Proteomic analysis of latex from Jatropha curcas L. stems and comparison of two classic proteomic sample isolation methods: the phenol extraction and the TCA/acetone extraction

Background: Jatropha curcas is a wide-spreading latex-rich biodiesel plant with high oil content in seeds that have always been under intense studies. However, studies are lacking on the latex component that is considered rich in proteins with potentially important physiological functions and secondary metabolites that are a promising source for new drugs. The proteomic analysis, which would be the first step to study these substances, was hampered by the presence of interfering components. Phenol extraction and Trichloroacetic acid (TCA)/ acetone extraction, two major plant proteomic isolation methods, were used and compared in this study. Results: We identified 459 proteins from the J. curcas latex proteome using the combination of the two extraction techniques. Although more number of latex proteins were identified by the phenol extraction (401 proteins vs. 123 proteins by the TCA/acetone extraction), only 65 proteins were commonly isolated by both methods. Analysis of the biochemical properties revealed that relatively more number of lower isoelectric point (pI) proteins were isolated by the TCA/acetone method (pI mode: 4.79, 6.51 for phenol). Moreover, GO, COG, and KEGG analyses showed that certain classes/categories/pathways annotated more number of proteins than others, and most of them had proportionally comparable protein counts by both the methods, however, with exemplified exceptions. Conclusions: A large number of proteins were found and exclusively identified by either method, indicating that a better proteome coverage of plant samples in a similar context needs the combined use of multiple isolation methods. In addition, the core biological function of the latex may be uncovered by certain GO, COG, and KEGG classes/categories/pathways that annotate more proteins.

Repair of amalgam restorations with composite resin and bonded amalgam: A microleakage study.

Background: Total replacement is the most common technique for defective amalgam restorations, and it represents a major part of restorative dental treatment. Repair is an alternative option for amalgam restorations with localized defects. Aims: This study compared microleakage of amalgam restorations repaired by bonded amalgam or composite resin. Materials and Methods: Thirty extracted human pre-molars were prepared and restored with class I amalgam. A simulated defect was prepared that included the cavosurface margin on restorations, and the pre-molars were assigned to two treatment groups (n=15): In group 1, premolars were treated by composite resin (34% Tooth Conditioner Gel + Adper Single Bond 2 + Z100) and in group 2, premolars were repaired by bonded amalgam (34% Tooth Conditioner Gel + Prime and Bond 2.1 + Permite C). The teeth were immersed in a 50% silver nitrate solution, thermocycled, sectioned longitudinally and then observed by three examiners using a stereomicroscope. Microleakage was evaluated using a 0-4 scale for dye penetration, and data was analyzed by Kruskal Wallis and Dunn tests. Results: Neither of the two methods eliminated microleakage completely. Composite resin was significantly the most effective for repair/tooth interface sealing (score 0 = 80.0%; P=0.0317). For the repair/restoration interface, composite resin was also statistically more effective as a sealant (score 0=66%; P=0.0005) when compared to the bonded amalgam technique (score 0=13%; P=0.0005). Conclusions: The use of adhesive systems significantly affected the ability to seal the repair/ tooth interface. However, at the level of the repair/restoration interface, the bonded amalgam technique may increase microleakage.

Comparación Histológica e Inmunohistoquimica de Muestras de Tejido Procesadas por la Técnica Convencional o por el Método Simplificado de Acetonas / Histological and Immunohistochemical Comparison of Tissue Samples Processed by the Conventional Technique or the Acetone Simplified Method

