Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein-free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II-S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris-based extender, containing either 20% egg yolk, 1% soybean lecithin Type II-S or 1% soybean lecithin Type IV-S. The samples were then frozen. Sperm motility was evaluated by computer-assisted sperm analysis (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (SYBR-14/PI) post-thaw, as well as after 2 and 4 hr incubation at 37°C. Post-thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin-based extenders. Individual motility post-thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean-based extenders. The number of high green stained spermatozoa were significantly higher in Type IV-S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin-based extenders to cryopreserve canine semen.

In vitro evaluation of Labrador dog spermatozoa cryopreserved in Tris-citric acid-fructose buffer supplemented with different combinations of extracellular and intracellular cryoprotectants.

Aim of this study was to compare different combinations of penetrating intracellular CPAs, i.e., glycerol (G), ethylene glycol (EG), propylene glycol (PG), dimethyl formamide (DM), and methyl acetamide (MA) and extracellular [egg yolk (EY), egg yolk plasma (EYP), low-density lipoproteins (LDL), and coconut water (CW)] in Tris-citric acid-fructose buffer (T) for Labrador dog semen cryopreservation. The study was conducted in two parts, first trial was conducted to assess optimum glycerol concentration (5-7%) in TEY and equilibration time (ET, 2-4 hrs) for Labrador dog semen cryopreservation. Secondly, compatibility of 15% TEY, 15% TEYP, 13% TLDL, and 25% TCW with G, DMF, MA, D + M, EG, and PG was evaluated for in vitro sperm function tests. Decline in sperm attributes, i.e., motility, viability, plasma membrane integrity (PMI), and acrosome integrity (AI)) was significantly (p < 0.05) less in 7% TEY-G and 4 h compared to other concentrations and ET at post-thaw. There was significantly (p < 0.05) less decline in sperm attributes in TEY-G, TEYP-G, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders compared to other combinations at post-thaw. However, these parameters were significantly (p < 0.05) high in TEY-G and TEYP-G compared to TEYP-D, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders at post-thaw. However, decline in motility, viability, PMI, and AI was identical in these seven extenders. This study concluded that glycerol at a concentration of 7% in TEY and 4 h ET were optimum for successful cryopreservation and besides TEY-G, other combinations of protectants may be an alternative for canine semen cryopreservation.

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa.

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 µ M l-tryptophan, D2 = 50 µ M l-tryptophan, D3 = 75 µ M l-tryptophan, and D4 = 100 µ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.

Effect of reduced glutathione (GSH) supplementation to Tris-egg yolk extender on chilled semen variables of dogs.

One reason for reduced longevity of chilled dog semen is oxidative stress. The antioxidant glutathione (GSH) improves viability of frozen-thawed dog sperm, but its effect on chilled dog semen has not been investigated. An experiment consisting of two parts was performed: Sperm rich fractions, SRF, were split, diluted with a Tris-egg yolk (TEY) extender containing 0, 5 or 10 mM GSH and stored at 4 °C for 10 days (Part 1; n = 19) or 4 days (Part 2; n = 11), respectively. For Part 1 of the study, percentage (%) of motile, viable, morphologically abnormal spermatozoa and % acrosomal deviations were assessed on days 0, 1, 2, 4 and 10 after dilution. For % sperm motility, samples from all three aliquots of each SRF (0/5/10 mM GSH) were pipetted simultaneously and analysed in a randomised order (time point of analysis, TPA). In Part 2 of the study, motility analysis was performed during 4 days storage and samples were analysed immediately after pipetting (part 2). Most investigated parameters were affected by storage time. For motility variables, there was an effect of GSH identified for circular, CM (ANOVA, Part 1: P = 0.05, Part 2: P < 0.0001) and local motility, LM (ANOVA, Part 2: P = 0.004). Furthermore, there was a trend for an interaction between time and sperm treatment for CM (Part 2: P = 0.077). In conclusion, in the present study there was not an overall positive effect of GSH addition (5/10 mM) on sperm motility in chilled dog semen samples that were characterised to be of good quality during 4- and 10-days of storage.

