RESUMO
Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40 MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.
Assuntos
Microscopia/instrumentação , Microscopia/métodos , Algoritmos , Calibragem , Linhagem Celular Tumoral , Corantes/química , Fluorescência , Análise de Fourier , Humanos , Soluções , Fatores de TempoRESUMO
The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reconhecimento Automatizado de Padrão/métodos , Inteligência Artificial , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Health Informatics World Wide, a Web index of research and educational institutions in the fields of Health Informatics and Medical Informatics has been available to WWW users since 1995. In this paper we report on the original conception of this service, its subsequent modifications and address its maintenance and related problems. Access statistics are presented which demonstrate its relevance as well as the geographical focus of Health Informatics research and development.
Assuntos
Indexação e Redação de Resumos , Internet , Informática Médica , HipermídiaRESUMO
A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.
Assuntos
Fármacos Anti-HIV , Delavirdina/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Pirimidinas , Inibidores da Transcriptase Reversa , Inibidores da Transcriptase Reversa/síntese química , Sulfetos , Substituição de Aminoácidos , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Resistência Microbiana a Medicamentos , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Leucina/genética , Camundongos , Prolina/genética , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade , Sulfetos/síntese química , Sulfetos/química , Sulfetos/farmacologiaAssuntos
Benzopiranos/farmacologia , Transtornos de Enxaqueca/tratamento farmacológico , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Vasoconstritores/farmacologia , Animais , Benzopiranos/química , Benzopiranos/metabolismo , Benzopiranos/toxicidade , Disponibilidade Biológica , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Gatos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Dura-Máter/irrigação sanguínea , Dura-Máter/efeitos dos fármacos , Gorilla gorilla , Cobaias , Hipotermia/metabolismo , Inflamação/fisiopatologia , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/toxicidade , Receptor 5-HT1D de Serotonina , Receptores de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/química , Agonistas do Receptor de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/toxicidade , Estereoisomerismo , Sumatriptana/metabolismo , Sumatriptana/farmacologia , Sumatriptana/toxicidade , Resistência Vascular/efeitos dos fármacos , Vasoconstritores/química , Vasoconstritores/metabolismo , Vasoconstritores/toxicidadeAssuntos
Fármacos Anti-HIV/farmacologia , HIV-1 , Piridinas/farmacologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Linhagem Celular , Resistência Microbiana a Medicamentos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Masculino , Mutação , Piridinas/síntese química , Piridinas/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacocinética , EstereoisomerismoRESUMO
Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Artrite Experimental/tratamento farmacológico , Difosfonatos/síntese química , Inflamação/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Difosfonatos/química , Difosfonatos/uso terapêutico , Desenho de Fármacos , Granuloma/tratamento farmacológico , Granuloma/imunologia , Hipersensibilidade Tardia , Indicadores e Reagentes , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
We have discovered two substituted 4-aminopiperidine compounds having high in vitro affinity and selectivity for the human dopamine D1 receptor. Both compounds, 3-ethoxy-N-methyl-N-[1-(phenylmethyl)-4-piperidinyl]-2-pyridinylamine (U-99363E), and its 3-isopropoxy analog (U-101958), were found through a routine receptor binding screen. The determined affinities (Ki) of these compounds for the cloned human dopamine D4 receptor were 2.2 and 1.4 nM, respectively. They exhibited at least 100-fold lower affinities for dopamine D2 and for other dopaminergic, serotonergic and adrenergic receptors. Both compounds were found to antagonize quinpirole-induced mitogenesis in Chinese hamster ovary cells expressing the human dopamine D4 receptor. In spite of their poor metabolic stability and low bioavailability. U-99363E and U-101958 appear to be among the first high-affinity, highly selective dopamine D4 receptor antagonists reported, and may have utility in in vitro investigations requiring selective tagging or blockade of dopamine D4 sites.
