La técnica de polarización fluorescente para el diagnóstico de Brucella en Chaetophractus villosus / Technique of fluorescence polarization for the diagnosis of Brucella in Chaetophractus villosus / A técnica de polarização fluorescente para o diagnóstico de Brucella em Chaetophractus villosus

La brucelosis es una enfermedad muy extendida por todo el mundo. El diagnóstico se realiza mediante el uso de diferentes técnicas, entre ellas el aislamiento del agente etiológico o por serología detectando anticuerpos específicos. Todos estos métodos están estandarizados y validados en general para las distintas especies animales de interés zootécnico. En animales silvestres pueden utilizarse los mismos procedimientos serológicos, pero cada uno debe ser validado para la especie animal estudiada. El objetivo de este trabajo fue comprobar si la técnica de Polarización Fluorescente (FPA) utilizada para la determinación de anticuerpos contra Brucella en bovinos puede ser usada para el diagnóstico de Brucella en el armadillo (Chaetophractus villosus). Para ello se tomaron 150 muestras de sangre de Chaetophractus villosus. Veinticuatro muestras fueron positivas por medio de las técnicas de antígeno bufferado en placa, seroaglutinación lenta en tubo, 2-mercaptoetanol y fijación del complemento. Las mismas muestras resultaron positivas a la técnica de Polarización Fluorescente, estableciéndose un límite de corte de 82 mP. El índice Kappa registrado fue de 1 para todos los tests de diagnóstico comparados con FPA (IC: 0,84-1).

Brucellosis is a disease widespread throughout the world. The diagnosis is made through the use of different techniques including the isolation of the etiological agent or by serology detecting specific antibodies. All these methods are standardized and validated in general for animal species of zoo-technical interest. In wild animals, serological procedures may be used, but each should be validated for the particular animal species studied. The objective of this work was to verify if the technique of Fluorescent Polarization used for the determination of antibodies against Brucella in ruminants can be used for the diagnosis of Brucella in Chaetophractus villosus. To this aim, s 150 blood samples of Chaetophractus villosus were taken. Twenty-four samples were positive by the techniques of Antigen Buffered in Plate, serum agglutination test (SAT), 2-mercaptoethanol (2-ME) agglutination test and complement fixation test (CFT). The same samples were also positive by Fluorescent Polarization, and a cut-off limit of 82 mP was established. The KAPPA index was 1 for all diagnostic tests compared to FPA (CI: 0.84-1).

A brucelose é uma doença muito espalhada pelo mundo. O diagnóstico é feito por meio de diferentes técnicas, incluindo o isolamento do agente etiológico ou por sorologia detectando anticorpos específicos. Todos esses métodos são padronizados e validados em geral para diferentes espécies animais de interesse zootécnico. Em animais silvestres podem ser utilizados os mesmos métodos serológicos, mas cada um tem de ser validado para a espécie de animal estudada. O objectivo deste estudo foi testar se a técnica de Polarização Fluorescente utilizada para a determinação de anticorpos contra Brucella em bovinos pode ser utilizada para o diagnóstico de Brucella em tatu (Chaetophractus villosus). Para isso, 150 amostras de sangue de Chaetophractus villosus foram coletadas. Vinte e quatro amostras foram positivas por técnicas de antígeno bufferado em placa, soroaglutinação lenta em tubo, 2-mercaptoetanol e fixação do complemento. As mesmas amostras foram positivas para a técnica de Polarização Fluorescente, estabelecendo um limite de corte de 82 mP. O índice Kappa registrado foi de 1 para todos os testes de diagnóstico em comparação com FPA (CI: 0,84-1).