La técnica convencional (TC) que usa alcohol como agente deshidratante, es un método útil en los laboratorios de Histología y Anatomía Patológica. Recientemente, un nuevo método simplificado con acetona (TA) fué diseñado en un intento por reducir el tiempo de procesamiento de los tejidos (de 12 a 4 horas), el costo de reactivos, y como un factor importante para tener un diagnóstico veraz y rápido. En este trabajo se compararon los parámetros histológicos e inmunohistoquímicos en muestras de cáncer de mama, colon y riñón tratadas con ambos métodos. Los cortes fueron teñidos con H & E o tricrómico de Masson. También se llevó a cabo inmunohistoquímica con anticuerpos especificos para la identificación de citoqueratinas AE1/AE3, el receptor de estrógeno y receptor de progesterona. Se realizó un estudio ciego por tres especialistas en morfológía, quienes evaluaron la tinción nuclear, tinción del citoplasma y friabilidad o cambios en la estructura tisular. La especificidad y sensibilidad de la unión de los anticuerpos también fueron evaluadas. Los valores obtenidos para cada parámetro se analizaron estadísticamente con la prueba t de Student. Nuestros resultados muestran que los métodos de TC y TA no modificaron características histológicas como el patrón de tinción, ni se detectaron cambios tisulares. La positividad de la inmunohistoquímica fué similar para ambos métodos. No se observó diferencia estadística entre TC y TA. Nuestros resultados sugieren que la aplicación de la TA no modificó las propiedades histológicas e inmunohistoquímicas y que podría ser un método útil en el análisis morfológico.

The conventional technique (CT) using alcohol as a drying agent, is a useful method of histology and pathology in the laboratory. Recently, a new simplified method with acetone (TA) was designed in an attempt to reduce the processing time of the tissues (12 to 4 hours), the cost of reagents, and as an important factor to have an accurate and prompt diagnosis . This study compared the histological and immunohistochemical parameters in breast cancer, colon and kidney specimens treated with both methods. The sections were stained with H & E or Masson trichrome. Immunohistochemistry study with antibodies specific for the identification of cytokeratin AE1/AE3, the estrogen receptor and progesterone receptor was also carried out. Blind study was conducted by three specialists in morphology, who evaluated the nuclear staining, staining of the cytoplasm and friability or changes in tissue structure. The specificity and sensitivity of antibody binding were also evaluated. The values obtained for each parameter were statistically analyzed with Student t test. Our results show that the methods of CT and TA did not alter histological features as the pattern of staining, and tissue changes were detected. Immunohistochemical positivity was similar for both methods. There was no statistical difference between TC and TA. Our results suggest that implementation of the TA did not alter the histological and immunohistochemical properties and could be a useful method for morphological analysis.

Antioxidant activity of acetone extract/fractions of Terminalia bellerica Roxb. fruit.

Terminalia bellerica Roxb. (Family: Combretaceae) has been valued in Indian system of medicine for treatment of wide range of diseases and reported to have antioxidant properties. In the present study, the free radical scavenging activity and antioxidant potential of acetone extract/fractions of its fruit was investigated using in vitro assays, including scavenging ability against 2,2′-diphenyl-2-picrylhydrazyl (DPPH), β-carotene bleaching inhibition, reducing power and chelating ability on Fe2+ ions. The fruit powder was extracted at room temperature with different solvents in the order of increasing and decreasing polarity to obtain crude acetone extract which was further partitioned with ethyl acetate and water (1:1). It was found that ethyl acetate fraction was more effective than crude acetone extract in all antioxidant assays, except chelating power which was highest in water fraction. Maximum antioxidant activities (expressed as EC50 values) observed were 14.56 μg/ml, 27.81 μg/ml and 67.8 μg/ml in DPPH, β-carotene bleaching and reducing power assays, respectively. The antioxidant potential was compared with known antioxidant (butylated hydroxyl toluene) and correlated with total phenolic and flavonoid content in crude extract and fractions. Fractions rich in polyphenolic content were more effective than the crude extract.

