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1.
Surg Endosc ; 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33475845

RESUMO

BACKGROUND: Prior to antireflux surgery, most patients with symptoms of gastroesophageal reflux disease (GERD) have been taking long-term proton pump inhibitors (PPIs). PPIs have been shown to cause changes to the intestinal microbiota, such as small intestinal bacterial overgrowth (SIBO), which is characterised by symptoms of gas bloating. Patients undergoing antireflux surgery are not routinely screened for SIBO, yet many patients experience gas-related symptoms postoperatively. METHODS: Data from consecutive patients (n = 104) referred to a speciality reflux centre were retrospectively assessed. Patients underwent a routine diagnostic workup for GERD including history, endoscopy, oesophageal manometry and 24-h pH-impedance monitoring off PPIs. Intestinal dysbiosis was determined by hydrogen and methane breath testing with a hydrogen-positive result indicative of SIBO and a methane-positive result indicative of intestinal methanogen overgrowth (IMO). RESULTS: 60.6% of patients had intestinal dysbiosis (39.4% had SIBO and 35.6% had IMO). Patients with dysbiosis were more likely to report bloating (74.6% vs 48.8%; P = 0.01) and belching (60.3% vs 34.1%; P = 0.01). The oesophageal acid exposure time and number of reflux episodes were similar between dysbiosis and non-dysbiosis groups, but patients with dysbiosis were more likely to have a positive reflux-symptom association (76.2% vs 31.7%; P < 0.001), especially for regurgitation in those with SIBO (P = 0.01). Hydrogen gas production was significantly greater in patients with a positive reflux-symptom association for regurgitation (228.8 ppm vs 129.1 ppm, P = 0.004) and belching (mean AUC 214.8 ppm vs 135.9 ppm, P = 0.02). CONCLUSIONS: The prevalence of intestinal dysbiosis is high in patients with GERD, and these patients are more likely to report gas-related symptoms prior to antireflux surgery. Independently, SIBO may be a contributory factor to refractory reflux symptoms and gas bloating in antireflux surgery candidates.

2.
Acta Neuropathol Commun ; 7(1): 218, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870439

RESUMO

Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only ß-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature ß-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated ß-glucocerebrosidase. Immunostaining revealed loss of neuronal ß-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on ß-glucocerebrosidase outside of the context of neurodegeneration, we investigated ß-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn-/- mice had lower ß-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs ß-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired ß-glucocerebrosidase processing.


Assuntos
Demência Frontotemporal/enzimologia , Demência Frontotemporal/genética , Glucosilceramidase/metabolismo , Córtex Pré-Frontal/enzimologia , Progranulinas/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Mutação com Perda de Função , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/enzimologia , Neurônios/patologia , Córtex Pré-Frontal/patologia
3.
Neurobiol Dis ; 124: 152-162, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30448285

RESUMO

Loss-of-function mutations in progranulin (GRN), most of which cause progranulin haploinsufficiency, are a major autosomal dominant cause of frontotemporal dementia (FTD). Individuals with loss-of-function mutations on both GRN alleles develop neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Progranulin is a secreted glycoprotein expressed by a variety of cell types throughout the body, including neurons and microglia in the brain. Understanding the relative importance of neuronal and microglial progranulin insufficiency in FTD pathogenesis may guide development of therapies. In this study, we used mouse models to investigate the role of neuronal and microglial progranulin insufficiency in the development of FTD-like pathology and behavioral deficits. Grn-/- mice model aspects of FTD and NCL, developing lipofuscinosis and gliosis throughout the brain, as well as deficits in social behavior. We have previously shown that selective depletion of neuronal progranulin disrupts social behavior, but does not produce lipofuscinosis or gliosis. We hypothesized that reduction of microglial progranulin would induce lipofuscinosis and gliosis, and exacerbate behavioral deficits, in neuronal progranulin-deficient mice. To test this hypothesis, we crossed Grnfl/fl mice with mice expressing Cre transgenes targeting neurons (CaMKII-Cre) and myeloid cells/microglia (LysM-Cre). CaMKII-Cre, which is expressed in forebrain excitatory neurons, reduced cortical progranulin protein levels by around 50%. LysM-Cre strongly reduced progranulin immunolabeling in many microglia, but did not reduce total brain progranulin levels, suggesting that, at least under resting conditions, microglia contribute less than neurons to overall brain progranulin levels. Mice with depletion of both neuronal and microglial progranulin failed to develop lipofuscinosis or gliosis, suggesting that progranulin from extracellular sources prevented pathology in cells targeted by the Cre transgenes. Reduction of microglial progranulin also did not exacerbate the social deficits of neuronal progranulin-insufficient mice. These results do not support the hypothesis of synergistic effects between progranulin-deficient neurons and microglia. Nearly complete progranulin deficiency appears to be required to induce lipofuscinosis and gliosis in mice, while partial progranulin insufficiency is sufficient to produce behavioral deficits.


Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Microglia/metabolismo , Neurônios/metabolismo , Progranulinas/metabolismo , Animais , Comportamento Animal , Feminino , Demência Frontotemporal , Gliose/metabolismo , Lipofuscina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Progranulinas/genética , Comportamento Social
5.
Bioorg Med Chem Lett ; 18(3): 999-1005, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18162396

RESUMO

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K(I)s of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K(I)s of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.


Assuntos
Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Sulfonamidas/química , Tiadiazóis/química , Animais , Sítios de Ligação , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Camundongos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Especificidade por Substrato , Sulfonamidas/farmacologia , Tiadiazóis/farmacologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-16820676

RESUMO

Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a -CF2- or -CHNH2- spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Inibidores Enzimáticos/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares
8.
Org Lett ; 8(2): 187-9, 2006 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-16408871

RESUMO

[reaction: see text] Since the first disclosure of difluoromethylenediphosphonate, 2, almost 40 years ago, interest in this compound has flourished in several research areas. In this paper, we present a convenient, high-yielding (99% overall) method for the preparation of milligram to multigram quantities of 2 (as the bis(tributylammonium salt, 2b) in a solid form that is easy to handle.

9.
BMC Genomics ; 6: 160, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16288651

RESUMO

BACKGROUND: Nonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. RESULTS: We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. CONCLUSION: This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.


Assuntos
Perfilação da Expressão Gênica , Técnicas Genéticas , Genoma Humano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Animais , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA/química , DNA Complementar/metabolismo , Éxons , Expressão Gênica , Genômica , Humanos , Macaca mulatta , Modelos Genéticos , Oligonucleotídeos/química , Papio , Reação em Cadeia da Polimerase , Primatas , RNA Mensageiro/metabolismo , Análise de Sequência de DNA
10.
J Med Chem ; 48(7): 2695-700, 2005 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-15801860

RESUMO

The triphosphates of antiviral 2',3'-dideoxynucleosides (ddNs) are the active chemical species that inhibit viral DNA synthesis. The inhibition involves incorporation of ddNMP into DNA and subsequent chain termination. A conceivable strategy for antiviral drugs is to employ nucleoside 5'-triphosphate mimics that can entirely bypass cellular phosphorylation. AZT 5'-alpha-R(P)-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaB-betagammaCF(2)TP) has been identified as a potent inhibitor of HIV-1 reverse transcriptase (HIV-1 RT). This work was aimed at confirming that 5'-alphaB-betagammaCF(2)TP is a useful generic triphosphate moiety and can render antiviral ddNs with potent inhibitory effects on HIV-1 RT. Thus, 10 ddNs were converted to their 5'-alphaB-betagammaCF(2)TPs via a sequence (one-pot) of reactions: formation of an activated phosphite, formation of a cyclic triphosphate, boronation, and hydrolysis. Other synthetic routes were also explored. All ddN 5'-alphaB-betagammaCF(2)TPs tested exhibited essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs. A conclusion can be made that 5'-alphaB-betagammaCF(2)TP is a generic and promising triphosphate mimic (P3M) concerning HIV-1 RT inhibition and serum stability. It is anticipated that use of 5'-alphaB-betagammaCF(2)TP as P3M moiety will lead to the discovery of a new class of anti-HIV agents.