Brucella spp. in equines slaughtered in the south region of Brazil / Anticorpos contra Brucella spp. em equídeos da região Sul do Brasil abatidos em matadouro-frigorífico

Bacteria of the genus Brucella are widespread in many countries. These microorganisms can infect humans and many wild and domestic animal species. These bacteria have zoonotic potential, and can cause economic and public health problems since they can be transmitted by direct contact with sick animals, through consumption of contaminated milk, raw meat and its derivatives (Soares et al., 2015). Brucellosis is considered a chronic evolving disease, unusual in horses, predominantly caused by Brucella abortus. However, it is not characterized by reproductive disorders in horses, but primarily by abscess in the cervical region, bursa, tendons, and joints. Transmission is likely to occur via ingestion of contaminated water and pastures, especially in areas endemic for bovine brucellosis (Ribeiro et al., 2008). The slaughterhouse is a strategic point for obtaining information about the animal and animal products, edible or not. This study investigated the presence of anti-Brucella spp. immunoglobulins in the serum samples from horses slaughtered in a slaughterhouse in southern Brazil, to estimate the frequency of Brucella spp. antibodies and determine the spatial distribution of the cases.(AU)

Objetivou-se investigar a presença de imunoglobulinas anti-Brucella spp. em amostras de soros sanguíneos de equídeos abatidos em matadouro-frigorífico, sob Serviço de Inspeção Federal, localizado na região Sul do Brasil. Utilizaram-se 767 amostras de sangue de equídeos adultos abatidos no período de abril a maio de 2013. Os animais foram provenientes de 45 municípios dos estados do Rio Grande do Sul, Santa Catarina e Paraná. Para diagnóstico, foram utilizados os testes do antígeno acidificado tamponado (AAT), sendo os resultados positivos confirmados pelos testes de polarização fluorescente (TPF), reação de fixação de complemento (RFC) e 2-mercaptoetanol (2-ME). Foram sororreagentes no AAT 65 (8,47%) animais. Destes, apenas dois (3,07%) foram positivos também na RFC e três (4,62%) animais foram positivos no TPF. Apesar da baixa frequência de animais positivos para Brucella spp., pode-se afirmar que a infecção em equinos está presente na área estudada, o que é demonstrado pela presença de animais sororreatores. No âmbito da saúde animal, pública e ocupacional, sugere-se a atenção a essa doença, visando diminuir o risco de infecção.(AU)

Effects of mercury on the structure and activity of BLM642-1290 recombinant helicase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Bloom's syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg(2+) on the structure and activity of BLM642-1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase.</p><p><b>METHODS</b>The effects of Hg(2+) on biological activity and structure of BLM642-1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free-phosphorus assay technologies, respectively.</p><p><b>RESULTS</b>The helicase activity, the DNA-binding activity, and the ATPase activity of BLM642-1290 recombinant helicase were inhibited by Hg(2+) treatment. The LMCT (ligand-to-metal charge transition) peaks of the helicase were enhanced with the increase of the Hg(2+) level. The LMCT peaks of the same concentration of helicase gradually increased over time.</p><p><b>CONCLUSION</b>The biological activity of BLM642-1290 recombinant helicase is inhibited by Hg(2+) treatment. The conformation of the helicase is significantly altered by Hg(2+). There exist two binding sites between Hg(2+) and the helicase, which are located in the amino acid residues 1063-1066 and 940-944 of the helicase, respectively.</p>

Avaliação da maturidade pulmonar fetal pela contagem dos corpos lamelares no líquido amniótico / Evaluation of fetal lung maturity by lamellar bodies counting in amniotic fluid

