RESUMO
Functional nucleic acids with the capability of generating fluorescence in response to hybridization events, microenvironment or structural changes are valuable as structural probes and chemical sensors. We now demonstrate the enzyme-assisted preparation of nucleic acids possessing multiple thiazole orange (TO) dyes and their fluorescent behavior, that show a spectral change from the typical monomer emission to the excimer-type red-shifted emission. We found that the fluorescent response and emission wavelength of the TO dyes were dependent on both the state of the DNA structure (single- or double-stranded DNA) and the arrangement of the TO dyes. We showed that the fluorescent behavior of the TO dyes can be applied for the detection of RNA molecules, suggesting that our approach for preparing the fluorescent nucleic acids functionalized with multiple TO dyes could be useful to design a fluorescence bioimaging and detection technique of biomolecules.
Assuntos
Benzotiazóis/química , DNA/química , Quinolinas/química , Aminação , Dicroísmo Circular , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico , Oxirredução , Espectrometria de Fluorescência , Temperatura de TransiçãoRESUMO
Brain-derived neurotrophic factor (BDNF) is a key player in synaptic plasticity, and consequently, learning and memory. Because of its fundamental role in numerous neurological functions in the central nervous system, BDNF has utility as a biomarker and drug target for neurodegenerative and neuropsychiatric disorders. Here, we generated a screening assay to mine inducers of Bdnf transcription in neuronal cells, using primary cultures of cortical cells prepared from a transgenic mouse strain, specifically, Bdnf-Luciferase transgenic (Bdnf-Luc) mice. We identified several active extracts from a library consisting of 120 herbal extracts. In particular, we focused on an active extract prepared from Ginseng Radix (GIN), and found that GIN activated endogenous Bdnf expression via cAMP-response element-binding protein-dependent transcription. Taken together, our current screening assay can be used for validating herbal extracts, food-derived agents, and chemical compounds for their ability to induce Bdnf expression in neurons. This method will be beneficial for screening of candidate drugs for ameliorating symptoms of neurological diseases associated with reduced Bdnf expression in the brain, as well as candidate inhibitors of aging-related cognitive decline.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/citologia , Luciferases/metabolismo , Programas de Rastreamento , Neurônios/metabolismo , Transcrição Gênica , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dopamina/metabolismo , Ginsenosídeos/farmacologia , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ratos Sprague-Dawley , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Many types of fermented food are consumed in Japan. Although some are produced by plant-origin lactic-acid bacteria (LAB) fermentation, the physiological functions of such bacteria remain unclear. We therefore isolated LAB of plant origin from Kyoto pickles and determined the immunological activity of heat-killed preparations of plant-origin LAB. METHODS: The Lactobacillus pentosus strain S-PT84 was selected from among 16 LAB of plant origin as the strongest interleukin (IL)-12-inducing strain. IL-12- and IL-10-inducing activities were determined with macrophages from BALB/c mice. The in vivo immunomodulating effect of S-PT84was determined with BALB/c mice fed S-PT84. The antiallergic activity of S-PT84 was examined in ovalbumin (OVA)/alum-administered BALB/c mice. RESULTS: The L. pentosus strain S-PT84 induced production of both IL-12 and IL-10 in vitro. S-PT84 enhanced splenic natural-killer activity and modulated the T helper (Th) type 1/type 2 balance toward a Th1-dominant state. In the OVA-induced allergy model, orally administered S-PT84 lowered serum IgE levels and suppressed active cutaneous anaphylaxis reaction and splenic IL-4 production. IL-10 production from splenocytes of OVA-immunized mice was upregulated by feeding S-PT84. CONCLUSIONS: Despite heat-killing, S-PT84 exhibited antiallergic effects by modulating the Th1/Th2 balance and inducing regulatory T cells. The L. pentosus strain S-PT84, which is of plant origin and isolated from a traditional Japanese food, is expected to be useful for treatment of many immune diseases including allergies, tumors, infectious diseases and auto-immune diseases.