Bonding agent underneath sealant: shear bond strength to oil-contaminated

This study evaluated in vitro the shear bond strength of a resin-based pit-and-fissure sealant (Fluroshield - F) associated with either an ethanol-based (Adper Single Bond 2 - SB) or an acetone-based (Prime & Bond - PB) adhesive system under conditions of oil contamination. Mesial and distal enamel surfaces from 30 sound third molars were randomly assigned to 2 groups (n=30): I - no oil contamination; II - oil contamination. Contamination (0.25 mL during 10 s) was performed after 37 percent phosphoric acid etching with an air/oil spray. The specimens were randomly assigned to subgroups, according to the bonding protocol adopted: subgroup A - F was applied to enamel without an intermediate bonding agent layer; In subgroups B and C, SB and PB, respectively, were applied, light-cured, and then F was applied and light-cured. Shear bond strength was tested at a crosshead speed of 0.5 mm/min in a universal testing machine. Means (± SD) in MPa were: IA-11.28 (±1.84); IIA-12.02 (±1.15); IB-9.73 (±2.38); IIB-9.62 (±2.29); IC-28.30 (±1.63); and IIC-25.50 (±1.91). It may be concluded that the oil contamination affected negatively the sealant bonding to enamel and the acetone-based adhesive system (PB) layer applied underneath the sealant was able to prevent its deleterious effects to adhesion.

Este estudo avaliou in vitro a resistência ao cisalhamento (RC) de um selante resinoso [Fluroshield (F); Dentsply/Caulk] em associação com um sistema adesivo com solvente a base de etanol [Adper Single Bond 2 (SB); 3M/ESPE] ou a base de acetona [Prime & Bond (PB); 3M/ESPE] após contaminação com óleo do esmalte. Superfícies mesiais e distais de esmalte de 30 terceiros molares hígidos foram aleatoriamente alocadas em 2 grupos (n=30): I - contaminação com óleo; II - sem contaminação. A contaminação foi realizada (0,25 mL;10 s) com um jato de ar/óleo após o condicionamento do esmalte com ácido fosfórico a 37 por cento. Os espécimes foram aleatoriamente alocados em subgrupos, de acordo com a técnica adesiva empregada: A - F foi aplicado sobre o esmalte condicionado sem sistema adesivo; B - SB + F; C - PB + F. RC foi testada em uma máquina universal de ensaios (0,5 mm/min; 50 kgf) e os dados analisados por ANOVA e t-teste (α=0,01). As médias de RC em MPa foram: IA-11,28 (±1,84); IIA-12,02(±1,15); IB-9,73 (±2,38); IIB-9,62 (±2,29); IC-28,30 (±1.63); e IIC-25,50 (±1,91). Conclui-se que a contaminação com o óleo afetou a adesão do selante resinoso ao esmalte e o sistema adesivo com solvente a base de acetona (Prime & Bond) aplicado sob o selante foi capaz de impedir os efeitos deletérios da contaminação com óleo.

Effect of repeated use on dentin bond strength of two adhesive systems: All-in-one and one-bottle.

Aims: To compare the effects of repeated use of two one-bottle adhesives with that of two all- in- one adhesives (with acetone solvent) on bond strength to dentin. Materials and Methods: A flat dentin surface was prepared on 120 bovine incisors using 600- grit abrasive pape. The teeth were randomly assigned into 12 equal groups. The four adhesive systems [Prime and Bond NT (P&B NT), One-Step Plus (OS), iBond (iB), and G-Bond (GB)] were used at baseline, after the lid of the container had been opened 30 times, and after it had been opened 60 times. Before each use of the adhesives, the lids of the containers were left open for 1 min. The resin composites were applied on the dentin in a cylindrical split mold. After thermocycling, shear bond strength test was performed with a universal testing machine at 1 mm/min. We used Kruskal-Wallis and Dunn tests for statistical analysis. Results: There was no statistically significant difference among bond strength (MPa) of the groups of P&B NT (31.9 ± 4.6, 31.8 ± 6.5, 26.1 ± 6.7) and OS (33.2 ± 5.1, 30.9 ± 7, 29.3 ± 5.9), respectively (P > 0.05). The mean of the bond strength of iB and GB after 60 times (15.3 ± 4.1 and 12.2 ± 3.9, respectively) was significantly lower than that of iB and GB at baseline (23.5 ± 4.8 and 22.2 ± 4.5, respectively) (P < 0.05). Conclusions: Repeated use (60 times) of the all-in-one adhesive led to a decline in the dentin bond strength. To avoid this problem it would be advisable to have containers with smaller amounts of adhesive or perhaps those with only a singe dose.