Objetivo: comparar o teste de contagem de corpos lamelares (CCL) no líquido amniótico com o teste da polarização fluorescente (PF) como parâmetro diagnóstico para avaliação da maturidade pulmonar fetal. Método: estudo transversal, analítico e controlado realizado com 60 gestantes atendidas no período de março de 2002 a dezembro de 2007. Foram colhidas amostras de líquido amniótico e realizados os testes de CCL e PF (TDxFLM II), considerados de referência, e comparados à presença ou ausência da Síndrome do Desconforto Respiratório (SDR). Foram estabelecidos valores de corte para maturidade de 30 mil corpos lamelares/µL para o teste da CCL e 55 mg/g de albumina para o PF. Foram avaliadas as características maternas e perinatais, a evolução neonatal e o desempenho dos testes diagnósticos para predição da maturidade pulmonar fetal. Na análise estatística, foram utilizadas medidas descritivas e calculados os valores referentes à sensibilidade, especificidade, valor preditivo positivo e negativo dos testes, considerando-se significativos valores de p<0,05. Resultados: a idade materna variou entre 15 e 43 anos, com média de 26,6 anos. A idade gestacional variou entre 24,3 e 41,6 semanas, com média de 35,1 semanas. A Síndrome do Desconforto Respiratório foi diagnosticada em 13,3 por cento dos neonatos. As características perinatais, como peso, índice de Apgar, incidência de SDR, foram comparadas aos resultados dos testes de CCL e PF, sendo observada uma correspondência, estatisticamente significativa (p<0,05), entre os grupos de neonatos clinicamente classificados como imaturos e maduros em ambos os testes. Os testes foram concordantes em 68,3 por cento dos casos. Quando se comparou o teste da PF com o teste da CCL, a sensibilidade foi de 100 por cento para ambos, e a especificidade do teste da CCL foi superior (73,1 por cento), quando comparado com o teste de PF (51,9 por cento). O padrão-ouro para determinação da maturidade fetal é a ocorrência da SDR. O valor...

Purpose: to compare the lamellar body number density (LBND) count in amniotic fluid using the fluorescent polarization (FP) test as a diagnostic parameter for the assessment of fetal pulmonary maturity. Method: this was an analytical, controlled cross-sectional study conducted on 60 pregnant women from March 2002 to December 2007. Amniotic fluid specimens were obtained by amniocentesis or at the time of caesarean section, and submitted to the LBND and FP tests (TDxFLM®, Abbott Laboratories), the latter considered to be a reference test, and compared in terms of the presence or absence of respiratory distress syndrome (RDS). Cut-off values for maturity were established at 30,000 lamellar bodies/µL for the LBND test and 55 mg/g albumin for the FP test. Maternal and perinatal characteristics and neonatal evolution were evaluated, and the performance of the diagnostic tests regarding fetal pulmonary maturity was determined. In the statistical analysis, descriptive measures were used and the sensitivity, specificity and positive and predictive values of the tests were determined with the level of significance set at p<0.05. Results: maternal age ranged from 15 to 34 years (mean: 26.6 years) and gestational age ranged from 24.3 to 41.6 weeks (mean: 35.1 weeks). RDS was diagnosed in 35.1 percent of neonates. Perinatal characteristics such as weight, Apgar score, and RDS incidence were compared to the results of the LBND and FP tests and a significant correspondence (p<0.05) was observed between the groups of neonates clinically classified as mature and immature in both tests. The tests were concordant in 68.3 percent of the cases. Comparison of the PF and LBND tests revealed 100 percent specificity for both and a higher specificity for the LBND test (73.1 percent as opposed to 51.9 percent for the PF test). The gold standard for the determination of fetal maturity is the occurrence of RDS. The positive predictive value of the LBND test was higher (36.4%) than that...

Expression, purification and activity analyses of three Bcl-2 family proteins / 生物医学工程学杂志

Bcel-2 family proteins (Bcl-x(L), Bcl-2, Mel-1 etc.) are key regulators of some life processes, including apoptosis and autophagy. They are currently considered as promising targets for developing new anti-tumor therapies. In our study, the human Bcl-2/Bcl-x(L) chimeric gene and the human/mouse Mel-1 chimeric gene were designed and cloned, and the prokaryotic expression vectors for expressing glutathione S-transferase (GST) fusion proteins and histidine tag fusion proteins were constructed respectively. These two proteins as well as the GST-Bcl-x(L) fusion protein were all successfully expressed in E. coli and subsequently purified. In addition, we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay. The dissociation constants (Kd) obtained by us were in general agreement with the data reported in literature. The Kd values of all three proteins with or without the GST tag were almost identical. All these results validate the biological functions of these Bcl-2 family proteins obtained by us. These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.

