Effect of alternative solvent evaporation techniques on mechanical properties of primer-adhesive mixtures / Efeito de técnicas alternativas utilizando evaporação de solvente nas propriedades mecânicas da mistura primer-adesivo

ABSTRACT This study evaluated the influence of the mode and time of solvent evaporation on the tensile strength (TS), flexural strength (FS) and elastic modulus (EM) of two adhesive systems: Scotchbond Multipurpose (SBMP) and Clearfil SE (CSEB). For this purpose, rectangular samples (2x1x7 mm) were prepared with 10 μL of primer and the solvents were evaporated with air spray at (23±1) ºC, (40±1) ºC and negative control (without spray). For each temperature, the times of 5, 20, 30, and 60 seconds were investigated. The statistical results showed that evaporation at 40±1ºC resulted in better EM for the two adhesives tested and all the evaporation times evaluated. However, there were no significant differences between the times and modes of evaporation for TS. The results of this study indicate that evaporation at a temperature of (40±1) °C could improve the elastic modulus of both adhesives tested, regardless of the evaporating time.

RESUMO Este estudo avaliou a influência do modo e do tempo de evaporação do solvente na resistência à tração (RT), resistência à flexão (RF) e módulo de elasticidade (MR) de dois sistemas adesivos: Scotchbond Multipurpose (SBMP) e Clearfil SE (CSEB). Para isso, amostras retangulares (2x1x7 mm) foram preparadas com 10 μL de primer e os solventes foram evaporados com aerossol a (23±1) ºC, (40±1) ºC e controle negativo (sem aerossol). Para cada temperatura, foram avaliados os tempos de 5, 20, 30 e 60 segundos. Os resultados estatísticos mostraram que a evaporação a (40±1) ºC resultou em melhor MR para os dois adesivos testados e todos os tempos de evaporação avaliados. No entanto, não houve diferenças significativas entre os tempos e modos de evaporação na RT. Os resultados deste estudo indicam que a evaporação a uma temperatura de (40±1) °C poderia melhorar o módulo de elasticidade de ambos os adesivos testados, independentemente do tempo de evaporação.

Antimicrobial activity of chitosan coatings and films against Listeria monocytogenes on black radish / Actividad antimicrobiana de coberturas y películas de quitosano contra Listeria monocytogenes en rábano negro

The antibacterial activity of chitosan coatings prepared with acetic or lactic acid, as well as of composite chitosan-gelatin films prepared with essential oils, was evaluated in fresh shredded black radish samples inoculated with Listeria monocytogenes ATCC 19115 and L. monocytogenes ATCC 19112 during seven days of storage at 4 °C. The chitosan coating prepared with acetic acid showed the most effective antibacterial activity. All tested formulations of chitosan films exhibited strong antimicrobial activity on the growth of L. monocytogenes on black radish, although a higher inhibition of pathogens was achieved at higher concentrations of chitosan. The antimicrobial effect of chitosan films was even more pronounced with the addition of essential oils. Chitosan-gelatin films with thyme essential oils showed the most effective antimicrobial activity. A reduction of 2.4 log10 CFU/g for L. monocytogenes ATCC 19115 and 2.1 log10 CFU/g for L. monocytogenes ATCC 19112 was achieved in the presence of 1% chitosan film containing 0.2% of thyme essential oil after 24 h of storage.

Se evaluó la actividad antimicrobiana de coberturas del quitosano y de películas compuestas de quitosano-gelatina en muestras frescas de rábano negro cortado inoculadas con las cepas de Listeria monocytogenes ATCC 19115 y ATCC 19112, almacenadas durante 7 días a 4 °C. Las primeras fueron preparadas con ácido acético o ácido láctico, las segundas con aceites esenciales. Las coberturas de quitosano preparadas con ácido acético mostraron la actividad antimicrobiana más eficaz. Todas las formulaciones de películas de quitosano exploradas mostraron una fuerte actividad antimicrobiana sobre el crecimiento de L. monocytogenes, aunque la mayor inhibición de estos patógenos se logró con las mayores concentraciones de quitosano. La actividad antimicrobiana de las películas de quitosano fue mayor con la adición de aceite esencial. Las películas de quitosano-gelatina con aceite esencial del tomillo fueron las que mostraron la actividad antimicrobiana más eficiente. A las 24 h de almacenamiento, la película con 1% de quitosano y 0,2% de aceite esencial de tomillo produjo una reducción de 2,4 log10 UFC/g en L. monocytogenes ATCC 19115, y de 2,1 log10 UFC/g en L. monocytogenes ATCC 19112.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Effectiveness of three solvents and two associations of solvents on gutta-percha and resilon