Supplementation of tris-based extender with plasma egg yolk of six avian species and camel skim milk for chilled preservation of dromedary camel semen.

The present study was conducted to investigate a suitable source (Expt. 1) and concentration (Expt. 2) of plasma egg yolk (PEY) and concentration of camel skim milk (CSM; Expt. 3) to supplement tris based extender for chilled storage of dromedary camel semen. In Expt. 1, PEY (20%) of six avian species (domestic chicken, domestic duck, Japanese quail, partridge, pigeon and guinea fowl) was added to semen extender. In Expt. 2, different concentrations (0, 10, 20, 30 and 40%) of selected PEY from Expt.1 were added to semen extender. In both Expt. 1 and 2, CSM remained constant (20%). In Expt. 3, semen extender was supplemented with different concentrations of CSM (0, 20, 40, 60 and 80%) while the concentration of PEY remained constant. The sperm viability parameters were assessed at 6, 12 and 24h following chilled storage. In Expt. 1, progressive forward motility (PFM) of diluted semen supplemented with pigeon PEY was similar to domestic duck and Japanese quail PEYs (P>0.05) and superior to other PEYs (P<0.05). In Expt. 2, PFM following the addition of 20% pigeon PEY was similar to 10 and 30% (P>0.05) and greater than 0 and 40% (P<0.05). In Expt. 3, total motility, PFM and live percentage of sperm were better in 20% compared to 40, 60 and 80% CSM (P<0.05). In the last experiment, PFM in 20% was better than 0% CSM (P<0.05). In conclusion, pigeon PEY at the concentration of 20% and CSM at the concentration of 20% could provide beneficial effect on some of the sperm viability parameters during chilled storage of dromedary camel semen.

Pseudomonas aeruginosa produces phosphatidyltris(hydroxymethyl)aminomethane and derivatives when grown in Tris-buffered medium.

For optimal growth of a microorganism, the pH of the culture medium should be set at an optimum value. For that reason, growth media require buffering agents. We show in this study that, when grown in a medium supplemented with tris(hydroxymethyl)aminomethane (Tris), Pseudomonas aeruginosa is able to use this organic compound to produce new phospholipids. We thus pointed out that phosphatidyltris(hydroxymethyl)aminomethane as well as diphosphatidyltris(hydroxymethyl)aminomethane was detected in membrane lipid extracts of bacteria grown in Tris-buffered medium. Moreover, the amounts of lysoglycerophospholipids in the lipidome of P. aeruginosa grown in Tris-buffered medium increased leading to the presence of lysophosphatidylglycerol and lysophosphatidyltris(hydroxymethyl)aminomethane as well as other lysophospholipid derivatives. Finally, we investigated the effect of the presence of these exogenous phospholipids on the susceptibility of P. aeruginosa to some antibiotics. We observed a decrease of the minimal inhibitory concentrations of different antibiotic families, i.e., fluoroquinolones, aminoglycosides, ß-lactams and polymyxins, proving the importance of the buffer choice for growth medium and its impact on the lipidome.

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk.

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal-protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris-based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5-ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (C-FDA) were evaluated 5 min post-thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin-based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin-based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS.

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P<0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12h of cooling for both extenders (P<0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.

The mechanism of antibacterial activity of tetrandrine against Staphylococcus aureus.

Tetrandrine (TET) is a bis-benzylisoquinoline alkaloid derived from the radix of Stephania tetrandra S. Moore. TET performs a wide spectrum of biological activities. The radix of S. tetrandrae has been used traditionally in Asia, including Korea, to treat congestive circulatory disorders and inflammatory diseases. The aim of this study was to examine the mechanism of antibacterial activity of tetrandrine against Staphylococcus aureus. The mechanism was investigated by studying the effects of TET in combination with detergent or membrane potential un-couplers. In addition, the direct involvement of peptidoglycan (PGN) was assessed in titration assays. TET activity against S. aureus was 125-250 µg/mL, and the minimum inhibitory concentration (MIC) of the two reference strains was 250 µg/mL. The OD(600) of each suspension treated with a combination of ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl) aminomethane (TRIS), and Triton X-100 (TX) with TET (0.25×MIC) had been reduced from 43% to 96%. Additional structure-function studies on the antibacterial activity of TET in combination with other agents may lead to the discovery of more effective antibacterial agents.