In vitro assessment of solvent evaporation from commercial adhesive systems compared to experimental systems

Solvents should be properly evaporated after application to dental substrates. The aim of this study was to assess the evaporation of commercial, experimental and neat solvents. The tested null hypotheses were that there are no differences in solvent evaporation regardless of its formulation and over time. Evaporation from commercial adhesive systems (Scotchbond Multipurpose Primer, Scotchbond Multipurpose Adhesive, Prime & Bond NT, Multi Bond, Excite, Single Bond 2, Adhese Primer, Adhese Bond, Xeno III A and Xeno III B) and experimental primers (35 percent HEMA plus 65 percent acetone or ethanol or water v/v) were compared to neat solvents (acetone, ethanol and water). Samples (10 µL) of these products were dripped into glass containers placed on a digital precision balance. Evaporation was assessed at 0, 5, 10, 15, 30, 60, 120, 300 and 600 s times to calculate mass loss. Data were analyzed statistically by ANOVA and Bonferroni's correction (a=0.05). Acetone-based products exhibited a remarkable capacity to evaporate spontaneously over time. Neat acetone evaporated significantly more than the HEMA-mixtures and the commercial formulations (p<0.05). The incorporation of monomers and other ingredients in the commercial formulations seem to reduce the evaporation capacity. Solvent evaporation was time and material-dependent.

O solvente deve ser adequadamente evaporado após aplicação ao substratos dentários. O objetivo deste estudo foi avaliar a evaporação de formulações comerciais, primers experimentais e solventes puros. As hipóteses nulas testadas foram de que não há diferenças da quantidade evaporada independentemente do material e tempo. Evaporação dos sistemas adesivos comerciais (Scotchbond multipurpose primer, Scotchbond multipurpose adhesive, Prime & Bond NT, Multi Bond, Excite, Single Bond 2, Adhese Primer, Adhese Bond, Xeno III A e Xeno III B) e primers experimentais (35 por cento HEMA associado com 65 por cento acetona, etanol ou água v/v) foram comparadas a solventes puros (acetona, etanol e água). Amostras (10 µL) de cada produto foram dispensadas em balança de precisão digital. As massas nos tempos 0, 5, 10, 15, 30, 60, 120, 300 e 600 s foram registradas. Os dados foram analisados estatisticamente por ANOVA e Bonferroni (a=0,05). Produtos a base de acetona exibiram maior capacidade de evaporação espontânea ao longo do tempo. Acetona pura evaporou significantemente mais que as misturas de HEMA e formulações comerciais (p<0,05). A incorporação de monômeros e outros ingredientes nas formulações comerciais reduzem a capacidade de evaporação. A evaporação é dependente do produto e do tempo.

Cytotoxic, mutagenic and antimutagenic screening of Arenosclera brasiliensis acetone and ethanol extracts

The marine environment is a rich source of biologically active compounds with pharmacological properties. Marine organisms often produce secondary metabolites with structural features different from those produced by terrestrial ones, and the Phylum Porifera seems to be one of the most productive in this sense. This study was undertaken to provide data on mutagenic and antimutagenic activities from an acetone (Areac) and an ethanol (Areet) extract obtained from Arenosclera brasiliensis, an endemic Brazilian sponge. A qualitative Salmonella reverse mutation test was performed with the TA97, TA98, TA100, and TA102 strains by incubating cells with Areac and Areet in the presence and absence of a known mutagen. A cytotoxic evaluation of the extracts was also performed. A. brasiliensis did not display any mutagenic activity, but Areac showed significant toxicity against test strains. In the antimutagenic assay, a reduction in the number of his+ revertants was observed for the TA97, TA100 and TA102 strains treated with Areac when compared to the positive controls. Areet treatment showed protective activity against DNA lesions only for the TA100. These results are in agreement with those obtained previously with other A. brasiliensis extracts, suggesting an antimutagenic activity.