The Effect of Tetracaine.HCl on Rotational Mobility of n-(9-Anthroyloxy) Stearic Acid in Outer Monolayers of Neuronal and Model Membranes

To provide a basis for studying the pharmacological actions of tetracaine.HCl, we analyzed the membrane activities of this local anesthetic. The n-(9-anthroyloxy) stearic and palmitic acid (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used previously to examine fluorescence polarization gradients. These probes can report the environment at a graded series of depths from the surface to the center of the membrane bilayer structure. In a dose-dependent manner, tetracaine.HCl decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes derived from total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. However, this compound increased the anisotropy of 2-AS at the membrane interface. The magnitude of the membrane rotational mobility reflects the carbon atom numbers of the phospholipids comprising SPMV, SPMVTL and SPMVPL and was in the order of the 16, 12, 9, 6, and 2 positions of the aliphatic chains. The sensitivity of the effects of tetracaine.HCl on the rotational mobility of the hydrocarbon interior or surface region was dependent on the carbon atom numbers in the descending order 16-AP, 12-AS, 9-AS, 6-AS and 2-AS and on whether neuronal or model membranes were involved in the descending order SPMV, SPMVPL and SPMVTL.

Brucelosis en el zorro gris pampeano (Pseudalopex gymnocercus) en la provincia de La Pampa (Argentina) / Brucelosis in the grey fox of the pampas (Pseudalopex gymnocercus) in La Pampa province (Argentina)

Brucella abortus (Br abortus) es una de las principales causas de abortos y pérdidas reproductivas en el ganado bovino. Los fetos abortados y sus fluidos fetales son la mayor fuente de infección. Es así como la enfermedad puede llegar a animales silvestres cuando ingieren esos tejidos. El objetivo de este trabajo fue determinar la prevalencia serológica de brucelosis en el zorro gris pampeano (Pseudalopex gymnocercus). Se tomaron muestras de sangre de 41 zorros de la región centro-este de la provincia de La Pampa, Argentina. Para determinar la presencia de anticuerpos específicos contra Br abortus se utilizaron las Pruebas de Polarización de la Fluorescencia (FPA), Seroaglutinación en Microplaca (SAP) y Seroaglutinación en Microplaca con 2-mercaptoetanol (2-ME). Sobre un total de 41 sueros procesados por FPA, en 7 (17,1%) se detectaron anticuerpos contra Br abortus. Sólo 34 sueros fueron analizados por las pruebas de SAP y 2-ME, encontrando 5 (14,7%) y 4 (11,8%) muestras positivas, respectivamente. Los resultados permiten inferir que el zorro gris pampeano es susceptible a infectarse con Br. abortus a una tasa de incidencia importante. Es necesario realizar futuros estudios para establecer el rol del zorro en la transmisión de la enfermedad y sus consecuencias en esta especie.

Brucellosis is produced by several species of Brucella. Brucella abortus causes abortion and reproductive loss in bovine cattle. In the epidemiology of brucellosis, aborted fetus and their fetal fluids are the main source of infection and dissemination. Although the biological cycle and the disease consequences to domestic cattle have been widely studied, it is not the case with wild fauna. The objective of this study was to determine the serologic prevalence to brucellosis in the grey fox of the pampas (Pseudalopex gymnocercus). To that purpose, 41 foxes were sampled in the centre-east area of La Pampa province (Argentina). Blood samples for serologic studies were collected. A test of agglutination in microplate (SAP), another SAP with the addition of 2-mercapto-etanol (2-ME) and a test of polarization of the fluorescence (FPA) were used for the diagnosis of antibodies against Brucella abortus. For this, 17.1%, 14.7% and 11.8% was the prevalence found by FPA, SAP and SAP with 2-ME. More studies will be necessary to know the role of foxes in disease transmission and maintenance as well as the consequence of the diseases in foxes.