Assuntos
Hipersensibilidade Alimentar/terapia , Interleucina-10/metabolismo , Lactobacillus/imunologia , Lactobacillus/metabolismo , Probióticos/administração & dosagem , Probióticos/metabolismo , Alérgenos , Anafilaxia/imunologia , Animais , Antialérgicos/imunologia , Células Cultivadas , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Hipersensibilidade Alimentar/sangue , Microbiologia de Alimentos , Temperatura Alta , Imunoglobulina E/sangue , Imunoglobulina E/metabolismo , Imunossupressores/administração & dosagem , Imunossupressores/metabolismo , Interleucina-12/metabolismo , Células Matadoras Naturais/imunologia , Lactobacillus/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Baço/imunologia , Linfócitos T Reguladores/citologia , Células Th1/imunologia , Células Th2/imunologia , Regulação para CimaRESUMO
An autosomal dystrophic epidermolysis bullosa (DDEB) is a hereditary mechanobullous disease characterized by blistering of the skin and the mucous membrane. DDEB is caused by a heterozygous mutation in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils, and phenotypically classified into several types. We experienced two boys with DDEB and examined the mutation analyses of the COL7A1 genes of the two patients and their fathers to clarify the relationship between the genotypes and phenotypes, that is, the mutation sites of COL7A1 gene and the clinical types of DDEB. The case 1 and 2 patients and their fathers revealed a heterozygous nucleotide G to A transition at position 6109 and 6082 in 73 exon of COL7A1, which resulted in a glycine to arginine substitution (G2037R and G2028R), respectively. G2037R found in the case 1 patient was a novel mutation. There was no clear relationship recognized between the two mutation sites in the COL7A1 gene and the clinical variations.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Arginina , Povo Asiático , Análise Mutacional de DNA , Enzimas de Restrição do DNA , Glicina , Humanos , Lactente , Recém-Nascido , Masculino , Mutação de Sentido IncorretoRESUMO
Avermectins were isolated as compounds possessing anthelmintic activity from the culture broth of Streptomycesavermitilis by Omura and co-workers. Owing to their potent anthelmintic and insecticidal activities, as well as their unique pentacyclic architecture, the avermectin family attracted keen interest from synthetic organic chemists. We have recently completed a more efficient and straightforward total synthesis of avermectin B1a, as compared with previous syntheses.
Assuntos
Anti-Helmínticos/síntese química , Ivermectina/análogos & derivados , Anti-Helmínticos/química , Ivermectina/síntese química , Ivermectina/química , Modelos Moleculares , Estrutura MolecularRESUMO
The ABCC6 gene encodes MRP6, a member of the multidrug resistance-associated protein (MRP) family. Interest in ABCC6/MRP6 derives, in part, from the fact that mutations in this gene/protein system have been identified in families with pseudoxanthoma elasticum (PXE). Early studies indicated that ABCC6 is expressed primarily in the liver and to a lesser extent in the kidney, but more recently a widespread distribution has been suggested. To explore the tissue-specific expression of ABCC6, we first examined various mouse tissues by RT-PCR. The results indicated high levels of mRNA in the liver, whereas low level of expression was noted in the kidney and small intestine. To explore other tissues in which initial RT-PCR was essentially negative, a second-round nested PCR was performed, which revealed expression also in the brain, tongue, stomach, and eye. Unexpectedly, however, distinct PCR products of smaller molecular weight were noted in these tissues. Subcloning and sequencing of these PCR products indicated that they reflected aberrant splicing in the 3' end of the ABCC6 mRNA, resulting in each case in a premature termination codon. Similar results were noted with RT-PCR analysis using RNA isolated from cultured human epidermal keratinocytes and dermal fibroblasts. Collectively, our results confirm high level of expression of ABCC6 in the liver and the kidney, whereas very low level of expression in a variety of other tissues was noted. The results have implications for mutation detection strategies in PXE by RT-PCR, and they further support the notion that PXE is a primary metabolic disorder.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Processamento Alternativo , Rim/metabolismo , Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Células Cultivadas , Humanos , Intestino Delgado/química , Intestino Delgado/metabolismo , Queratinócitos/metabolismo , Rim/química , Fígado/química , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Pseudoxantoma Elástico/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
Acrodermatitis enteropathica is an autosomal recessive disease characterized by skin involvement due to defective intestinal zinc absorption. Usually, the skin lesions include erythema, erosions, and small blisters in perioral, perianal regions, and hands and feet, which develop soon after weaning from the breast. The acrodermatitis enteropathica gene has been localized to chromosomal region 8q24.3 and subsequently the SLC39A4 gene has been disclosed as the acrodermatitis enteropathica gene. SLC39A4 mutations have been demonstrated in several acrodermatitis enteropathica families, and in this study we have examined two Japanese acrodermatitis enteropathica families for SLC39A4 mutations. The mutation detection strategy consisted of polymerase chain reaction amplification of all 12 exons and flanking intronic sequences, followed by direct nucleotide sequencing. It revealed three novel mutations, 1017ins53, which creates a premature termination codon, and two mis-sense mutations, R95C and Q303H.