Bond strength of adhesive systems to human tooth enamel

The purpose of this study was to evaluate in vitro three adhesive systems: a total etching single-component system (G1 Prime & Bond 2.1), a self-etching primer (G2 Clearfil SE Bond), and a self-etching adhesive (G3 One Up Bond F), through shear bond strength to enamel of human teeth, evaluating the type of fracture through stereomicroscopy, following the ISO guidance on adhesive testing. Thirty sound premolars were bisected mesiodistally and the buccal and lingual surfaces were embedded in acrylic resin, polished up to 600-grit sandpapers, and randomly assigned to three experimental groups (n = 20). Composite resin cylinders were added to the tested surfaces. The specimens were kept in distilled water (37°C/24 h), thermocycled for 500 cycles (5°C-55°C) and submitted to shear testing at a crosshead speed of 0.5 mm/min. The type of fracture was analyzed under stereomicroscopy and the data were submitted to Anova, Tukey and Chi-squared (5 percent) statistical analyses. The mean adhesive strengths were G1: 18.13 ± 6.49 MPa, (55 percent of resin cohesive fractures); G2: 17.12 ± 5.80 MPa (90 percent of adhesive fractures); and G3: 10.47 ± 3.14 MPa (85 percent of adhesive fractures). In terms of bond strength, there were no significant differences between G1 and G2, and G3 was significantly different from the other groups. G1 presented a different type of fracture from that of G2 and G3. In conclusion, although the total etching and self-etching systems presented similar shear bond strength values, the types of fracture presented by them were different, which can have clinical implications.

O objetivo deste estudo foi avaliar in vitro três sistemas adesivos: um monocomponente com condicionamento ácido total (G1 Prime & Bond 2.1), um "primer" autocondicionante (G2 Clearfil SE Bond) e um adesivo autocondicionante (G3 One Up Bond F), através de resistência ao cisalhamento ao esmalte de dentes humanos, avaliando o tipo de fratura por estereomicroscopia, seguindo as normas ISO para testes adesivos. Trinta pré-molares hígidos foram seccionados ao meio em sentido mésio-distal, incluídos em resina acrílica, polidos até lixa d'água de granulação 600 e aleatoriamente divididos em três grupos (n = 20). Cilindros de resina composta foram adicionados às superfícies de teste. Os espécimes foram armazenados em água destilada (37°C/24 h), termociclados por 500 ciclos (5°C-55°C) e submetidos ao teste de cisalhamento com velocidade de 0,5 mm/min, sendo o tipo de fratura analisado sob estereomicroscopia e os dados submetidos à análise estatística Anova, Tukey e Qui-quadrado (5 por cento). As médias de resistência adesiva foram: G1: 18,13 ± 6,49 MPa, (55 por cento de fraturas coesivas em resina); G2: 17,12 ± 5,80 MPa (90 por cento de fraturas adesivas) e G3 10,47 ± 3,14 MPa (85 por cento de fraturas adesivas). Em termos de resistência adesiva, não houve diferenças significantes entre G1 e G2, tendo G3 apresentado diferença significante em relação aos demais grupos. G1 apresentou tipo de fratura diferente de G2 e G3. Em conclusão, apesar de os sistemas adesivos com condicionamento ácido total e "primer" autocondicionante terem apresentado valores de resistência adesiva similares, o tipo de fratura apresentado por eles foi diferente, o que pode ter implicações clínicas.