Assuntos
Aminopiridinas/farmacologia , Piperidinas/farmacologia , Receptores de Dopamina D2/efeitos dos fármacos , Aminopiridinas/metabolismo , Animais , Células CHO , Cricetinae , Humanos , Mitose/efeitos dos fármacos , Piperidinas/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4 , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transdução de SinaisAssuntos
Antipsicóticos/síntese química , Antagonistas de Dopamina/síntese química , Antagonistas dos Receptores de Dopamina D2 , Piperazinas/síntese química , Sulfonamidas/síntese química , Animais , Antipsicóticos/química , Antipsicóticos/metabolismo , Antipsicóticos/farmacologia , Disponibilidade Biológica , Células CHO , Cricetinae , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/química , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Ergolinas/antagonistas & inibidores , Humanos , Estrutura Molecular , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Ligação Proteica , Quimpirol , Receptores Adrenérgicos/metabolismo , Receptores de Dopamina D4 , Receptores de Serotonina/metabolismo , Esquizofrenia/tratamento farmacológico , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologiaRESUMO
A study of the decomposition of the pyrazoline bisphosphonate ester 2 identified 3 as the sole bisphosphonate component. Evaluation in a delayed-type hypersensitivity granuloma model of chronic inflammation in mice (DTH-GRA) showed 3 to be a potent inhibitor of granuloma formation (sc, 10 mg/kg, 45%), but in a murine model of antigen-induced arthritis (AIA), no significant inhibition was observed. As a result, new ketonic bisphosphonate tetraethyl esters were synthesized from vinylidenebisphosphonic acid tetraethyl ester 4 and activated carbonyl compounds in 13-84% yield. 6 significantly inhibited the pathology of both the DTH-GRA (sc, 25 mg/kg, 45%) and AIA models (sc, 25 mg/kg, 55%). Other compounds in the series were not as potent. Our results show that bisphosphonate ester 6 can inhibit the chronic inflammatory response associated with cutaneous granuloma formation and erosive arthritis.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Artrite/tratamento farmacológico , Difosfonatos/síntese química , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Difosfonatos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-AtividadeRESUMO
Dopamine receptors play an important role in neurotransmission within the central nervous system, as evidenced by their affinity to and apparent site of action of a variety of psychotropic agents. The cloning of dopamine receptor subtypes makes possible the discovery of subtype-selective agonist and antagonist compounds useful for the study of receptor physiology and for the development of more effective treatments for disturbances stemming from aberrant dopamine neurotransmission. We describe in this report the receptor pharmacology of a human D4-dopamine receptor expressed in HEK293 cells from a synthetic gene. The affinities of monoaminergic antagonists in competition with [3H]spiperone-labeled sites in these cells matched very closely their rank order potency measured by other investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell line thus bears the earmarks of a human D4-dopamine receptor. Competition of several agonists best fit a two-site binding model. This result, along with the measured 10-fold GTP-shift for dopamine, strongly suggests functional coupling of this receptor to endogenous G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Rim/ultraestrutura , Receptores de Dopamina D2 , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores Dopaminérgicos/metabolismo , Animais , Ligação Competitiva , Células CHO/metabolismo , Células CHO/ultraestrutura , Células Cultivadas , Cricetinae , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Embrião de Mamíferos , Humanos , Rim/química , Receptores Dopaminérgicos/genética , Receptores de Dopamina D4 , Sensibilidade e EspecificidadeRESUMO
Diphosphonates (DP) are synthetic pyrophosphates with a P-C-P backbone and are predominantly used for the treatment of bone diseases. Several DP have also been shown to exert significant antiarthritic effects in the rat adjuvant-induced polyarthritis model; however, there is no direct evidence for the anti-inflammatory effects of these compounds. We therefore tested the effects of dichloromethylene diphosphonate on delayed-type hypersensitivity granuloma elicited by s.c. implantation of antigen-soaked hydroxyapatite disks in antigen-sensitized mice. Dichloromethylene diphosphonate induced a dose-related inhibition of the delayed-type hypersensitivity granuloma response (38-64% at 25-100 mg/kg/day s.c. or p.o.); novel DP analogs, U-81581, U-82579 and U-84849 were also effective in the same dose range. In contrast, all DP failed to suppress 24-hr delayed-type hypersensitivity paw edema in mice. In addition to rat adjuvant-induced polyarthritis, mouse antigen-induced erosive arthritis was also significantly suppressed by s.c. administration of all four DP. Toxicity was minimal for each DP (> 600 mg/kg p.o. or s.c.). We conclude that DP represent a novel class of anti-inflammatory agents with excellent therapeutic potential for chronic inflammatory diseases including rheumatoid arthritis.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Difosfonatos/farmacologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/tratamento farmacológico , Feminino , Granuloma de Corpo Estranho/tratamento farmacológico , Hipersensibilidade Tardia/tratamento farmacológico , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Ratos , Ratos WistarRESUMO
Vinylidenebisphosphonic acid tetraethyl ester (1) and diazo ketones 7a-1 in ether at 22 degrees C yield pyrazoline bisphosphonate tetraethyl esters 8a-1 in moderate to good yield. These compounds were evaluated in animal models of arthritis: rat adjuvant-induced polyarthritis (AIP) and murine antigen-induced arthritis (AIA) and a murine model of chronic inflammation, the delayed type hypersensitivity granuloma reaction (DTH-GRA). (5-Benzoyl-2,4-dihydro-3H-pyrazol-3-ylidene)-bisphosphonic acid tetraethyl ester (8a), and [5-(3-fluorobenzoyl)-2,4-dihydro-3H-pyraxol-3-ylidene]- bisphosphonic acid tetraethyl ester (8d) significantly inhibited the arthritis models, AIP (15 mg/kg) and AIA (25 mg/kg), as well as the DTH-GRA (25 mg/kg). Conversion of 8a to the corresponding bisphosphonic acid, 10a, resulted in loss of activity. Compounds with alkyl substituents on the pyrazoline nitrogen, 9a-d, were inactive in the DTH-GRA. These results show that 8a and 8d have novel antiinflammatory activity and are capable of inhibiting chronic arthritis and inflammation in animals. Such compounds might be useful in man for treating chronic tissue injury associated with arthropathies such as inflammatory joint disease as well as other chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Difosfonatos/síntese química , Pirazóis/síntese química , Animais , Artrite/tratamento farmacológico , Difosfonatos/química , Difosfonatos/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/química , Pirazóis/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
In 1974 the University of Kentucky was faced with two problems: 1) overburdening of local clinical facilities by students and 2) a large exodus of graduates from the state. The advent of the Area Health Education System offered the opportunity for the clinical education program to develop nontraditional clinical sites across the state. The development and use of these nontraditional sites in predominantly rural areas has become an integral part of the clinical education program. Local facilities are no longer inundated with students. The retention rate of graduates has improved in the seven years of the program from 36 percent to 1972 to 81 percent in 1979.