A gold nanoparticles-modified aptamer beacon for urinary adenosine detection based on structure-switching/fluorescence-"turning on" mechanism.

A novel small molecule probe, aptamer beacon (AB), was introduced for adenosine (Ade) recognition and quantitative analysis. The Ade aptamer was engineered into an aptamer beacon by adding a gold nanoparticle-modified nucleotide sequence which is complementary to aptamer sequence (FDNA) at the 3'-end of FDNA. The fluorescence signal "turning on" was observed when AB was bound to Ade, which is attributed to a significant conformational change in AB from a FDNA/QDNA duplex to a FDNA-Ade complex. The Ade measurement was carried out in 20 mmol L(-1) Tris-HCl buffer solution of pH 7.4, ΔF signal linearly correlated with the concentration of Ade over the range of 2.0×10(-8) to 1.8×10(-6) mol L(-1). The limit of detection (LOD) for Ade is 6.0×10(-9) mol L(-1) with relative standard deviations (R.S.D) of 3.64-5.36%, and the recoveries were 98.6%, 100%, 102% (n=6), respectively. The present method has been successfully applied to determine Ade in human urine samples, and the obtained results were in good agreement with those obtained by the HPLC method. Our investigation shows that the unique properties of the AB could provide a promising potential for small molecules detection, and be benefit to extend the application of aptamer beacon technique.

Critical evaluation and further development of methods for testing ecotoxicity at multiple pH using Daphnia magna and Pseudokirchneriella subcapitata.

To meet the requirements of risk assessment legislature regarding the ecotoxicity of ionizing compounds, the present study attempts to establish easy, robust methods for testing ecotoxicity at various pH levels. An overview is given of the buffering methods found in the literature. This is supplemented by a series of experiments where toxicity and ability to stabilize pH of seven common buffering compounds was tested on Daphnia magna and Pseudokirchneriella subcapitata. We consider a buffer applicable at a given concentration if the pH drift is below 0.2 pH units, and if there are no toxic effects. Twenty-four- and 48-h acute toxicity tests with D. magna were carried on a series of organic buffers with pH monitoring. Based on the experimental results it is possible to give recommendations for buffer concentrations for use in toxicity testing with D. magna at pH levels in the range of pH 6.0-7.8 for 48 h exposure, and pH 6.0-9.5 for 24 h exposure. Forty-eight- and 72-h growth inhibition tests with P. subcapitata were carried out, and recommendations for buffer concentrations at pH 7.5 and 8.0 are made for both 48 and 72 h of exposure.

Optimisation of a capillary zone electrophoresis methodology for simultaneous analysis of organic aliphatic acids in extracts of Brachiaria brizantha.

INTRODUCTION: Aluminum toxicity is commonly verified in acidic soils, and poses a severe limitation to plant growth and development. Therefore, Al complexation by the root system mucilage, Al complexation by organic compounds that are exuded by the roots and internal metabolic processes must be monitored by organic acids (OA), since they play a central role in these aluminum tolerance mechanisms. OBJECTIVE: To optimise a capillary zone electrophoresis method able to perform simultaneous separation of acetic, citric, formic, lactic, malic, oxalic, pyruvic, succinic, tartaric and aspartic acid in plant extract solutions. METHODOLOGY: Method optimisation was achieved by a chemometric approach through experimental designs. The optimal condition found was: 20 mmol/L phthalic acid buffer; 0.8 mmol/L cetyltrimethyl-ammonium bromide; pH 3.4 adjusted with tris(hydroxymethyl)aminomethane (around 16 mmol/L); -15 kV of voltage; 25 °C of cartridge temperature; indirect ultraviolet detection at 240 nm; and 25 mbar injection for 2 s, within an analysis time of 4 min. RESULTS: As a repeatability test of the optimal condition, 30 replicates were carried out with the same working electrolyte, where the relative standard deviation of each peak ranged from 0.081 to 0.36% (for migration time) and from 2.4 to 4.6% (for peak area). CONCLUSION: The methodology was successfully applied to simultaneously determine citric, malic and aspartic acid in roots and leaves extract solutions of Brachiaria brizantha, demonstrating its usefulness to study aluminum tolerance.

