Validation of ACB in vitro and in vivo as a biomagnetic method for measuring stomach contraction

Background The aim of this study was to validate a biomagnetic method (alternate current biosusceptometry, ACB) for monitoring gastric wall contractions in rats. Methods In vitro data were obtained to establish the relationship between ACB and the strain-gauge (SG) signal amplitude. In vivo experiments were performed in pentobarbital-anesthetized rats with SG and magnetic markers previously implanted under the gastric serosa or after ingestion of magnetic material. Gastric motility was quantified from the tracing amplitudes and frequency profiles obtained by Fast Fourier Transform. Key Results The correlation between in vitro signal amplitudes was strong (R = 0.989). The temporal cross-correlation coefficient between the ACB and SG signal amplitude was higher (P < 0.0001) in the postprandial (88.3 +/- 9.1 V) than in the fasting state (31.0 +/- 16.9 V). Irregular signal profiles, low contraction amplitudes, and smaller signal-to-noise ratios explained the poor correlation between techniques for fasting-state recordings. When a magnetic material was ingested, there was also strong correlation in the frequency and signal amplitude and a small phase-difference between the techniques. The contraction frequencies using ACB were 0.068 +/- 0.007 Hz (postprandial) and 0.058 +/- 0.007 Hz (fasting) (P < 0.002) and those using SG were 0.066 +/- 0.006 Hz (postprandial) and 0.059 +/- 0.008 Hz (fasting) (P < 0.005). Conclusions & Inferences In summary, ACB is reliable for monitoring gastric wall contractions using both implanted and ingested magnetic materials, and may serve as an accurate and sensitive technique for gastrointestinal motility studies.

Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro: Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction

Purpose: Eicosapentaenoic acid has been tested in bladder cancer as a synergistic cytotoxic agent in the form of meglumine-eicosapentaenoic acid, although its mechanism of action is poorly understood in this cancer. The current study analyzed the mechanisms by which eicosapentaenoic acid alters T24/83 human bladder cancer metabolism in vitro. Materials and Methods: T24/83 human bladder cancer cells were exposed to eicosapentaenoic acid for 6 to 24 hours in vitro and incorporation profiles were determined. Effects on membrane phospholipid incorporation, energy metabolism, mitochondrial activity, cell proliferation and apoptosis were analyzed Reactive oxygen species and lipid peroxide production were also determined. Results: Eicosapentaenoic acid was readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. Energy metabolism was significantly altered by eicosapentaenoic acid, accompanied by decreased mitochondrial membrane potential, and increased lipid peroxide and reactive oxygen species generation. Subsequently caspase-3 activation and apoptosis were detected in eicosapentaenoic acid exposed cells, leading to decreased cell numbers. Conclusions: These findings confirm that eicosapentaenoic acid is a potent cytotoxic agent in bladder cancer cells and provide important insight into the mechanisms by which eicosapentaenoic acid causes these changes. The changes in membrane composition that can occur with eicosapentaenoic acid likely contribute to the enhanced drug cytotoxicity reported previously in meglumine-eicosapentaenoic acid/epirubicin/mitomycin studies. Dietary manipulation of the cardiolipin fatty acid composition may provide an additional method for stimulating cell death in bladder cancer. In vivo studies using intravesical and dietary manipulation of fatty acid metabolism in bladder cancer merit further attention.

In vitro evidence for immune evasion activity by human plasmin associated to pathogenic Leptospira interrogans

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host`s innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLC), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement Ob and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date. (C) 2011 Elsevier Ltd. All rights reserved.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives: Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. Study design: Immature oocytes obtained from cows slaughtered in a slaughterhouse (n = 840) were divided into the following groups: (1) control (n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h (n = 208) submitted to meiotic blockage with 100 mu M BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h (n = 307), pre-matured with 100 mu m BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. Results: Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively). Conclusion: Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Life history, morphological variability and growth rates of the life phases of Gracilaria tenuistipitata (Rhodophyta: Gracilariales) in vitro

Gracilaria tenuistipitata, a species of commercial interest, is becoming a model organism for studies on red algal physiology and molecular biology as it can be grown easily in vitro under a broad range of conditions. Most of the experiments carried out around the world have been based on a tetrasporophytic clone isolated in our laboratory from a specimen collected in China. Here we describe the life history of this species, give anatomic details of the reproductive structures, illustrate the morphological variability of tetraspore progeny and compare the growth rate of gametophytic and sporophytic thalli. Tetrasporophytic branches showed higher growth rates than gametophytic branches.

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

A new nitrosyl ruthenium complex: Synthesis, chemical characterization, in vitro and in vivo antitumor activities and probable mechanism of action

This study describes the synthesis of a new ruthenium nitrosyl complex with the formula [RuCl(2)NO(BPA)] [BPA = (2-hydroxybenzyl)(2-methylpyridyl)amine ion], which was synthesized and characterized by spectroscopy, cyclic voltammetry, X-ray crystallography, and theoretical calculation data. The biological studies of this complex included in vitro cytotoxic assays, which revealed its activity against two different tumor cell lines (HeLa and Tm5), with efficacy comparable to that of cisplatin, a metal-based drug that is administered in clinical treatment. The in vivo studies showed that [RuCl2NO(BPA)] is effective in reducing tumor mass. Also, our results suggest that the mechanism of action of [RuCl(2)NO(BPA)] includes binding to DNA, causing fragmentation of this biological molecule, which leads to apoptosis. (C) 2011 Elsevier Masson SAS. All rights reserved.

