1 expression was detectable in 41 of 43 AML samples and also in all control samples; no significant differences between these groups were observed and no correlation of GATA-1 expression with WT1 expression levels was apparent. In conclusion, PAX2, and possibly PAX8, appears to be a candidate for the upregulation of WT1 in a proportion of AML, whereas GATA-1 expression cannot be explained as an inducer of WT1. In two-thirds of leukaemias from our series, the basis of WT1 upregulation cannot be explained by the simple upregulation of the known WT1 activators.


Assuntos
Regulação Leucêmica da Expressão Gênica , Genes do Tumor de Wilms , Leucemia Mieloide/genética , Proteínas Nucleares , Fatores de Transcrição/genética , Proteínas WT1/genética , Doença Aguda , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Fator de Transcrição PAX2 , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , RNA Mensageiro/genética , RNA Neoplásico/genética , Transativadores/genética , Células Tumorais Cultivadas , Regulação para Cima/genética
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