Kinetics of fluoride release from glass components of glass ionomers.

OBJECTIVES: To compare the F release from glass ionomer glasses (GICs) into water changed periodically with release into an unchanged "sink" [Williams JA, Billington RW, Pearson GJ. The glass ionomer cement: the sources of soluble fluoride. Biomaterials 2002;23:2191-200]. To evaluate the effect of replacing Ca wholly or partially by Sr. To compare two different methods of decomplexing F containing species. METHODS: All four glasses contained Al, Si, P, O and F. Glass AH2 (Advanced Healthcare Ltd.) had Na and Ca, LG26 (Advanced Healthcare Ltd.) had Ca, LG26Sr (ULTRASET project) had Ca and Sr, and LG125 (ULTRASET project) had Sr. Glasses were tested: after ball-milling, after washing in dilute acid, and W mixed with 35% acetic acid to form a "pseudo-cement". F(-) release was from 130mg samples into 10ml of deionised water changed at 1, 3, 7, 14, 21, 28, 35, and 42 days. Analysis was carried out: (a) using ISE without decomplexing, (b) using TISAB buffer, and (c) acid hydrolysis+TISAB (after Hattab). RESULT: The cumulative release rates from all glasses and treatments are linear with respect to t(1/2) with r=0.97 or greater (p=<0.001). F release into a "sink" showed no such correlation. The higher release rate from AH2 is more than accounted for by its higher F content. Most F release is not complexed except of AH2. AH2 has 20% of complex fluoride from raw glass, 44% from acid-washed glass and 51% from pseudo-cement. More fluoride is released after acid treatment from all 4 glasses, these are on average 4.4 times higher from acid-washed glass and 5.3 times higher from pseudo-cement. For TISAB fluoride release, LG26Sr is 26% more than LG26. Hattab fluoride>TISAB fluoride only for raw glasses. CONCLUSIONS: Changing water produces diffusion controlled kinetics. Acid treated will increase the complex fluoride from AH2. Replacing Ca by Sr enhances F release rate slightly.

Microhardness and fluoride release of restorative materials in different storage media.

This study evaluated the surface microhardness and fluoride release of 5 restorative materials - Ketac-Fil Plus, Vitremer, Fuji II LC, Freedom and Fluorofil - in two storage media: distilled/deionized water and a pH-cycling (pH 4.6). Twelve specimens of each material, were fabricated and the initial surface microhardness (ISM) was determined in a Shimadzu HMV-2000 microhardness tester (static load Knoop). The specimens were submitted to 6- or 18-h cycles in the tested media. The solutions were refreshed at the end of each cycle. All solutions were stored for further analysis. After 15-day storage, the final surface microhardness (FSM) and fluoride release were measured. Fluoride dose was measured with a fluoride-specific electrode (Orion 9609-BN) and digital ion analyzer (Orion 720 A). The variables ISM, FSM and fluoride release were analyzed statistically by analysis of variance and Tukey's test (p<0.05). There was significant difference in FSM between the storage media for Vitremer (pH 4.6 = 40.2 +/- 1.5; water = 42.6 +/- 1.4), Ketac-Fil Plus (pH 4.6 = 73.4 +/- 2.7; water = 58.2 +/- 1.3) and Fluorofil (pH 4.6 = 44.3 +/- 1.8; water = 38.4 +/- 1.0). Ketac-Fil Plus (9.9 +/- 18.0) and Fluorofil (4.4 +/- 1.3) presented higher fluoride release in water, whereas Vitremer (7.4 +/- 7.1), Fuji II LC (5.7 +/- 4.7) and Freedom (2.1 +/- 1.7) had higher fluoride release at pH 4.6. Microhardness and fluoride release of the tested restorative materials varied according to the storage medium.

