Influence of solvent and a supplementary step with a finishing instrument on filling material removal from canals connected by an isthmus.

AIM: To evaluate the effectiveness of a solvent (eucalyptol) in improving filling material removal from canals connected by isthmuses, and the additional cleaning effect of a finishing instrument. METHODOLOGY: The mesial canals from 32 mandibular molars (Vertucci's type II morphology) were instrumented and filled with the single-cone technique using Reciproc R25 gutta-percha points (VDW, Munich, Germany) combined with Sealer 26 (Dentsply, Petrópolis, RJ, Brazil). Each root was then subjected to retreatment using the Mtwo instrument system (VDW), with or without a solvent (n = 16 per group). The volume of filling material in the canals was assessed by micro-computed tomographic (micro-CT) scans taken before and after retreatment. Canals with remnants of filling material received a supplementary procedure with the XP-endo Finisher R instrument (FKG Dentaire, La Chaux-de-Fonds, Switzerland), with or without eucalyptol, and another micro-CT scan was taken. All retreatment procedures were performed inside a cabinet under a controlled temperature (37 °C). Filling material removal was evaluated in the 5-mm apical canal system for the canal+isthmus space or the isthmus alone. Statistical analyses were performed to compare the removal of filling material with and without eucalyptol, and after a supplementary approach with XP-endo Finisher R. The level of significance was set at 5% for all statistical tests (P < 0.05). RESULTS: The amount of filling material removed from the canal+isthmus with Mtwo instruments was 83.2% when no solvent was used and 83.8% using the solvent (P > 0.05). When the isthmus area was evaluated separately, most specimens were associated with a reduction in the filling material, with no significant difference between the groups with or without using a solvent (P > 0.05). The supplementary step with XP-endo Finisher R significantly improved removal of filling material from both canal and isthmus area (P < 0.05), regardless of the use of a solvent (P > 0.05). CONCLUSION: The use of eucalyptol did not improve filling material removal from Vertucci's type II molar mesial canals and isthmuses. XP-endo Finisher R significantly enhanced removal of filling material from the canals and isthmuses.

Effects of preparation with the Self-Adjusting File, TRUShape and XP-endo Shaper systems, and a supplementary step with XP-endo Finisher R on filling material removal during retreatment of mandibular molar canals.

AIM: To compare the effects of three instrumentation systems, and a supplementary approach with a finishing instrument, on filling material removal during retreatment of mandibular molar canals. METHODS: Sixty mesial canals from mandibular molars (Vertucci's type IV anatomy) were instrumented, filled and subjected to retreatment. After initial removal of the root canal filling material using the D-RaCe system, the canals were randomly distributed into three groups (n = 20) according to the instrument system used for preparation: the Self-Adjusting File (SAF), TRUShape or XP-endo Shaper. The filling material volume in the apical 5 mm of the canals was assessed by means of micro-computed tomography (micro-CT) before and after retreatment. All specimens with residual filling material were subjected to a supplementary approach with the XP-endo Finisher R instrument and another micro-CT scan was taken. Data on the volumes of filling material and incidence of total removal were compared between groups by the general linear model for paired data and the Fisher's exact test. The effects of the refinement step were evaluated by the Wilcoxon Signed Ranks test. RESULTS: The amount of removed material was 92.4%, 96.9% and 96.9% for the SAF, TRUShape and XP-endo Shaper, respectively. There were no significant differences between them (P > 0.05). Canals were completely cleaned of filling material in 70% of the specimens for XP-endo Shaper, 55% for SAF and 30% for TRUShape; the difference between XP-endo Shaper and TRUShape was significant (P = 0.03). The supplementary step with the XP-endo Finisher R instrument was associated with additional filling material removal of 38% (P < 0.001). Six more canals were rendered free of filling material after using this finishing instrument. CONCLUSIONS: The tested systems were equally effective in removing the mass of filling material from the apical 5 mm of molar canals. The supplementary step with the XP-endo Finisher R instrument enhanced filling material removal.

