RESUMO
Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process.
Assuntos
Arabidopsis , Modelos Biológicos , Morfogênese , Mutação , Pólen , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Biologia Computacional , Simulação por Computador , Cinética , Morfogênese/genética , Morfogênese/fisiologia , Mutação/genética , Mutação/fisiologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/fisiologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease caused by selective motor neuron death. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) belong to one of the four major mutation clusters responsible for pathogenesis of ALS. Toxic gain-of-function (not loss-of-function) of SOD1 mutants causes motor neuron degeneration. Aberrant protein-protein interactions (PPI) between mutant SOD1 and other proteins are involved in this toxic gain-of-function. Therefore, PPI inhibitors of mutant SOD1 not only increase understanding of ALS pathogenesis, but can also be used as novel therapeutics for ALS. Although it is challenging to identify PPI inhibitors, prior knowledge of the binding site can increase success probability. We have previously reported that tubulin interacts with N-terminal residues 1-23 of mutant SOD1. In the present study, we performed virtual screening by docking simulation of 32,791 compounds using this N-terminal binding site as prior knowledge. An established assay system for interaction inhibition between mutant SOD1-tubulin was used as an in-house model system to identify mutant SOD1 PPI inhibitors, with the goal of developing novel therapeutics for ALS. Consequently, five of six assay-executable compounds among top-ranked compounds during docking simulation inhibited the mutant SOD1-tubulin interaction in vitro. Binding mode analysis predicted that some inhibitors might bind the tubulin binding site of G85R SOD1 by pi electron interaction with the aromatic ring of the Trp32 residue of G85R SOD1. Our screening methods may contribute to the identification of lead compounds for treating ALS.
Assuntos
Mutação/fisiologia , Superóxido Dismutase-1/química , Superóxido Dismutase-1/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Células COS , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Mutação/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Superóxido Dismutase-1/antagonistas & inibidores , Superóxido Dismutase-1/genética , Tubulina (Proteína)/genéticaRESUMO
The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant ß2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing ß2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying ß2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing ß2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of ß2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.
Assuntos
Epilepsia/metabolismo , Prosencéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Sono/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/biossíntese , Simportadores/biossíntese , Animais , Animais Recém-Nascidos , Epilepsia/genética , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Mutação/fisiologia , Neocórtex/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/genética , Tálamo/metabolismo , Cotransportadores de K e Cl-RESUMO
INTRODUCTION: c.250G>A (p.Ala84Thr) in ETFDH is the most common mutation that causes later-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD) in the southern Chinese population. No functional study has targeted this mutation. METHODS: Using cells expressing ETFDH-wild-type (WT) or ETFDH-mutant (p.Ala84Thr), reactive oxygen species (ROS) production and neurite length were analyzed, followed by pathomechanism exploration and drug screening. RESULTS: Increased ROS production and marked neurite shortening were observed in the cells expressing the ETFDH-mutant, compared with WT. Further studies demonstrated that suberic acid, an accumulated intermediate metabolite in MADD, could significantly impair neurite outgrowth of NSC34 cells, but neurite shortening could be restored by supplementation with carnitine, riboflavin, or Coenzyme Q10. CONCLUSIONS: Neurite shortening caused by the c.250G>A mutation in ETFDH suggests that neural defects could be underdiagnosed in human patients with MADD. This impairment might be treatable with mitochondrial cofactor supplementation. Muscle Nerve 56: 479-485, 2017.
Assuntos
Flavoproteínas Transferidoras de Elétrons/biossíntese , Flavoproteínas Transferidoras de Elétrons/genética , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação/fisiologia , Crescimento Neuronal/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Linhagem Celular , Humanos , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Neuritos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
No disponible
Assuntos
Humanos , Masculino , Feminino , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Mutação/imunologia , Mutação/fisiologia , NADPH Oxidases/imunologia , NADPH Oxidases/uso terapêutico , Burkholderia cepacia/patogenicidade , Staphylococcus aureus/patogenicidade , Salmonella/patogenicidade , Serratia marcescens/patogenicidade , Nocardia/patogenicidade , Aspergillus/patogenicidade , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/patologia , Rifampina/uso terapêutico , Isoniazida/uso terapêutico , Clotrimazol/uso terapêutico , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.