Substituted phenols as pollutants that affect membrane fluidity.

In a toxicological context, the cellular effects of a variety of molecular compounds interacting with membranes may be understood in terms of their ability to affect and modulate lipid-membrane physical properties and even slight changes in membrane fluidity may cause aberrant function and pathological processes. Different model systems (mice splenocytes and liposomes) have been used in modelling studies of the physical effects on lipid bilayers underlying the action of membrane active phenolic compounds, considered by EPA (Environmental Protection Agency) as priority pollutants (phenol; 2-chlorophenol; 2,4-dichlorophenol; 2,4,6-trichlorophenol; pentachlorophenol; 2-nitrophenol; 2,4-dinitrophenol; 2-methyl-4,6-dinitrophenol). Membrane fluidity was assessed by fluorescence steady-state anisotropy of a fluorescent probe 1,6-diphenil-1,3,5-hexatriene (DPH). The substituted phenols increased the fluidity of cells and liposome membranes in a concentration dependent manner and the nitro substituted phenols were the most efficient perturbing the biophysical properties of the membrane. A good parallelism has been established between the results obtained with cell models and artificial liposome model systems, implying that liposomes are useful alternative systems in membrane modification studies and can be conveniently used in order to evaluate the potential toxic effect of phenol derivatives that are common environmental pollutants.

A study of changes in homocysteine levels during normal pregnancy and pre-eclampsia.

To find out changes in homocysteine levels that occur during normal pregnancy and pregnancy with pre-eclamptic toxaemia and also to find out correlation between homocysteine concentration and preeclamptic toxaemia a study was carried out among 90 women of which 30 were control which included normotensive non-pregnant women and the study group I comprised 30 pregnant normotensive women and the study group II comprised 30 pregnant women with pre-eclamptic toxaemia. Serum homocysteine was measured in all subjects using fluorescence polarisation immuno-assay. Control group had highest mean homocysteine levels while the study group I had least mean homocysteine levels (p < 0.001). Levels were significantly higher in subjects with BP > 146/100 mm Hg as compared to subjects with BP >140/90 and <146/100 mm Hg (p=0.017). There was significant difference between study group I and II at same gestational age. Hyperhomocysteinaemia was observed in pre-eclamptic females, also it was found that homocysteine levels were directly correlated with severity of pre-eclampsia.

Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients / Fluidez e composição lipídica da membrana de cepas de Candida albicans resistentes e sensíveis ao fluconazol, isoladas de pacientes diabéticos

Ten clinical isolates of Candida albicans, five strains belonging to each of fluconazole resistant and susceptible groups isolated from diabetic patients, were studied for the membrane fluidity and lipid composition. Compared to fluconazole susceptible strains, fluconazole resistant ones exhibited enhanced membrane fluidity as measured by fluorescence polarization technique. The increased membrane fluidity was reflected in the decreased p-values exhibited by the resistant strains. On the other hand, susceptible isolates contained higher amount of ergosterol, almost twice as compared to resistant isolates which might have contributed to their lower membrane fluidity. However, no significant alteration was observed in the phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of the exposed aminophospholipid, phosphatidylethanolamine was highest in the resistant strains as compared to the susceptible strains, indicating a possible overexpression of CDR1 and CDR2 genes in resistant strains. The results presented here suggest that the changes in the ergosterol content and overexpression of ABC transporter genes CDR1 and CDR2 could contributeto fluconazole resistance in C. albicans isolated from diabetic patients.