Effects of increased apical enlargement on the amount of unprepared areas and coronal dentine removal: a micro-computed tomography study.

AIM: To evaluate the effects of progressive apical enlargement on the amount of unprepared root canal surface area and remaining dentine thickness. METHODOLOGY: The root canals of 30 extracted mandibular incisors with Vertucci's type I configuration were instrumented with rotary HyFlex CM instruments (Coltene-Whaledent, Altstätten, Switzerland) up to 4 instruments larger than the first one that bound at the working length (WL). Teeth were scanned in a micro-computed tomography (micro-CT) device before canal preparation and after instrumentation with the 2nd, 3rd and 4th larger instruments. The amount of unprepared surface area in the full canal or in the apical 4 mm as well as the remaining dentine thickness at 10 mm from the WL were calculated and compared. The general linear model for repeated measures adjusted by Bonferroni's post hoc test was used for statistic analysis. RESULTS: There was a significant reduction in the amount of unprepared areas after each increase in preparation size (P < 0.01). This was observed for both the full canal length and the 4-mm apical segment. The amount of remaining dentine was also significantly reduced after each file size (P < 0.01). However, dentine thickness always remained greater than 1 mm, even after using the largest instrument. CONCLUSION: Apical preparations up to 4 instruments larger than the first one to bind at the WL caused a significant progressive reduction in the unprepared canal area.

What happens to unprepared root canal walls: a correlative analysis using micro-computed tomography and histology/scanning electron microscopy.

AIM: To microscopically examine the cleanliness of root canal walls that remained unprepared as revealed by micro-CT. METHODOLOGY: The root canals of 10 freshly extracted mandibular premolars with necrotic pulps and apical periodontitis along with the mesiobuccal canals of 11 mandibular molars with vital pulps were prepared using Reciproc instruments R40 and R25, respectively, and 2.5% sodium hypochlorite irrigation. Specimens were scanned in micro-CT before and after preparation, and the unprepared areas were identified. The outer root surface corresponding to the untouched areas was marked on each root third to guide further analysis using histological (for teeth with vital pulps) and scanning electron microscopic (SEM; for necrotic teeth) examination. In the teeth with vital pulps, the root canal area occupied by tissue remnants was calculated. In SEM analysis of teeth with necrotic pulps, scores were attributed for the amount of debris on the untouched areas. RESULTS: The proportion of unprepared areas in the mesiobuccal molar canals was 18.1% and 9.6% over the full canal length and apical canal, respectively. In premolars, corresponding figures were 34.6% and 17.6%, respectively. Histological analysis of canals with vital pulps revealed tissue remnants over the untouched walls almost exclusively in the apical canal. SEM analysis of the canals with necrotic pulps revealed debris along the untouched walls in all root canal thirds. CONCLUSION: The areas that remain untouched by Reciproc instruments used with 2.5% NaOCl irrigation as revealed by micro-CT analysis were usually covered with debris, in the form of pulp tissue remnants, bacteria and dentine chips, especially in the apical root canal.

Micro-CT evaluation of the efficacy of hard-tissue removal from the root canal and isthmus area by positive and negative pressure irrigation systems.

AIM: To evaluate the removal of accumulated hard-tissue debris (AHTD) from the root canal system of mandibular molars by positive and negative pressure irrigation systems, using micro-CT imaging analysis. METHODOLOGY: Mandibular molars with a single canal in the distal root and 2 canals connected by an isthmus in the mesial root were matched based on similar morphological dimensions using micro-CT evaluation and assigned to 2 experimental groups (n = 20 mesial and 10 distal canals), according to the irrigation protocol: apical positive (conventional irrigation) or negative (EndoVac system) pressure. Changes in root canal volume and surface area as well as percentage of uninstrumented canal wall surface and accumulated hard-tissue debris (AHTD) after canal preparation were compared statistically using the independent sample t-test and Mann-Whitney U-test, with the significance level set at 5%. RESULTS: Volume, surface area and percentage of static voxels in either mesial or distal root canal systems were not significantly different between groups before or after root canal preparation (P > 0.05). After preparation, AHTD was not observed in the distal canal of both groups. However, in the mesial root canal system, the conventional irrigation group was associated with a significantly higher median percentage of AHTD (11.48%; IQR: 5.9-22.6; range: 1.86-41.98) than the EndoVac group (3.40%; IQR: 1.5-7.3; range: 0.82-12.84) (P < 0.05). CONCLUSIONS: Neither irrigation protocol succeeded in rendering the mesial canal system free of AHTD; however, apical negative pressure irrigation resulted in lower levels of AHTD than conventional irrigation.