Assuntos
Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , DNA Complementar/genética , Ligantes , Modelos Moleculares , Mutação/fisiologia , Técnicas de Patch-Clamp , Conformação ProteicaRESUMO
Candidemia is the fourth most common kind of microbial bloodstream infection, with Candida albicans being the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding ß-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate the in vivo outcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans compared with the resistance seen with heterozygous single mutations. The various FKS1 hot spot mutations differ in the degree of their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with the in vivo response when a standard dosing regimen is used but cannot predict the in vivo response after a dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida albicans/genética , Candidemia/tratamento farmacológico , Candidemia/microbiologia , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Equinocandinas/uso terapêutico , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Mutação/genética , Mutação/fisiologia , Adulto , Animais , Candida albicans/metabolismo , Quitina/metabolismo , Impressões Digitais de DNA , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único/genética , Virulência/genéticaRESUMO
It has been argued that the in vitro activity of caspofungin (CSP) is not a good predictor of the outcome of echinocandin treatment in vivo. We evaluated the in vitro activity of CSP and the presence of FKS mutations in the hot spot 1 (HS1) region of the FKS1 and FKS2 genes in 17 Candida glabrata strains with a wide range of MICs. The efficacy of CSP against systemic infections from each of the 17 strains was evaluated in a murine model. No HS1 mutations were found in the eight strains showing MICs for CSP of ≤ 0.5 µg/ml, but they were present in eight of the nine strains with MICs of ≥ 1 µg/ml, i.e., three in the FKS1 gene and five in the FKS2 gene. CSP was effective for treating mice infected with strains with MICs of ≤ 0.5 µg/ml, showed variable efficacy in animals challenged with strains with MICs of 1 µg/ml, and did not work in those with strains with MICs of >1 µg/ml. In addition, mutations, including one reported for the first time, were found outside the HS1 region in the FKS2 gene of six strains with different MICs, but their presence did not influence drug efficacy. The in vitro activity of CSP was compared with that of another echinocandin, anidulafungin, suggesting that the MICs of both drugs, as well as mutations in the HS1 regions of the FKS1 and FKS2 genes, are predictive of outcome.
Assuntos
Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Candidíase/tratamento farmacológico , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Mutação/fisiologia , Animais , Candida glabrata/genética , Candidíase/microbiologia , Caspofungina , Farmacorresistência Fúngica/genética , Rim/microbiologia , Lipopeptídeos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Taxa de SobrevidaRESUMO
Absence epilepsy accompanies the paroxysmal oscillations in the thalamocortical circuit referred as spike and wave discharges (SWDs). Low-threshold burst firing mediated by T-type Ca(2+) channels highly expressed in both inhibitory thalamic reticular nuclei (TRN) and excitatory thalamocortical (TC) neurons has been correlated with the generation of SWDs. A generally accepted view has been that rhythmic burst firing mediated by T-type channels in both TRN and TC neurons are equally critical in the generation of thalamocortical oscillations during sleep rhythms and SWDs. This review examined recent studies on the T-type channels in absence epilepsy which leads to an idea that even though both TRN and TC nuclei are required for thalamocortical oscillations, the contributions of T-type channels to TRN and TC neurons are not equal in the genesis of sleep spindles and SWDs. Accumulating evidence revealed a crucial role of TC T-type channels in SWD generation. However, the role of TRN T-type channels in SWD generation remains controversial. Therefore, a deeper understanding of the functional consequences of modulating each T-type channel subtype could guide the development of therapeutic tools for absence seizures while minimizing side effects on physiological thalamocortical oscillations.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo T/fisiologia , Epilepsia Tipo Ausência/fisiopatologia , Tálamo/fisiopatologia , Animais , Eletroencefalografia/tendências , Epilepsia Tipo Ausência/diagnóstico , Epilepsia Tipo Ausência/genética , Humanos , Mutação/fisiologiaRESUMO