Dez isolados clínicos, sendo cinco resistentes e cinco sensíveis ao fluconazol, obtidos de pacientes diabéticos, foram estudados quanto à fluidez e composição química da membrana. Quando comparados aos isolados sensíveis ao fluconazol, os isolados resistentes apresentaram fluidez de membrana aumentada, conforme mensurado pela técnica de polarização fluorescente. A fluidez de membrana aumentada refletiu-se pelos valores mais baixos de p. Por outro lado, os isolados sensíveis continham quantidades mais elevadas de ergosterol, quase o dobro dos isolados resistentes, o que pode ter contribuído para a fluidez de membrana mais baixa. Entretanto, não se observou alteração significativa na composição fosfolipídica e de ácidos graxos nesses isolados. Experimentos de marcação com corante fluorescamina indicaram que a porcentagem de aminofosfolípides e fosfatidiletanolamina expostos foi mais elevada nos isolados resistentes do que nos sensíveis, indicando uma possível superexpressão dos genes CDR1 e CDR2 nos isolados resistentes. Os resultados aqui apresentados sugerem que alterações no teor de ergosterol e superexpressão dos genes ABC transportadores CDR1 e CDR2 podem contribuir na resistência ao fluconazol em isolados de C. albicans de pacientes diabéticos.

Polarização fluorescente do líquido amniótico para avaliação da maturidade pulmonar fetal em gestações de alto risco / Fluorescence polarization of amniotic fluid to assess fetal lung maturity in high risk pregnancies

O objetivo deste trabalho foi verificar a acurácia, sensibilidade, especificidade, valores preditivos positivo e negativo da polarização fluorescente do líquidoamniótico em gestações de alto risco ante a presença ou ausência de síndrome de desconforto respiratório neonatal. Estudo prospectivo descritivo que incluiu 54 gestantes de alto risco para avaliação da maturidade pulmonar fetal por meio da polarização fluorescente do líquido amniótico obtido por amniocentese, cujos partos ocorreram em até 72 horas após a coleta de líquido amniótico. A ocorrência de síndrome de desconforto respiratóriofoi o desfecho primário analisado, sendo estratificado pela idade gestacional ao nascimento. O resultado negativo(indicador de maturidade pulmonar fetal) da polarização fluorescente do líquido amniótico foi considerado para valores maiores ou iguais a 50 mg/g. Resultados: A idade gestacionalmédia ao nascimento foi de 35 semanas (DP 2.0). A síndrome de desconforto respiratório foi identificada em 14 recém-nascidos (24%). A polarização fluorescente do líquido amniótico apresentou alta sensibilidade (86%) e especificidade (81%) com 14% de falsosnegativos e 19% de falsos-positivos. O valor preditivo positivofoi de 60% e o valor preditivo negativo de 94%. A Curva ROC apontou como melhor ponto de corte a relação albumina/surfactante de 50 mg/g (sensibilidade de 85% e especificidade de 81%). A polarização fluorescente do líquido amniótico comvalor negativo (resultado maior ou igual a 50 mg/g) confirma a maturidade pulmonar com um risco muito baixo de o recémnascido desenvolver SDR em gestações de alto-risco.

Identification of ligands for human LOX-1 through fluorescence polarization-based high throughput screening / 药学学报

<p><b>AIM</b>To develop a fluorescence polarization-based high throughput screening and identify ligands for human Lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1).</p><p><b>METHODS</b>Sequential ultracentrifugation at 4 degrees C from normolipidemic fasting volunteers to obtain low density lipoprotein (LDL), which was modified by CuSO4 (5 micromol x L(-1)) at 37 degrees C for 24 h. The assay was based on the interaction between receptor and ligand, and hLOX-1 was labeled by FITC and bound to its specific ligand, oxLDL. Different reaction time and DMSO concentration were optimized to determine the stability and tolerance of fluorescence polarization (FP) assay. 3 200 compounds were screened in black 384-well microplate by FP-based competitive displacement assay, at excitation filter of 485 nm and emission filter of 530 nm. Z' was used to assess the assay quality.</p><p><b>RESULTS</b>The FP-based HTS was formatted in a 384-well microplate with a Z' factor of 0. 75, and three active compounds for hLOX-1 were identified with IC50 below 40 micromol x L(-1) from total 3 200 compounds.</p><p><b>CONCLUSION</b>The results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.</p>