Assuntos
Fármacos Anti-HIV/síntese química , Compostos de Boro/síntese química , Desoxirribonucleotídeos/síntese química , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/síntese química , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Bovinos , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Técnicas In Vitro , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/metabolismo , Estereoisomerismo
11.
Org Biomol Chem ; 3(2): 222-4, 2005 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-15632962

RESUMO

Preparation of the key intermediate carboxydifluoromethanesulfonamide provides direct synthetic access to a wide range of novel difluoromethanesulfonamides, including the acetazolamide analogue (2-ethanoylamino-1,3,4-thiadiazol-5-yl)-difluoromethanesulfonamide. Their water solubility and stability, ether partition coefficient, pK(a) and submicromolar dissociation constants for human carbonic anhydrase isozyme II (HCA II) make them promising candidates for topical glaucoma therapy.


Assuntos
Anidrase Carbônica II/química , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/síntese química , Hidrocarbonetos Fluorados/química , Sulfonamidas/química , Sulfonamidas/síntese química , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Estrutura Molecular , Solubilidade , Estereoisomerismo , Sulfonamidas/farmacologia
12.
Nucleosides Nucleotides Nucleic Acids ; 24(10-12): 1651-64, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16438041

RESUMO

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs which require three intracellular phosphorylation steps to yield their corresponding, biologically active, nucleoside triphosphate. In order to circumvent this often inefficient phosphorylation cascade, a plausible approach is to provide the active species directly in the form of a stabilized nucleoside triphosphate mimic. We have previously shown that such a mimic, namely 5'-alpha-Rp-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaBCF2TP) is a generic triphosphate mimic that is biologically stable and can render antiviral ddNs with potent inhibitory activity against HIV-1 RT. Herein we report the synthesis and activity against HIV-1 RT of several ddN 5'-alpha-modified-beta,gamma(difluoromethylene)triphosphate mimics with either a non-bridging calphaP-thio (5'-alphaSCF2TP) or alpha-P-seleno (5'-alpha SeCF2TP) modification. One compound, namely, AZT-5'-alpha-P-seleno-beta,gamma-(difluoromethylene)triphosphate (diastereomer I), was identified as a potent inhibitor of HIV-1 RT (Ki = 64 nM) and represents the first report of HIV-1 RT inhibition data for a nucleotide bearing an alpha-P-seleno modification. These triphosphate mimics may be useful in the investigation of enzyme mechanism and may have interesting properties with respect to drug resistance and polymerase selectivity.


Assuntos
Fármacos Anti-HIV/síntese química , Didesoxinucleosídeos/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , Compostos Organosselênicos/síntese química , Inibidores da Transcriptase Reversa/síntese química , Sulfetos/síntese química , Fármacos Anti-HIV/química , Didesoxinucleosídeos/química , Transcriptase Reversa do HIV/química , Compostos Organosselênicos/química , Inibidores da Transcriptase Reversa/química , Sulfetos/química
13.
J Med Chem ; 47(27): 6902-13, 2004 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-15615539

RESUMO

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/síntese química , Zidovudina/análogos & derivados , Estabilidade de Medicamentos , Humanos , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade
16.
Mini Rev Med Chem ; 2(5): 463-73, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12370047

RESUMO

The replacement of the amide bond in a peptide backbone is a widely used form of peptide mimicry. Several of the most common amide bond surrogates, including peptidomimetic work done in this laboratory, and their biological applications are presented in this review.


Assuntos
Mimetismo Molecular , Peptídeos/síntese química , Aminoácidos/química , Indicadores e Reagentes , Peptídeos/química
17.
Curr Opin Chem Biol ; 6(3): 339-46, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12023115

RESUMO

Methods for combinatorial and parallel synthesis continue to evolve in order to meet the demands of modern synthetic organic chemistry. The nature of the support, while typically overlooked, is a key consideration for successful combinatorial organic synthesis. Developments in combinatorial synthesis technologies such as the 'lab-on-a-chip' concept and 96-well-plate-compatible resin plugs have been reported, which should contribute to meeting the increasing challenges of this field.


Assuntos
Técnicas de Química Combinatória/métodos
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