Regulation of sexual reproduction and energy homeostasis are closely interconnected, but only few efforts were made to explore the impact of gonadotropic neurons on metabolic processes. We have used Nscl-2 mutant mice suffering from adult onset of obesity and hypogonadotropic hypogonadism to study effects of gonadotropin releasing hormone (GnRH) neurons on neuronal circuits controlling energy balance. Inactivation of Nscl-2 in GnRH neurons but not in pro-opiomelanocortin (POMC) neurons reduced POMC neurons and increased visceral fat mass, suggesting a critical role of GnRH cells in the regulation of POMC neurons. In contrast, absence of POMC processing in the majority of Nscl-2-deficient POMC neurons had no effect on energy homeostasis. Finally, we investigated the cellular basis of the reduction of GnRH neurons in NSCL-2 mutants using a lineage tracing approach. We found that loss of Nscl-2 results in aberrant migration of GnRH neurons in Nscl-2 mutant mice causing a lineage switch of ectopically located GnRH neurons.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/fisiologia , Hipotálamo/fisiologia , Neurônios/fisiologia , Obesidade/genética , Pró-Opiomelanocortina/fisiologia , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/fisiologia , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/fisiologia , Animais , Western Blotting , Divisão Celular/fisiologia , Estradiol/sangue , Feminino , Homeostase/genética , Homeostase/fisiologia , Hipotálamo Posterior/fisiologia , Infertilidade/genética , Camundongos , Camundongos Knockout , Mutação/genética , Mutação/fisiologia , Área Pré-Óptica/fisiologia , Pró-Opiomelanocortina/biossíntese , Pró-Opiomelanocortina/genética , Reprodução/genética , Reprodução/fisiologiaRESUMO
Hearing over a wide range of sound intensities is thought to require complementary coding by functionally diverse spiral ganglion neurons (SGNs), each changing activity only over a subrange. The foundations of SGN diversity are not well understood but likely include differences among their inputs: the presynaptic active zones (AZs) of inner hair cells (IHCs). Here we studied one candidate mechanism for causing SGN diversity-heterogeneity of Ca(2+) influx among the AZs of IHCs-during postnatal development of the mouse cochlea. Ca(2+) imaging revealed a change from regenerative to graded synaptic Ca(2+) signaling after the onset of hearing, when in vivo SGN spike timing changed from patterned to Poissonian. Furthermore, we detected the concurrent emergence of stronger synaptic Ca(2+) signals in IHCs and higher spontaneous spike rates in SGNs. The strengthening of Ca(2+) signaling at a subset of AZs primarily reflected a gain of Ca(2+) channels. We hypothesize that the number of Ca(2+) channels at each IHC AZ critically determines the firing properties of its corresponding SGN and propose that AZ heterogeneity enables IHCs to decompose auditory information into functionally diverse SGNs.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Nervo Coclear/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Audição/fisiologia , Animais , Canais de Cálcio/fisiologia , Cóclea/crescimento & desenvolvimento , Cóclea/inervação , Nervo Coclear/crescimento & desenvolvimento , Núcleo Coclear/citologia , Núcleo Coclear/fisiologia , Simulação por Computador , Fenômenos Eletrofisiológicos , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos Neurológicos , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Receptores Pré-Sinápticos/fisiologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/crescimento & desenvolvimento , Gânglio Espiral da Cóclea/fisiologia , Frações Subcelulares/fisiologiaRESUMO
BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral small vessel disease, clinically characterized by migraine, recurrent transient ischemic attacks or strokes, psychiatric disorders and cognitive decline. Strokes are typically ischemic, while hemorrhagic events have been only sporadically described. However, cerebral microbleeds have been found in 31-69% of CADASIL patients. METHODS: We describe four unrelated CADASIL patients who had hemorrhagic strokes. We also briefly review the literature on intracerebral hemorrhage (ICH) in CADASIL. RESULTS: Three patients had a thalamo-capsular hemorrhage (age at onset: 54, 67, 77) and one of these had a second hemispheric cerebellar hemorrhage. Another patient experienced an interpeduncular cistern subarachnoid hemorrhage when he was 39. None of these patients was receiving antiplatelets, anticoagulants or statins at the time of hemorrhage; all were hypertensive. NOTCH3 gene analysis revealed mutations on exons 14, 22 (two patients presenting the same mutation), and 24. MRI signs of previous hemorrhages were present in all these patients. CONCLUSIONS: Hemorrhagic stroke can occur in CADASIL similarly to sporadic cerebral small vessel diseases; this finding expands the phenotype of the disease. A diagnosis of CADASIL should probably be considered also in patients with ICH. These data bear potential implications in terms of need of better control of risk factors, particularly hypertension, and raise relevant questions about the use of antiplatelets as prevention measures in CADASIL patients.