This study evaluated the effectiveness of 3 solvents (Citrol orange oil, Eucalyptol and Tetrachloroethylene) and 2 associations of solvents (Citrol orange oil+Tetrachloroethylene and Eucalyptol+Tetrachloroethylene) on 3 types of gutta-percha (conventional, thermoplastic and EndoREZ) and Resilon. Ten discs (10 mm diameter x 1 mm thick) from each material were prepared using standard metallic molds. Each specimen was weighed to determinate its initial mass. The specimens were immersed in the solvents for 10 min, followed by immersion in distilled water for 20 min, and were then reweighed to obtain the final mass. The mean weight loss determined the solvent capacity. Data were analyzed by ANOVA and Tukey's test at 5 percent significance level. Tetrachloroethylene was the most effective on conventional gutta-percha (p<0.05). Tetrachloroethylene was also the most effective on thermoplastic gutta-percha, but it was not significantly different (p>0.05) from Eucalyptol+Tetrachloroethylene, Citrol+Tetrachloroethylene, and Citrol. All solvents and associations presented little effectiveness on Resilon. The association Eucalyptol+Tetrachloroethylene was the most effective on EndoREZ, but it did not differ significantly (p>0.05) from Citrol+Tetrachloroethylene and Tetrachloroethylene. All evaluated substances presented solvent action. Tetrachloroethylene improved the effectiveness of both Citrol and Eucalyptol.

Este estudo avaliou a efetividade de 3 solventes (Citrol, Eucaliptol e Tetracloroetileno) e 2 associações (Citrol+Tetracloroetileno e Eucaliptol+Tetracloroetileno) sobre 3 tipos de guta-percha (convencional, termoplástica e EndoREZ) e Resilon. Dez discos (10 mm x 1 mm) de cada material foram preparados utilizando moldes metálicos. Cada espécime foi pesado para determinar a massa inicial. Os mesmos foram imersos nas soluções testadas por 10 min e em água destilada por 20 min. Os espécimes foram novamente pesados, agora para determinar a massa final. A perda média de peso determinou a capacidade solvente. Os dados foram analisados pelos testes ANOVA e Tukey. O tetracloroetileno foi o mais efetivo sobre a guta-percha convencional (p<0,05). Ele também foi o mais efetivo sobre a guta-percha termoplástica, mas sem diferença significativa para o Eucaliptol+Tetracloroetileno, Citrol+Tetracloroetileno e o Citrol (p>0,05). Todos os solventes e associações apresentaram pequena ação sobre o Resilon. A associação Eucaliptol+Tetracloroetileno obteve o melhor resultado sobre o EndoREZ, mas sem diferença significativa para o Citrol+Tetracloroetileno e o Tetracloroetileno (p>0,05). Todas as soluções apresentam ação solvente. O Tetracloroetileno melhorou a efetividade do Citrol e do Eucaliptol.

Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study.

Aim: To determine if Tulsi (Ocimum sanctum) extract has an antimicrobial activity against Streptococcus mutans and to determine which concentration of Tulsi (Ocimum sanctum) extract among the 15 concentrations investigated has the maximum antimicrobial activity. Setting and Design: Experimental design, in vitro study, Lab setting. Materials and Methods: Ethanolic extract of Tulsi was prepared by the cold extraction method. The extract was then diluted with an inert solvent, dimethyl formamide, to obtain 15 different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7% 8%, 9%, 10%) of the extract. 0.2% chlorhexidine was used as a positive control and dimethyl formamide was used as a negative control. The extract, along with the controls, was then subjected to microbiological investigation to determine which concentration among the 15 different concentrations of the extract gave a wider inhibition zone against Streptococcus mutans. The zones of inhibition were measured in millimeters using a vernier caliper. Results: At the 4% concentration of Tulsi extract, a zone of inhibition of 22 mm was obtained. This was the widest zone of inhibition observed among all the 15 different concentrations of Tulsi that were investigated. Conclusion: Tulsi extract demonstrated an antimicrobial property against Streptococcus mutans.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Ethanolic Zingiber officinale R. extract pretreatment alleviates isoproterenol-induced oxidative myocardial necrosis in rats.

Ethanolic Z. officinale (ZO) extract (200 mg/kg) pretreatment for 20 days in isoproterenol (ISO)-treated rats significantly increased the levels of endogenous myocardial antioxidants (catalase, superoxide dismutase and tissue glutathione), decreased the levels of serum marker enzymes (lactate dehydrogenase, creatine kinase, aspartate transaminase and alanine transaminase) and increased myocardial lipid peroxides. Histological examination of rat's heart section confirmed myocardial injury with ISO administration and near normal pattern with ethanolic ZO extract pretreatment. The results of the present study, for the first time, provide clear evidence that the ethanolic ZO extract pretreatment enhances the antioxidant defense against ISO-induced oxidative myocardial injury in rats and exhibit cardioprotective property.