Analysis of genetic diversity of a native population of Myrcia lundiana Kiaersk. plants using ISSR markers.

Myrcia lundiana Kiaersk. is a tree of the family Myrtaceae found in tropical and subtropical areas of the southern hemisphere that produces essential oil. The aim of this study was to characterize the genetic diversity of M. lundiana plants from a native population of Parque Nacional de Itabaiana, using inter-simple sequence repeat molecular markers. Thirty-five primers were tested, 20 of which were polymorphic, resulting in 135 polymorphic and informative bands. Results of the cluster analysis, obtained using the unweighted pair group method with arithmetic mean, grouped plants into three clusters: Cluster I - MLU001, MLU002, MLU003, MLU004, MLU005, MLU006, MLU018, MLU019, MLU020, MLU021, MLU022; MLU008, MLU011, MLU012, MLU014, MLU015, MLU017, MLU026, and MLU028; Cluster II - MLU007, MLU009, MLU010, MLU013, and MLU016; and Cluster III - MLU023, MLU024, MLU025, and MLU027. Jaccard similarity coefficients for pair-wise comparisons of plants ranged between 0.15 and 0.87. MLU014 and MLU015 presented low genetic diversity, with a similarity index of 0.87. Conversely, MLU007 and MLU019 presented high diversity, with a similarity index of 0.15. According to the structure analysis, three distinct clusters were formed. Genetic diversity of M. lundiana plants was intermediate, and expansion of its genetic diversity is necessary. MLU026 and MLU028 are the most suitable for selection in breeding programs, since they clearly represent all of the diversity present in these plants. Moreover, these results provide important information on the existing genetic variability, highlighting the importance of Parque Nacional de Itabaiana for the conservation of this species.

Shaping ability of single-file reciprocating and heat-treated multifile rotary systems: a micro-CT study.

AIM: To investigate changes in three-dimensional geometry, in various cross-sectional morphological parameters and in the centring ability of root canals prepared with different preparation systems using microcomputed tomographic imaging technology. METHODOLOGY: Sixty-four mesial canals of mandibular molars were matched based on similar morphological dimensions using micro-CT evaluation and assigned to four experimental groups (n = 16), according to the canal preparation technique: Reciproc, WaveOne, Twisted File and HyFlex CM systems. Changes in several 2D (area, perimeter, form factor, roundness, minor and major diameter) and 3D [volume, surface area, structure model index (SMI)] morphological parameters, as well as canal transportation, were compared with preoperative values using Kruskal-Wallis and anovapost hoc Tukey's tests with the significance level set at 5%. RESULTS: Preparation significantly increased all tested parameters in the experimental groups. No significant differences were observed between groups regarding changes in volume, surface area, SMI, form factor and roundness of the root canal after preparation (P > 0.05). In the apical third, the Reciproc group had significantly greater changes in canal area, perimeter, major and minor diameters than the other groups (P < 0.05). Overall, the Twisted File and HyFlex CM systems were associated with significantly less transportation than the reciprocating instruments, Reciproc and WaveOne (P < 0.05). CONCLUSIONS: Shaping procedures led to the enlargement of the root canal space with no evidence of significant preparation errors. Changes in 3D parameters were not different between groups whilst, in the apical third, Reciproc was associated with significantly greater changes in several 2D parameters compared to the other groups. Twisted File and HyFlex CM systems were able to maintain the original canal anatomy with less canal transportation than Reciproc and WaveOne; however, these differences are unlikely to be of clinical significance.