Shear bond strength of acetone-based one-bottle adhesive systems

O objetivo deste estudo foi avaliar a resistência ao cisalhamento de quatro sistemas adesivos de frasco único com solvente acetona e um sistema com solvente etanol em esmalte e à dentina. Cinqüenta molares humanos foram seccionados ao meio no sentido mesiodistal e incluídos com resina acrílica em tubos de PVC. As superfícies vestibulares foram desgastadas até atingir dentina plana, enquanto as superfícies linguais foram desgastadas até atingir esmalte plano. Os espécimes foram polidos até lixa d'água de granulação 600 e divididos aleatoriamente entre os 5 grupos (n=20; 10 espécimes de dentina e 10 espécimes de esmalte), de acordo com o sistema adesivo utilizado: One-Step (Bisco); Gluma One Bond (Heraeus Kulzer), Solobond M (Voco), TenureQuik w/F (Den-Mat) and OptiBond Solo Plus (Kerr) (controle). Os adesivos foram aplicados conforme as instruções dos fabricantes. Uma cápsula cilíndrica gelatinosa (d=4,5mm), preenchida com compósito híbrido de cada fabricante foi posicionada sobre a superfície dental e fotopolimerizada durante 40 s. Após 24 h, os corpos-de-prova foram submetidos a ensaios de cisalhamento em uma máquina de ensaios Instron com velocidade de 5 mm/min. Os dados foram analisados estatisticamente pela ANOVA a um critério e teste de Duncan post hoc (p£0.05). As médias de resistência ao cisalhamento resistência em esmalte e dentina (MPa) (±SD) foram: Esmalte: One-Step=11,3(±4,9); Gluma One Bond=16,3(±10,1); Solobond M=18,9(±4,5); TenureQuik w/F=18,7(±4,5) e OptiBond Solo Plus=16,4(±3,9); Dentina: One-Step=6,4(±2,8); Gluma One Bond=3,0(±3,4); Solobond M=10,6(±4,9); TenureQuik w/F=7,8(±3,9) e OptiBond Solo Plus=15,1(±8,9). Em esmalte, todos os agentes adesivos tiveram resultados estatisticamente semelhantes entre si (p>0.05). Entretanto, em dentina, o sistema adesivo a base de etanol apresentou maior resistência de união que todos os sistemas a base de acetona (p£0.0001). O tipo de solvente presente nos sistemas adesivos de frasco único não exerceu influência na sua capacidade de união ao esmalte, mas se mostrou um componente importante na adesão dentinária, o que deve ser levado em conta quando da escolha de um sistema adesivo.

One-bottle adhesives: in vitro analysis of solvent volatilization and sealing ability

O objetivo deste estudo foi comparar a taxa de volatilização do solvente e a capacidade de selamento de diferentes adesivos de frasco único que estavam em constante uso clínico – um à base de etanol/água (Single Bond, 3M/ESPE) e um à base de acetona (Prime & Bond 2.1, Dentsply/Caulk). Nove frascos de cada agente foram coletados das clínicas da faculdade e outros novos foram utilizados como controle. Os pesos de todos os frascos e de frascos vazios foram determinados em uma balança analítica. Uma gota de cada solução foi dispensada na balança, tomando-se seu peso inicial (PI) e, após 10 min, seu peso final (PF). A razão PI/PF foi utilizada para determinar a taxa de volatilização do solvente. Os frascos com os maiores níveis de evaporação (SB Controle e PB Controle) e com os menores níveis de evaporação (SB Teste e PB Teste) foram aplicados em restaurações Classe V com margens em dentina. Os espécimes foram termociclados e imersos em solução de fucsina básica a 0,5%. A penetração do corante foi avaliada sob magnificação e os dados foram submetidos ao teste de Kruskal-Wallis. A volatilização do solvente foi mais rápida para o adesivo à base de acetona. As razões PI/PF variaram de 1,239 a 1,515 para SB e de 3,488 a 6,476 para PB. PB-Controle e SB-Controle exibiram vedamento similar. Os maiores escores de penetração foram encontrados para o PB-Teste (p < 0,05). Os resultados indicam que a habilidade de selamento pode ser afetada pela repetida utilização dos frascos de adesivo à base de acetona.

Influence of pH of different adhesive systems on the polymerization of a chemically cured composite resin