Effects of Chlorhexidine digluconate on Rotational Rate of n- (9-Anthroyloxy)stearic acid in Model Membranes of Total Lipids Extracted from Porphyromonas gingivalis Outer Membranes

The purpose of this study was to provide a basis for studying the molecular mechanism of pharmacological action of chlorhexidine digluconate. Large unilamellar vesicles (OPGTL) were prepared with total lipids extracted from cultured Porphyromonas gingivalis outer membranes (OPG). The anthroyloxy probes were located at a graded series of depths inside a membrane, depending on its substitution position (n) in the aliphatic chain. Fluorescence polarization of n- (9-anthroyloxy)stearic acid was used to examine effects of chlorhexidine digluconate on differential rotational mobility, while changing the probes' substitution position (n) in the membrane phospholipids aliphatic chain. Magnitude of the rotational mobility of the intact six membrane components differed depending on the substitution position in the descending order of 16- (9-anthroyloxy)palmitic acid (16-AP), 12, 9, 6, 3 and 2- (9-anthroyloxy)stearic acid (12-AS, 9-AS, 6-AS, 3-AS and 2-AS). Chlorhexidine digluconate increased in a dose-dependent manner the rate of rotational mobility of hydrocarbon interior of the OPGTL prepared with total lipids extracted from cultured OPG, but decreased the mobility of membrane interface of the OPGTL. Disordering or ordering effects of chlorhexidine digluconate on membrane lipids may be responsible for some, but not all of its bacteriostatic and bactericidal actions.

A high throughout assay for human papillomavirus genotypes with fluorescence polarization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA.</p><p><b>METHODS</b>Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence.</p><p><b>RESULTS</b>Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B, 11 and 16 (4), HPV11, 16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11, 16 and 18 (1)] and one HPV quadruple infection (HPV6B, 11, 16 and 18) were detected in cervical cancer scrapes.</p><p><b>CONCLUSIONS</b>The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.</p>

Effects of Chlorhexidine Digluconate on Rotational Rate of n- (9-Anthroyloxy) stearic Acid in Porphyromonas ginginvalis Outer Membranes

The aim of this study was to provide a basis for studying the molecular mechanism of pharmacological action of chlorhexidine digluconate. Fluorescence polarization of n- (9-anthroyloxy) stearic acid was used to examine the effect of chlorhexidine digluconate on differential rotational mobility of different positions of the number of membrane bilayer phospholipid carbon atoms. The six membrane components differed with respect to 2, 3, 6, 9, 12, and 16- (9-anthroyloxy) stearic acid (2-AS, 3-AS, 6-AS, 9-AS, 12-AS and 16-AP) probes, indicating different membrane fluidity. Chlorhexidine digluconate increased the rate of rotational mobility of hydrocarbon interior of the cultured Porphyromonas gingivalis outer membranes (OPG) in a dose-dependent manner, but decreased the mobility of surface region (membrane interface) of the OPG. Disordering or ordering effects of chlorhexidine digluconate on membrane lipids may be responsible for some, but not all of its bacteriostatic and bactericidal actions.