Secretory structures in Baccharis platypoda DC. inflorescences (Asteraceae) and characterization of the chemical composition of its secretion.

Rocky outcrop environments at high altitudes have nutrient-poor soil, where species are exposed to water scarcity and high solar radiation. Baccharis platypoda DC. occurs in such an environment and has a rigid and transparent secretion that covers the entire inflorescence. We analysed and compared the secretory structures and their chemical composition in female and male inflorescences of B. platypoda, a dioecious species, to explore chemodiversity within this species and assess potential differences between individuals. Our investigation also aims to understand the occurrence of these substances in the genus Baccharis L. Chemical compounds and secretory structures were similar in female and male inflorescences. There are glandular trichomes on the epidermis of the abaxial surface of bracts, and secretory ducts in the axis of the inflorescence, as well as in sepals, petals, and bracts. Histochemical tests were positive for phenolic compounds, flavonoids, proteins, pectin, and lipids, but not for mucilage. Flavonoid content varied between 6.24% and 9.81%, being higher in female inflorescences. Chromatography revealed the presence of several phenolic compounds, some terpenes, and other less frequent classes in both female and male inflorescences. We highlight that trichomes found on these surfaces produce abundant phenolic compounds. These act as natural defence agents, absorbing UV radiation and minimizing oxidative stress to plant cells. The chemical composition of the secretion covering the inflorescences may reflect adaptation and survival mechanisms of these organisms under extreme sun exposure.

Diversity and genetic structure among subpopulations of Gossypium mustelinum (Malvaceae).

Gossypium mustelinum is the only cotton species native to Brazil; it is endemic to the semi-arid region of the northeast. The populations are found near perennial and semi-perennial sources of water, such as ponds or pools in intermittent streams. Problems with in situ conservation derive from human interference in its habitat, mainly because of excessive cattle grazing and deforestation. Establishing efficient strategies for in situ conservation requires knowledge of the genetic structure of the populations. We evaluated the structure and genetic variability of populations of G. mustelinum in the Tocó and Capivara Rivers (State of Bahia). Two hundred and eighteen mature G. mustelinum plants were genotyped with SSR markers. The molecular data were used to estimate the allelic frequencies, the heterozygosity, the F statistics, and the genetic distance among the populations and among individuals. We found high genetic diversity among the populations. The FST indexes for each population were also high and strongly correlated with physical distance. The high estimated level of endogamy and the low observed heterozygosity are indicative that the populations reproduce mainly by self-fertilization and crosses between related individuals. Consequently, strategies for in situ preservation should include at least three occurrence sites of G. mustelinum from each population. For ex situ conservation, the collections should include as many sites as possible.

Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri.

Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K(m)) of 47 µM(-1)∙s(-1). The purified ((HIS))CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K(i) values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.

Antibacterial activity improvement of dental glass-ceramic by incorporation of AgVO3 nanoparticles.