Assuntos
Acrodermatite/genética , Proteínas de Transporte de Cátions/genética , Genes Recessivos , Mucosa Intestinal/metabolismo , Síndromes de Malabsorção/genética , Mutação , Absorção , Acrodermatite/patologia , Sequência de Bases/genética , Criança , Feminino , Pé , Mãos , Humanos , Lactente , Síndromes de Malabsorção/patologia , Masculino , Dados de Sequência Molecular , Linhagem , Pele/patologia , Zinco/farmacocinéticaRESUMO
Herlitz junctional epidermolysis bullosa (H-JEB) is a hereditary bullous disease caused by absent expression of laminin-5, a component of anchoring filaments within the dermal-epidermal basement membrane zone. Affected individuals usually die during the first 1 year of life. We studied an infant with H-JEB who presented with nephrotic syndrome, a previously unreported complication that may contribute to early death in this disease. DNA analysis revealed a compound heterozygote for mutations 2379delG and Q995X in the LAMB3 gene. The patient had massive albuminuria, attributable to failure of the glomerular filtration barrier, and high urinary N-acetylglucosaminidase levels, indicating renal tubular involvement. Electron-microscopic examination of the renal tissue revealed diffuse fusion of the foot processes, irregular swelling of the lamina rara interna, and disappearance of endothelial cell fenestrations. Immunohistopathologic analysis of the patient's renal tissue revealed compositional changes in laminin isoforms of the glomerular basement membrane and no detectable laminin-5 in the renal tubular basement membrane, which suggests that laminin-5 may play an important role in renal function. Our findings strongly suggest that H-JEB should be considered in the spectrum of congenital nephrotic syndromes. Combination therapy with meticulous skin care and treatment strategies established for congenital nephrotic syndromes may rescue patients with this disease.
Assuntos
Epidermólise Bolhosa Juncional/complicações , Membrana Basal Glomerular/metabolismo , Laminina/análise , Síndrome Nefrótica/complicações , Membrana Basal/metabolismo , Análise Mutacional de DNA , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Túbulos Renais/metabolismo , Laminina/genética , Masculino , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Isoformas de Proteínas/análise , ProteinúriaRESUMO
Epidermolysis bullosa (EB) is a group of inherited disorders characterized by increased skin fragility, resulting in blisters and erosions after minor trauma. Mutations in 10 structural genes expressed in the cutaneous basement membrane zone have been reported. The DebRA Molecular Diagnostics Laboratory at Jefferson Medical College has performed 144 DNA-based prenatal diagnoses since 1993 in families at risk for recurrence of the most severe forms of EB, including the recessive dystrophic EB (RDEB), junctional EB (JEB), EB with pyloric atresia (EB-PA), and EB simplex (EBS). A mutation-detection strategy using either conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC) scanning analysis, followed by nucleotide sequencing, was applied to most cases with DEB and to all JEB, EB-PA, and EBS families. For some RDEB families, linkage analysis was performed, either alone when the inheritance pattern was clear or in combination with one mutation. Among the 144 prenatal diagnoses, 63 were for RDEB, 69 for JEB, 6 for EB-PA, and 6 for EBS. Twenty-eight normal, 73 heterozygous carrier, and 28 affected RDEB, JEB, and EB-PA pregnancies were reported in these recessively inherited diseases. Two affected and four normal pregnancies were predicted in dominantly inherited EBS. Among the 144 pregnancies, 9 were terminated without confirmation, 13 cases were lost to follow-up, and 6 pregnancies are ongoing. There were 6 families with inconclusive results due either to recombination events between flanking markers, absence of informative markers for one allele, or lack of sample from the previously affected child. There were three discordant results, one that was explained by maternal contamination of the chorionic villus sample and two that were unresolved. Overall, the availability, relative ease, and over 98% success rate make molecular DNA-based prenatal diagnosis a viable option for EB families at risk.