Microhardness and fluoride release of restorative materials in different storage media

This study evaluated the surface microhardness and fluoride release of 5 restorative materials - Ketac-Fil Plus, Vitremer, Fuji II LC, Freedom and Fluorofil - in two storage media: distilled/deionized water and a pH-cycling (pH 4.6). Twelve specimens of each material, were fabricated and the initial surface microhardness (ISM) was determined in a Shimadzu HMV-2000 microhardness tester (static load Knoop). The specimens were submitted to 6- or 18-h cycles in the tested media. The solutions were refreshed at the end of each cycle. All solutions were stored for further analysis. After 15-day storage, the final surface microhardness (FSM) and fluoride release were measured. Fluoride dose was measured with a fluoride-specific electrode (Orion 9609-BN) and digital ion analyzer (Orion 720 A). The variables ISM, FSM and fluoride release were analyzed statistically by analysis of variance and Tukey's test (p<0.05). There was significant difference in FSM between the storage media for Vitremer (pH 4.6 = 40.2 ± 1.5; water = 42.6 ± 1.4), Ketac-Fil Plus (pH 4.6 = 73.4 ± 2.7; water = 58.2 ± 1.3) and Fluorofil (pH 4.6 = 44.3 ± 1.8; water = 38.4 ± 1.0). Ketac-Fil Plus (9.9 ± 18.0) and Fluorofil (4.4 ± 1.3) presented higher fluoride release in water, whereas Vitremer (7.4 ± 7.1), Fuji II LC (5.7 ± 4.7) and Freedom (2.1 ± 1.7) had higher fluoride release at pH 4.6. Microhardness and fluoride release of the tested restorative materials varied according to the storage medium.

Este estudo avaliou as propriedades de microdureza de superfície e liberação de flúor de 5 materiais restauradores (Ketac-Fil Plus, Vitremer, Fuji II LC, Freedom e Fluorofil) em dois meios de imersão: água destilada/deionizada e modelo de ciclagem de pH (4,6). Doze corpos-de-prova de cada material foram confeccionados e tiveram a microdureza de superfície inicial (MSI) determinada utilizando o microdurômetro Shimadzu HMV-2000 Micro Hardness Tester (carga estática Knoop). Os corpos-de-prova foram submetidos a ciclos de 6 e 18 h para os dois meios de imersão. A cada final de ciclo as soluções foram substituídas e armazenadas. Após 15 dias de imersão, a microdureza de superfície final (MSF) e a liberação de flúor foram determinadas. A dosagem de flúor foi feita com um eletrodo específico combinado para íon flúor (9609 BN - Orion) e analisador de íons digital (Orion 720 A). As variáveis MSI, MSF e liberação de flúor foram submetidas à análise de variância e teste de Tukey (p<0,05). Houve diferença estatisticamente significante na MSF entre os meios de imersão para o Vitremer (pH 4,6 = 40,2 ± 1,5; água = 42,6 ± 1,4), Ketac-Fil Plus (pH 4,6 = 73,4 ± 2,7; água = 58,2 ± 1,3) e Fluorofil (pH 4,6 = 44,3 ± 1,8; água = 38,4 ± 1,0). O Ketac-Fil Plus (9,9 ± 18,0) e o Fluorofil (4,4 ± 1,3) liberaram mais flúor na água; o Vitremer (7,4 ± 7,1), Fuji II LC (5,7 ± 4,7) e o Freedom (2,1 ± 1,7) no pH 4,6. A microdureza e liberação de flúor dos materiais restauradores estudados variaram de acordo com o meio de imersão.

Novel and simple nonaqueous capillary electrophoresis separation and determination bioactive triterpenes in Chinese herbs.

Three bioactive triterpenes ursolic acid, oleanolic acid and 2alpha,3beta,24-trihydroxy-urs-12-en-28-oic acid were simultaneously separated by nonaqueous capillary electrophoresis (NACE) with methanol:acetonitrile (65:35 v/v) mixture containing 90 mm trishydroxymethylaminomethane (Tris) at an applied voltage of +25 kV and a hydrodynamic injection of 5s. The effect of solvent composition, electrolyte nature and concentration on the electrophoretic behavior of the analytes were systematically studied. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation and correlation coefficients were obtained. Meanwhile, the method was applied to separation and determination the three components in six Chinese herbs extraction. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive triterpenes in Chinese herbs.