Assuntos
CADASIL/complicações , Hemorragias Intracranianas/etiologia , Adulto , Idoso , CADASIL/genética , Feminino , Hemiplegia/etiologia , Humanos , Cápsula Interna/patologia , Hemorragias Intracranianas/genética , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Mutação/fisiologia , Receptor Notch3 , Receptores Notch/genética , Acidente Vascular Cerebral/etiologia , Hemorragia Subaracnóidea/etiologia , Tálamo/patologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Mutations within the gene encoding the skeletal muscle calcium channel ryanodine receptor can result in malignant hyperthermia. Although it is important to characterize the functional effects of candidate mutations to establish a genetic test for diagnosis, ex vivo methods are limited because of the low incidence of the disorder and sample unavailability. More than 250 candidate mutations have been identified, but only a few mutations have been functionally characterized. METHODS: The human skeletal muscle ryanodine receptor complementary DNA was cloned with or without a disease-related variant. Wild-type and mutant calcium channel proteins were transiently expressed in human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40, and functional analysis was carried out using calcium imaging with fura-2 AM. Six human malignant hyperthermia-related mutants such as R44C, R163C, R401C, R533C, R533H, and H4833Y were analyzed. Cells were stimulated with a specific ryanodine receptor agonist 4-chloro-m-cresol, and intracellular calcium mobility was analyzed to determine the functional aspects of mutant channels. RESULTS: Mutant proteins that contained a variant linked to malignant hyperthermia showed higher sensitivity to the agonist. Compared with the wild type (EC50=453.2 µM, n=18), all six mutants showed a lower EC50 (21.2-170.4 µM, n=12-23), indicating susceptibility against triggering agents. CONCLUSIONS: These six mutations cause functional abnormality of the calcium channel, leading to higher sensitivity to a specific agonist, and therefore could be considered potentially causative of malignant hyperthermia reactions.
Assuntos
Hipertermia Maligna/genética , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cálcio/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Imunofluorescência , Células HEK293 , Humanos , Hipertermia Maligna/fisiopatologia , Mutação/fisiologia , Neuroimagem , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologiaRESUMO
Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2(mut)), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe(-/-) mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p=5.4×10(-7)) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p=0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients.
Assuntos
Química Encefálica/genética , Química Encefálica/fisiologia , Hemocromatose/genética , Hemocromatose/patologia , Ferro/toxicidade , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Receptores da Transferrina/genética , Receptores da Transferrina/fisiologia , Transcriptoma/genética , Animais , Western Blotting , Ferritinas/metabolismo , Glioma/metabolismo , Glioma/patologia , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/fisiologia , Ferro da Dieta/farmacologia , Fígado/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Mutação/genética , Mutação/fisiologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria AtômicaRESUMO
OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.
Assuntos
Carcinoma Endometrioide/patologia , Doxorrubicina/farmacologia , Neoplasias do Endométrio/patologia , Paclitaxel/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/genética , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Feminino , Humanos , Mutação/fisiologia , Paclitaxel/administração & dosagem , Pirimidinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Células Tumorais CultivadasRESUMO
CONTEXT: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. OBJECTIVE: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. METHODS AND PATIENTS: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. RESULTS: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. CONCLUSIONS: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.