Assuntos
Epidermólise Bolhosa/diagnóstico , Diagnóstico Pré-Natal , Adulto , Amniocentese , Amostra da Vilosidade Coriônica , Cromatografia Líquida de Alta Pressão , DNA/análise , Análise Mutacional de DNA , Primers do DNA/química , Eletroforese em Gel Bidimensional , Epidermólise Bolhosa/classificação , Epidermólise Bolhosa/genética , Feminino , Ligação Genética , Humanos , Padrões de Herança , Reação em Cadeia da Polimerase , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Pele/ultraestruturaRESUMO
Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.
Assuntos
Moléculas de Adesão Celular/genética , Epidermólise Bolhosa Juncional/genética , Adolescente , Adulto , Processamento Alternativo , Criança , Códon de Terminação/genética , Primers do DNA , Epidermólise Bolhosa Juncional/classificação , Epidermólise Bolhosa Juncional/patologia , Éxons , Feminino , Deleção de Genes , Variação Genética , Impressão Genômica , Humanos , Lactente , Masculino , Linhagem , Fenótipo , Subunidades Proteicas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologia , CalininaRESUMO
BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of inherited blistering diseases characterized by epidermal-dermal separation resulting from mutations that affect the function of critical components of the basement membrane zone. This group of autosomal recessive diseases is especially prevalent in regions where consanguinity is common, such as the Middle East. However, the clinical and genetic epidemiology of JEB in this region remains largely unexplored. OBJECTIVE: The aim of the present study was to assess a series of consanguineous JEB families originating from the Middle East. METHODS: We identified 7 families referred to us between 1998 and 1999 and originating from the United Arab Emirates, Saudi Arabia, Sudan, Yemen, and Israel. Histologic, immunofluorescence, and electron microscopy studies were performed to direct the subsequent molecular analysis. DNA obtained from all family members was amplified by means of polymerase chain reaction and analyzed by conformation-sensitive gel electrophoresis with subsequent direct sequencing. RESULTS: In 6 families presenting with the clinical and histologic features distinctive for JEB, mutations in genes encoding 1 of the 3 subunit polypeptides of laminin-5 were identified. Two families each had mutations in LAMB3, 2 in LAMA3, and 2 in LAMC2. Out of 7 distinct mutations, 5 were novel and 2 were recurrent. No relationship was found between the presence of nonsense/frameshift mutations in laminin-5 genes and perinatal mortality, contradicting a major genotype-phenotype correlation previously reported in the European and US literature. Similarly, none of the recurrent LAMB3 hot spot mutations previously described in other populations was found in our series. Finally, in a family with the clinical diagnosis of generalized atrophic benign epidermolysis bullosa, a homozygous non-sense mutation in Col17A1 gene (encoding the BPAG2 antigen) was identified. CONCLUSION: The present report suggests (1) the existence of a unique spectrum of mutations in the Middle East populations and (2) the need for the implementation of a diagnostic strategy tailored to the genetic features of JEB in this region.