Assembly of DNA nanostructures with branched tris-DNA.

Branched tris-DNA, in which two oligonucleotides of the same sequence and one other oligonucleotide of a different sequence are connected with a rigid central linker, was prepared chemically by using a DNA synthesizer. Two branched tris-DNA molecules with complementary DNA sequences form dimer and tetramer as well as linear and spherical oligomer complexes. The complex formation was studied by UV/thermal denaturation, enzyme digestion, gel electrophoresis, and AFM imaging.

Formation of a nanostructured layer on bioglass particles of different sizes immersed in tris-buffered solution. N2 adsorption and HR-TEM/EDS analysis.

Morphological and structural variations of particles of Bioglass with two different grain sizes reacted in Tris-buffered solution were analyzed by means of N(2) adsorption/desorption at 77 K and HR-TEM/EDS. A remarkable increase in specific surface area (ssa) was observed after the first hour of dissolution. A plateau value corresponding to an increase of at least 2 orders of magnitude was reached after 2 days of dissolution. The ssa increase was faster for the smaller particle size sample, and the ratio between the ssa of the starting samples was not maintained during dissolution. Both micro- and mesopores were formed at different stages of the reaction for the two samples. Increasing ssa was also connected to the formation of a microcrystalline structure rich in Ca and P, as shown by TEM images. The segregation of both a SiO(2)-rich amorphous phase and a Ca/P-rich crystalline phase was observed after the first hour of dissolution. After 2 days of reaction, Ca/P-rich particles made of fine aciculate crystals were found either in close contact with SiO(2) particles or deposited on a small SiO(2)-rich core. A preliminary analysis of TEM data showed the formation, together with hydroxy carbonate apatite, of different types of calcium phosphates not detectable by powder X-ray diffraction.

Ouabain at nanomolar concentration promotes synthesis and release of angiotensin II from the endothelium of the tail vascular bed of spontaneously hypertensive rats.

The effects of 1 nM ouabain (OUA) on the contractile actions of phenylephrine (PHE, 0.001-100 microg) and functional activity of the sodium pump (NKA) in isolated-perfused tail vascular beds from WKY and SHR were investigated. In preparations from SHR, perfusion with OUA in the presence of endothelium (E+) increased the sensitivity (pED50) of PHE (before: 2.14 +/- 0.06 versus after: 2.47 +/- 0.07; P < 0.05) without altering the maximal response (Emax). After endothelial damage, OUA reduced the Emax of PHE in SHR (before: 350 +/- 29 versus after: 293 +/- 25 mm Hg; P < 0.05). In SHR/E+, pretreatment with losartan (10 microM) or enalaprilat (1 microM) prevented the increased sensitivity to PHE induced by OUA. OUA increased NKA activity in SHR/E+ (before: 45 +/- 6 versus after: 58 +/- 5%, P < 0.05). Losartan (10 mg/Kg, i.v.) also abolished the increment in systolic and diastolic blood pressure induced by OUA (0.18 microg/Kg, i.v.) in anesthetized SHR. OUA did not alter the actions of PHE in either anesthetized WKY rats or vascular preparations. Results suggest that 1 nM OUA increased the vascular reactivity to PHE only in SHR/E+. This effect is mediated by OUA-induced activation of endothelial angiotensin converting enzyme that promotes the local formation of angiotensin II, which sensitizes the vascular smooth muscle to the actions of PHE.

Synthesis of small multifunctional molecules having nucleic acid binding property.

We have previously reported that a multifunctional conjugate having the acridine derivative as an intercalative molecule and the polyamine moiety as an RNA cleaving molecule bound to a double helical RNA and cleaved the target efficiently. Along the study to develop a sequence and site specific artificial RNA cleaving molecule, we have development a novel intercalative molecule having polyamine moiety and DNA connecting function. The trisamine-acridine-tethered DNA is expected to bind to the complementary RNA and cleave the phosphodiester bond of the RNA near the position of the built-in interecalator.