Effects of Local Anesthetics on Rotational Mobility of n- (9-Anthroyloxy) stearic Acid in Neuronal Membranes

To elucidate the molecular mechanism of pharmacological action of local anesthetics, we studied membrane actions of tetracaine, bupivacaine, lidocaine, prilocaine and procaine. Fluorescence polarization of n- (9-anthroyloxy) stearic acid (n-AS) was used to examine the effects of these local anesthetics on differential rotational mobility of different positions of the number of synaptosomal plasma membrane vesicle (SPMV) phospholipid carbon atoms. The four membrane components differed with respect to 3, 6, 9 and 16- (9-anthroyloxy) stearic acid (3-AS, 6-AS, 9-AS and 16-AP) probes, indicating that differences in the membrane fluidity might be present. Degrees of the rotational mobility of 3-AS, 6-AS, 9-AS and 16-AP were different depending on depth of hydrocarbon interior. In a dose-dependent manner, tetracaine, bupivacaine, lidocaine, prilocaine and procaine decreased anisotropy of 3-AS, 6-AS, 9-AS and 16-AP in the hydrocarbon interior of the SPMV. These results indicate that local anesthetics have significant disordering effects on hydrocarbon interior of the SPMV, thus affecting the transport of Na+ and K+ in nerve membranes and leading to anesthetic action.

Time-resolved fluorescence anisotropy measurement and its application in the research of biomacromolecule / 生物医学工程学杂志

In this symposium, we reviewed the principle and development of time-resolved fluorescence anisotropy measurement. Its method of measurement, characteristics and applications in the research of biomacromolecule, configuration and molecular structure have been discussed. Its potential applications are also illustrated.

Correlation Between Cyclosporine Trough Levels and 2-Hour Postdose Blood Levels and Target C2 Levels in Patients after 12 Months Post Kidney Transplantation / 대한신장학회잡지

BACKGROUND: Cyclosporine dosing is traditionally based on trough levels(C0 level) rather than area under the concentration-time curve(AUC), although AUC correlates better with post transplantation acute rejection and acute toxicity. It is reported that C2 levels(2-hour postdose blood levels) are single sampling point that best reflects AUC0-4. But there has been no recommended C2 levels for patients after 12 months post kidney transplantation. The purpose of this study was to evaluate the correlation between C0 levels and C2 levels and define recommended target C2 levels in patients after 12 months post kidney transplantation. METHODS: Seventy three patients after 12 months post transplantation were studied. 83 data were obtained from 73 renal transplant patients. Blood C0 levels, blood C2 levels, body weight and serum creatinine level were measured. Blood cyclsporine levels were measured by monoclonal fluorescence polarization immunoassay(mFPIA)(TDX, Abbot). The data of C0 levels were divided into three groups : low group (mean+SD, 197.1 ng/mL). RESULTS: There was a positive correlation between C0 levels and C2 levels, but no correlation between C0 levels and C2 levels when C0 levels were divided into three groups. There was a positive correlation between cyclosporine/body weight and C2 levels in normal C0 group. Recommended C2 levels in normal C0 group is 724.7+/-210.1 ng/mL. CONCLUSION: It is assumed that cyclosporine doses can be individualized by using C2 levels rather than C0 levels in renal transplant patients. However, prospective study may be needed to confirm the improvement of longterm renal allograft survival by individualizing cyclosporine doses based on C2 levels.

Mecanismo de ação do metotrexato e avalição clínica de sua associação com a cloroquina no controle de artrite reumatóide juvenil / Methotrexate action mechanism and clinical assessment of its association with chroquine in juvenile rheumatoid arthritis control

Estudaram-se a eficácia clínica do uso do metotrexato (MTX) e sua associação com a cloroquina (CQ) para o controle da artrite reumatóide juvenil, e o mecanismo bioquímico de inibição celular do MTX. A resposta clínica foi obtida através da avaliação de dor, edma e número de articulações acometidas;a avaliação cinética do MTX no plasma e urina, bem como o estudo dos indicadores de atividade da doença: proteína C-reativa (PCR), substância amilóide A (SAA), velocidade de hemossedimentação (VHS), fibrinogênio e "alfa"-glicoproteína ácida foram feitos em pacientes, cuja idade variou de 12 a 25 anos. Todos os pacientes apresentaram relativo controle da doença e nao mostraram diferença significativa, com relação...