Evaluation of aqueous leaves extract of Moringa oleifera Linn for wound healing in albino rats.

Aqueous extract of leaves of M. oleifera was investigated and rationalised for its wound healing activity. The aqueous extract was studied at dose level of 300 mg/kg body weight using resutured incision; excision and dead space wound models in rats. Significant increase in wound closure rate, skin-breaking strength, granuloma breaking strength, hydroxyproline content, granuloma dry weight and decrease in scar area was observed. The prohealing actions seem to be due to increased collagen deposition as well as better alignment and maturation. From the results obtained, it may be concluded that the aqueous extract of M. oleifera has significant wound healing property.

Potentiation of pentobarbital hypnosis by Rosa damascena in mice.

Rosa damascena has been found to act on central nervous system including brain. It inhibits the reactivity of the hypothalamous and pituitary systems in rat. In traditional medicine hypnotic effect of Rose is also suggested. In the present study hypnotic effect of ethanolic, aqueous and chloroformic extracts of R. damascena was investigated in mice. Hypnotic method was based on potentiation of pentobarbital induced sleeping time by extracts. Three doses of extracts (100, 500 and 1000 mg/kg) were injected i.p. in comparison with diazepam (3mg/kg) as positive control and saline as negative control. After 30 min of injection of extracts, pentobarbital (30mg/kg) was injected and increase in sleeping time by extracts was recorded. The results showed that the ethanolic and aqueous extracts in 500 and 1000 mg/kg doses significantly increased pentobarbital induced sleeping time which was comparable to diazepam. The chloroformic extract had no hypnotic effect.

Effectiveness of rotary and hand files in gutta-percha and sealer removal using chloroform or chlorhexidine gel

O objetivo deste trabalho foi avaliar in vitro a eficácia de limas rotatórias K3 e limas manuais na remoção de guta-percha e cimento obturador de canais radiculares, utilizando clorofórmio ou clorexidina como solventes. Sessenta dentes bovinos extraídos unirradiculares com canais amplos e retos foram instrumentados, obturados e divididos aleatoriamente em 3 grupos (n=20). Os dentes foram armazenados a 37°C por 1 mês e em seguida os canais foram desobturados empregando diferentes técnicas. Grupo I: brocas Gates-Glidden #3 + limas Kerr e Hedström #30 + clorofórmio; Grupo II: limas rotatórias K3 + clorofórmio; e Grupo III: limas rotatórias K3 + gel de clorexidina a 2%. Após a desobturação, radiografadas dos espécimes foram feitas, escaneadas e as imagens obtidas foram digitalizadas. A área total do canal e a área com remanescente de material obturador foram medidas em milímetros por meio do software ImageLab. Os dados foram analisados estatisticamente por meio da ANOVA e do teste de Tukey. Os grupos diferiram estatisticamente (p<0,05) com relação à média percentual de material obturador remanescente, apresentado a seguinte ordem de efetividade (do mais para o menos efetivo): Grupo I (15,48%), Grupo II (28,42%) e Grupo III (35,96%). Os achados deste estudo demonstraram que, a despeito da técnica empregada remoção do material obturador, os canais retratados não se mostraram completamente livres de remanescentes de guta-percha e cimento. O uso de limas de aço manuais resultou em menor quantidade de material obturador nos canais radiculares do que o uso de instrumentos rotatórios de níquel-titânio.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80°C) and under high pressure conditions at low temperature (3.75 kbar, -13°C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Impact of feeding ethanolic extracts of Achyranthes aspera Linn. on reproductive functions in male rats.

Feeding 50% ethanolic extract of A. aspera to male rats resulted in reduced sperm counts, weight of epididymis, serum level of testosterone and testicular activity of 3beta-hydroxysteroid dehydrogenase, while motility of the sperm and activity of the HMG CoA reductase were not affected. Cholesterol level in the testis, incorporation of labelled acetate into cholesterol, 17-ketosteroids in urine and hepatic and fecal bile acids were increased. The results suggest that ethanolic extract of A. aspera caused reproductive toxicity in male rats and the action may be by suppressing the synthesis of androgen.