Assuntos
Proteínas de Homeodomínio/genética , Hormônio do Crescimento Humano/deficiência , Adolescente , Idade de Início , Células Cultivadas , Criança , Pré-Escolar , Sequência Conservada , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Variação Genética , Vetores Genéticos , Hormônios/sangue , Humanos , Hipotálamo/patologia , Lactente , Fator de Crescimento Insulin-Like I/deficiência , Íntrons/genética , Luciferases/genética , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Mutação/fisiologia , Penetrância , Hipófise/patologia , Hormônios Hipofisários/sangue , Polimorfismo de Nucleotídeo Único/genética , Transfecção , Adulto JovemAssuntos
Terapias Complementares/métodos , Doenças Raras/terapia , Síndrome da Deleção 22q11/genética , Síndrome da Deleção 22q11/terapia , Distonia/genética , Distonia/terapia , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/terapia , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Mutação/fisiologia , Doenças Raras/complicações , Doenças Raras/genética , SíndromeAssuntos
Imidazóis/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridazinas/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Duplicação Gênica , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Mutação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína/genética , Piridazinas/farmacologia , Especificidade por Substrato/genética , Sequências de Repetição em Tandem/genética , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
OBJECTIVE: To establish a rapid detection method for identifying rpoB mutations associated with rifampin (RIF) resistance in sputum specimens. METHODS: We detected rpoB mutations directly in 90 sputum specimens collected from suspected tuberculosis patients using PCR-based denaturing gradient gel electrophoresis (DGGE) and compared these results with those obtained by rpoB sequencing and conventional drug susceptibility testing. RESULTS: The positive detection rate of Mycobacterium tuberculosis (M. tuberculosis) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional mycobacterial culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the rpoB gene in the same specimens. Furthermore, 75% of the tested specimens presented abnormal patterns compared with the wild-type pattern (standard H37Rv strain) analysed by DGGE. A total of 12 different patterns, representing 12 different rpoB mutations, were observed in the 63 abnormal patterns. The match rate of rpoB mutations detected by DGGE reached 96.9% when compared to DNA sequencing. CONCLUSION: Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of rpoB mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.
Assuntos
Proteínas de Bactérias/genética , Análise Mutacional de DNA/métodos , Eletroforese em Gel de Gradiente Desnaturante/métodos , Resistência Microbiana a Medicamentos/genética , Mycobacterium tuberculosis/genética , Rifampina/uso terapêutico , Escarro/microbiologia , Proteínas de Bactérias/análise , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , RNA Polimerases Dirigidas por DNA , Humanos , Testes de Sensibilidade Microbiana , Mutação/fisiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Escarro/química , Escarro/metabolismoRESUMO
Wild apples (Malus sp.) have been a major food source in the northern Alpine region since prehistory and their use is well understood. The onset of deliberate fruit tree growing in the area is, however, less clear. It is generally assumed that horticulture was practised in Roman times, but it might be even earlier. In the archaeological record seed testa and pericarp remains are particularly frequent at sites with waterlogged preservation such as lakeshore settlements or wells, pits and ditches, but the distinction between wild and domestic plants is not morphologically possible. With waterlogged remains being one main source of information about past fruit cultivation, we have tested the feasibility of analysing ancient DNA from waterlogged preserved bulk samples of testa fragments. We studied apple seeds from three Neolithic and three Roman sites with waterlogged preservation in the Alpine foreland. Chloroplast markers failed in all samples, but nuclear ITS1 (internal transcribed spacer region 1) of the ribosomal DNA was successfully typed in two Roman samples from the site Oedenburg/Biesheim-Kunheim (Haut-Rhin, F). The retrieved ITS1 sequences are identical to each other and are shared with wild Malus sylvestris and Malus sieversii, and with domestic apple cultivars, supporting the potential of using waterlogged remains for identifying the genetic status of apple diachronically.