Interactions of Diorganolead(IV) with 3-(2-Thienyl)-2-sulfanylpropenoic Acid and/or Thiamine: Chemical and in Vitro and in Vivo Toxicological Results

The reactions of PbR(2)(OAc)(2) (R=Me, Ph) with 3-(2-thienyl)-2-sulfanylpropenoic acid (H(2)tSpa) in methanol or ethanol afforded complexes [PbR(2)(tspa)] that electrospray ionization-mass spectrometry (ESI-MS) and IR data suggest are polymeric. X-ray studies showed that [PbPh(2)(tspa)(dmso)] center dot dmso, crystallized from a solution of [PbPh(2)(tspa)] in dmso, is dimeric, and that [HQ](2)[PbPh(2)(tspa)(2)] (Q=diisopropylamine), obtained after removal of [PbPh(2)(tspa)] from a reaction including Q, contains the monomeric anion [PbPh(2)(tSpa)(2)](2-). In the solid state the lead atoms are O,S-chelated by the tspa ligands in all these products, and in the latter two have distorted octahedral coordination environments. NMR data suggest that tspa(2-) remains coordinated to PbR(2)(2+) in solution in dmso. Neither thiamine nor thiamine diphosphate reacted with PbMe(2)(NO(3))(2) in D(2)O. Prior addition of H(2)tSpa protected LLC center dot PK1 renal proximal tubule cells against PbMe(2)(NO(3))(2); thiamine had no statistically significant effect by itself, but greatly potentiated the action of H(2)tSpa. Administration of either H(2)tspa or thiamine to male albino Sprague-Dawley rats dosed 30 min previously with PbMe(2)(NO(3))(2) was associated with reduced inhibition of delta-ALAD by the organolead compound, and with lower lead levels in kidney and brain, but joint administration of both H(2)tspa and thiamine only lowered lead concentration in the kidney.

Effect of propolis gel on the in vitro reduction of dentin permeability

OBJECTIVE: The aim of this study was to evaluate the capacity of potassium oxalate, fluoride gel and two kinds of propolis gel to reduce the hydraulic conductance of dentin, in vitro. MATERIAL AND METHODS: The methodology used for the measurement of hydraulic conductance of dentin in the present study was based on a model proposed in literature. Thirty-six 1-mm-thick dentin discs, obtained from extracted human third molars were divided into 4 groups (n=9). The groups corresponded to the following experimental materials: GI-10% propolis gel, pH 4.1; GII-30% propolis gel; GIII-3% potassium oxalate gel, pH 4,1; and GIV-1.23% fluoride gel, pH 4.1, applied to the dentin under the following surface conditions: after 37% phosphoric acid and before 6% citric acid application. The occluding capacity of the dentin tubules was evaluated using scanning electron microscopy (SEM) at ×500, ×1,000 and ×2,000 magnifications. Data were analyzed statistically by two-way ANOVA and Tukey's test at 5% significance level. RESULTS: Groups I, II, III, IV did not differ significantly from the others in any conditions by reducing in hydraulic conductance. The active agents reduced dentin permeability; however they produced the smallest reduction in hydraulic conductance when compared to the presence of smear layer (P<0.05). The effectiveness in reducing dentin permeability did not differ significantly from 10% or 30% propolis gels. SEM micrographs revealed that dentin tubules were partially occluded after treatment with propolis. CONCLUSIONS: Under the conditions of this study, the application of 10% and 30% propolis gels did not seem to reduce the hydraulic conductance of dentin in vitro, but it showed capacity of partially obliterating the dentin tubules. Propolis is used in the treatment of different oral problems without causing significant great collateral effects, and can be a good option in the treatment of patients with dentin sensitivity.

In vitro evaluation of an alternative method to bond molar tubes

Despite the advances in bonding materials, many clinicians today still prefer to place bands on molar teeth. Molar bonding procedures need improvement to be widely accepted clinically. OBJECTIVE: The purpose of this study was to evaluate the shear bond strength when an additional adhesive layer was applied on the occlusal tooth/tube interface to provide reinforcement to molar tubes. MATERIAL AND METHODS: Sixty third molars were selected and allocated to the 3 groups: group 1 received a conventional direct bond followed by the application of an additional layer of adhesive on the occlusal tooth/tube interface, group 2 received a conventional direct bond, and group 3 received a conventional direct bond and an additional cure time of 10 s. The specimens were debonded in a universal testing machine. The results were analyzed statistically by ANOVA and Tukey's test (α=0.05). RESULTS: Group 1 had a significantly higher (p<0.05) shear bond strength compared to groups 2 and 3. No difference was detected between groups 2 and 3 (p>0.05). CONCLUSIONS: The present in vitro findings indicate that the application of an additional layer of adhesive on the tooth/tube interface increased the shear bond strength of the bonded molar tubes.