Açäo antimicrobiana de solventes de guta-pecha / Antimicrobial action of gutta-percha solvents

Estudou-se a açäo antimicrobiana de solventes de guta-percha (halotano, óleo de laranja, eucaliptol e xilol) sobre os microrganismos Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans e uma mistura destes. Duzentos e quarenta cones de papel contaminados foram mantidos em contato com os solventes por períodos de 5, 10, 15 e 30 minutos e, a seguir, transportados para 7mL de Brain Heart Infusion e incubados a 37ºC por 48 horas. Posteriormente, foi analisada a presença ou ausência de turvaçäo, indicativa ou näo de crescimento e multiplicaçäo dos microrganismos. Para a confirmaçäo dos resultados, empregou-se a coloraçäo de Gram. O halotano mostrou efetividade antimicrobiana em todos os tempos de análise para a C. albicans; a partir de 10 minutos para o E. faecalis e P. aeuruginosa; a partir de 15 minutos para o S. aureus e foi inefetivo para o B. subtilis e sobre a mistura. Os demais solventes foram enefetivos sobre todos os microrganismos testados, em todos os períodos de observaçäo

Interference of propylene glycol with the hole-board test

Experimental drugs and/or plant extracts are often dissolved in solvents, including propylene glycol. Nevertheless, there is evidence for psychoactive properties of this alcohol. In this study we found that in the hole-board test 10 percent propylene glycol did not modify the head-dipping behavior. However, 30 percent propylene glycol induced an increase in the number of head-dips (46.92 + or - 2.37 compared to 33.83 + or - 4.39, P<0.05, ANOVA/Student-Newman-Keuls), an effect comparable to that obtained with 0.5 mg/kg diazepam (from 33.83 + or - 4.39 to 54 + or - 3.8, P<0.01, ANOVA/Student-Newman-Keuls). These results demonstrate that 30 percent propylene glycol has significant anxiolytic effects in this model and therefore cannot be used as an innocuous solvent

Efetividade de solventes e de técnicas na desobturaçäo dos canais radiculares: estudo in vitro / Effectiveness of solvents and techniques for removal of filling material from root canals: a study in vitro

Neste estudo avaliou-se a efetividade de cinco solventes de guta-percha agindo por cinco e 10 minutos, e de quatro técnicas de desobturaçäo de canais radiculares. Nas duas partes do experimento procurou-se empregar metodologias que mais se aproximassem das aplicaçöes clínicas, bem como os materiais e técnicas mais usuais. Testou-se também, um procedimento complementar de desobturaçäo, no intuito de abreviar e melhorar a remoçäo dos remanescentes de materiais nos retratamentos, antes do início da reinstrumentaçäo. Verificou-se que, entre os solventes testados, o clorofórmio foi o mais eficiente e a técnica de desobturaçäo com o uso da broca de Gates-Glidden e limas endodônticas foi a mais rápida e eficaz. O procedimento complementar, com algodäo envolto na lima e embebido em solvente, melhorou os resultados de todas as técnicas testadas

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4 percent, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2 percent DMSO (59.4 9.1 and 77.0 9.1 nmol h-1 mg protein-1, controls without and with 2 percent DMSO, respectively; 47.7 5.2 and 55.8 4.1 nmol h-1 mg protein-1, NPC without and with 2 percent DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 0.049, 0.659 0.041, 0.688 0.063 and 0.733 0.088 mg/mg protein, without DMSO, 1 percent DMSO, 2 percent DMSO and 4 percent DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients

Improved cycle sequencing of GC-rich DNA template

Even when DNA sequencing of purified DNA template failed under the optimal condition, it can be generally contributed to high GC content. GC-rich region of template causes a secondary structure to produce shorter readable sequence. To solve this problem, the sequencing reaction was modified by using dimethyl sulfoxide (DMSO). It was found that 5% (v/v) of DMSO in the reaction mixture recovers sequencing signal intensity with reduced frequency of ambiguous bases. When DMSO was added to sequencing reaction of DNA template with normal GC content, it did not show any adverse effect. Sequencing accuracy and unambiguous base frequency were significantly improved at concentration of 2% to 5% (v/v) DMSO in GC-rich DNA template. DMSO has been empirically introduced to enhance the efficiency of PCR in GC-rich templates. However, the underlying mechanism of improved cycle sequencing by DMSO is unknown. Thus, cycle sequencing reaction was remodified with other additives such as N-methyl imidazole, N-methyl2-pyrrolidone, N-methyl-2-pyridone and glycerol, possessing the similar chemical properties as DMSO. Most of methyl nitrogen ring-containing chemicals did not improve sequencing accuracy, whereas only glycerol mimicked the positive effect of DMSO by the same extent. In the present study, we suggest that the treatment of DMSO improve cycle sequencing by the alteration of structural conformation of GC-rich DNA template.