O objetivo desse estudo foi investigar o efeito do pH de diferentes sistemas adesivos na polimerização de uma resina composta quimicamente ativada (Adaptic - AD), por meio do teste de resistência à tração. Os sistemas adesivos utilizados foram: ARM, Prime & Bond 2.1 (PB), Scotchbond Multi Purpose (SMP) e Single Bond (SB). Cinco grupos (n=5) foram formados: G1: AD/ARM/AD; G2: AD/PB/AD; G3: AD/SMP/AD; G4: AD/SB/AD; G5: AD/AD (sem interposição de adesivo). Uma matriz de aço inoxidável com abertura central no formato de cone (1 mm de espessura; 4 mm de diâmetro), foi utilizada para obter dois discos de resina composta. A resina AD foi inserida em uma primeira metade da matriz em incremento único. Após a polimerização da resina, uma camada de adesivo foi aplicada na superfície de união e foto-ativada. A segunda metade da matriz foi justaposta à primeira e preenchida com a resina. Após 10 min, o conjunto foi adaptado em uma máquina universal de ensaios para determinar a resistência à tração, com velocidade de 0,5 mm/min. Os dados foram submetidos a ANOVA e teste de Tukey (p<0,05). As médias (kgf) obtidas em cada grupo foram: G1: 15,23 ± 4,1; G2: 0,00 ± 0,0; G3: 16,96 ± 2,4; G4: 10,08 ± 2,7; G5: 15,44 ± 0,9. Houve diferença estatisticamente significante (p<0.05) entre os grupos G2-G1; G2-G3; G2-G4; G4-G1; G4-G3. Os sistemas com pHs mais baixos (PB e SB) apresentaram as menores médias de resistência à tração. Os achados desse estudo in vitro sugerem que o pH dos sistemas adesivos influencia na polimerização e na resistência adesiva de materiais resinosos quimicamente ativados. Os sistemas adesivos simplificados testados, com valores de pH mais baixos, mostraram graus distintos de incompatibilidade com a resina quimicamente ativada, quando comparados aos sistemas adesivos convencionais.

Loss of virus specific epitopes on JE virus 'E' glycoprotein by acetone treatment.

BACKGROUND & OBJECTIVES: Some Japanese encephalitis (JE) virus strains have been placed in group II based on the loss of reactivity against Hs (H = HI positive; s = JE virus specific) group of monoclonal antibodies (MAbs) in haemagglutination-inhibition (HI) test employing sucrose acetone (SA) extracted antigens. Also acetone-fixation of cells infected with some of the virus strains results in the loss of immunofluorescence (IF) against virus specific MAbs. The present study was undertaken to elucidate the effect of acetone on virus specific haemagglutination (HA) epitopes expressed on 'E' glycoprotein of group II strains of JE virus. METHODS: Porcine kidney (PS) cells were infected with JE virus strains (2 group I Indian strains, 5 group II strains and one neutralization-escape variant of 733913 group I strain). HI and complement fixation (CF) tests were carried out employing both polyethylene glycol (PEG) precipitated and SA extracted antigens of JE virus. RESULTS: Employing PEG precipitated antigens, Indian strain G9473 showed titres ranging from 1:40 to 1:160 against all the four virus specific HsMAbs and strain 641686 (1:160) with one of the four MAbs (Hs-1) by HI test whereas their SA extracted antigens did not react at all. In contrast, CF was positive employing both SA and PEG antigens in the presence of all four HsMAbs. The reactivity shown by PEG antigens in the HI test was confirmed by blocking the HA activity with the respective MAb. SA antigens, though negative in the HI test, were positive by the blocking assay. Interestingly, some of the non-HI MAbs which were negative against SA antigens, showed positive HI reaction with PEG antigens. Also, additional epitopes on Japanese (Yoken), Sri Lankan (691004) and two Indian (755468 and 641686) JE virus strains were detected either by blocking HA or surface IF. INTERPRETATION & CONCLUSIONS: It seems that the acetone treatment might result in HA property of the antigen which is no longer inhibited by an antibody in the HI test. The characterization of such labile and conformation-dependent epitopes is currently been undertaken to elucidate their role either in protection or immunopathogenesis of JE.

Solute-solvent interaction in methanol acetone system at different temperatures

Solute-solvent interactions were studied from density and viscosity measurements in methanol-acetone mixtures in the temperature range 25 to 40C with increments of 5C as a function of acetone composition [X1]. The contraction in volume [delta] is calculated at different temperatures which gave a pronounced minimum in all cases at X1 indicating the existence of a molecular complex. The calculated excess volume delta VE indicates association of both types of solvent molecules. The deviation of the viscosity delta eta M12 measurements in the same temperature range showed the presence of a molecular complex with the mole ratio [1 MeOH: 1 Acetone]. Finally, the energy of activation of viscous flow Ea was calculated from the Arrhenius equation