Estudo in vitro da resistência ao cisalhamento da colagem direta de tubos ortodônticos em molares

INTRODUÇÃO: apesar da colagem direta despender menor tempo clínico, com maior preservação da integridade gengival, ainda hoje se observa uma alta incidência de bandagem dos molares. Portanto, torna-se interessante a idealização de recursos para o aumento da eficiência desse procedimento para dentes submetidos a maiores impactos mastigatórios, como, por exemplo, os molares. OBJETIVO: esse estudo teve o propósito de avaliar se a resistência à adesão com a aplicação de uma camada de resina adicional na região oclusal da interface tubo/dente aumenta a qualidade do procedimento de colagem direta de tubos em molares. MÉTODOS: selecionou-se uma amostra composta por 40 terceiros molares inferiores, que foram aleatoriamente divididos em 2 grupos: Grupo 1 - colagem direta convencional, seguida pela aplicação de uma camada de resina na oclusal da interface tubo/dente; e Grupo 2 - colagem direta convencional. O teste de resistência ao cisalhamento foi realizado 24 horas após a colagem, utilizando-se uma máquina de ensaio universal, operando a uma velocidade de 0,5mm/min. Os resultados foram analisados por meio do teste t independente. RESULTADOS: os valores médios obtidos nos testes de cisalhamento foram: 17,08MPa para o Grupo 1 e 12,60MPa para o Grupo 2. O Grupo 1 apresentou uma resistência ao cisalhamento estatisticamente significativa mais alta do que o Grupo 2. CONCLUSÃO: a aplicação de uma camada adicional de resina na oclusal da interface tubo/dente aumenta a qualidade da adesão do procedimento de colagem direta de tubos ortodônticos em molares.

In vitro wear resistance of three types of polymethyl methacrylate denture teeth

The wear resistance of denture teeth is important to the longevity of removable prostheses of edentulous patients. The ability of denture teeth to maintain a stable occlusal relationship over time may be influenced by this property. The purpose of this in vitro study was to evaluate the wear resistance of polymethyl methacrylate (PMMA) denture teeth based on their chemical composition when opposed by a ceramic antagonist. The maxillary canines (n=10) of 3 PMMA denture teeth (Trubyte Biotone, cross-linked PMMA; Trilux, highly cross-linked IPN (interpenetrating polymer network)-PMMA; and Vivodent, highly cross-linked PMMA) were secured in an in vitro 2-body wear-testing apparatus that produced sliding contact of the specimens (4.5 cycles/s, sliding distance of 20 mm, under 37°C running water) against glazed or airborne particle abraded ceramic. Wear resistance was measured as height loss (mm) under 300 g (sliding force) after 100,000 cycles, using a digital measuring microscope. Mean values were analyzed by 2-way ANOVA and Tukey's test (a=0.05). The wear of Trubyte Biotone (0.93 ± 0.14 mm) was significantly higher than that of both other types of teeth tested against abraded ceramic (p<0.05). The Vivodent tooth (0.64 ± 0.17 mm) exhibited the best wear resistance among the denture teeth tested against airborne particle abraded ceramic. There were no statistically significant differences (p>0.05) in wear among the 3 denture teeth evaluated against glazed ceramic. Trilux and Vivodent teeth tested against either glazed or airborne particle abraded ceramic did not differ significantly from each other (p<0.05). All teeth showed significantly more wear against airborne particle abraded ceramic than against glazed ceramic (p<0.05). In conclusion, the three types of PMMA denture teeth presented significantly different wear resistance against the abraded ceramic. The high-strength PMMA denture teeth were more wear-resistant than the conventional PMMA denture tooth.

Validation of an experimental polyurethane model for biomechanical studies on implant-supported prosthesis: compression tests

OBJECTIVES: The complexity and heterogeneity of human bone, as well as ethical issues, most always hinder the performance of clinical trials. Thus, in vitro studies become an important source of information for the understanding of biomechanical events on implant-supported prostheses, although study results cannot be considered reliable unless validation studies are conducted. The purpose of this work was to validate an artificial experimental model based on its modulus of elasticity, to simulate the performance of human bone in vivo in biomechanical studies of implant-supported prostheses. MATERIAL AND METHODS: In this study, fast-curing polyurethane (F16 polyurethane, Axson) was used to build 40 specimens that were divided into five groups. The following reagent ratios (part A/part B) were used: Group A (0.5/1.0), Group B (0.8/1.0), Group C (1.0/1.0), Group D (1.2/1.0), and Group E (1.5/1.0). A universal testing machine (Kratos model K - 2000 MP) was used to measure modulus of elasticity values by compression. RESULTS: Mean modulus of elasticity values were: Group A - 389.72 MPa, Group B - 529.19 MPa, Group C - 571.11 MPa, Group D - 470.35 MPa, Group E - 437.36 MPa. CONCLUSION: The best mechanical characteristics and modulus of elasticity value comparable to that of human trabecular bone were obtained when A/B ratio was 1:1.