OBJECTIVE: This study aimed to investigate the role of the incorporation of an antibacterial nanoceramic (AgVO3) on the properties of a restorative dental glass-ceramic. METHOD: A commercially available restorative glass-ceramic, commonly designated as porcelain (IPS d.SIGN) was functionalized with an antibacterial agent (nanostructured ß-AgVO3), synthesized by a hydrothermal route. Both functionalized and pristine samples were processed according to the manufacturer's instructions. All samples were characterized by X-ray diffraction, Rietveld refinement, particle size distribution, Scanning Electron Microscopy, chemical solubility, and Inductively Coupled Plasma Spectroscopy. Their antibacterial potential (Mueller-Hinton test) was analyzed against gram-positive (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). RESULTS: The commercial glass-ceramic showed leucite (KAlSi2O6) as the only detectable crystalline phase, and, for both strains, no antibacterial activity could be detected in the Mueller-Hinton agar plates test. A monophasic, needle-shaped, and nanometric ß-AgVO3 powder was successfully synthesized by a simple hydrothermal route. After thermal treatment, glass-ceramic samples containing different percentages of ß-AgVO3 showed a second crystalline phase of microline [K0.95(AlSi3O8)]. For modified samples, inhibition halos were easily visible on the Mueller-Hinton test, which ranged from 11.1 ± 0.5 mm to 16.6 ± 0.5 mm and 12.7 ± 0.3 mm to 15.5 ± 0.3 mm in the S. aureus and E.coli cultures, respectively, showing that the halos formed were dose-dependent. Also, increasing the percentage of ß-AgVO3 promoted a significant increase in chemical solubility, from 72 µg/cm2 (samples with 1 wt% of ß-AgVO3) to 136 µg/cm2 (samples with 2 wt% of ß-AgVO3), which was associated with the silver and vanadium ions released from the glass matrix. SIGNIFICANCE: Our in vitro results indicate that IPS d.SIGN, as most of the dental glass-ceramics, do not exhibit antibacterial activity per se. Nonetheless, in this concept test, we demonstrated that it is possible to modify dental veneering materials giving them antibacterial properties by adding at least 2 wt% of ß-AgVO3, a nanomaterial easily synthesized by a simple route.

Reproductive biology of pequira Bryconamericus stramineus (Eigenmann, 1908) in Funil Reservoir-MG, Brazil.

In order to evaluate aspects of reproductive biology of the "pequira" ( Bryconamericus stramineus) in the elevator of the Funil Dam - MG, fish capture was carried out from November 2008 to January 2009 and 317 individuals were collected. The mean standard length (SL) of the population was 4.96 cm and mean weight 1.80 g. The females had SL of 5.0 cm, while males had a SL of 4.6 cm. A sex ratio of 2.20: 1 (females: male) was observed. Our results show that 73% of the individuals analyzed were considered adults. The species presented low fecundity, mean of 470.9 oocytes per female and a mean diameter of 221.08 µm, with an increase in oocyte diameter over the evaluation period. The length of the first gonadal maturation was estimated at 5.0 cm. The results obtained in this work suggest that the reproductive cycle of the species occurs in the analyzed period. Although this species does not have migratory reproductive habits, the presence of adults in the reproductive stage was observed in the transposition area, which suggests a search for new environments for spawning.

High titers of Chlamydia trachomatis antibodies in Brazilian women with tubal occlusion or previous ectopic pregnancy.

OBJECTIVE: To evaluate serum chlamydia antibody titers (CATs) in tubal occlusion or previous ectopic pregnancy and the associated risk factors. METHODS: The study population consisted of 55 women wih tubal damage and 55 parous women. CAT was measured using the whole-cell inclusion immunofluorescence test and cervical chlamydial DNA detected by PCR. Odds ratios were calculated to assess variables associated with C. trachomatis infection. RESULTS: The prevalence of chlamydial antibodies and antibody titers in women with tubal occlusion or previous ectopic pregnancy was significantly higher (P < .01) than in parous women. Stepwise logistic regression analysis showed that chlamydia IgG antibodies were associated with tubal damage and with a larger number of lifetime sexual partners. CONCLUSIONS: Chlamydia antibody titers were associated with tubal occlusion, prior ectopic pregnancy, and with sexual behavior, suggesting that a chlamydia infection was the major contributor to the tubal damage in these women.

Mixed pollen load and late-acting self-incompatibility flexibility in Adenocalymma peregrinum (Miers) L.G. Lohmann (Bignonieae: Bignoniaceae).

Mixed cross and self-pollen load on the stigma (mixed pollination) of species with late-acting self-incompatibility system (LSI) can lead to self-fertilized seed production. This "cryptic self-fertility" may allow selfed seedling development in species otherwise largely self-sterile. Our aims were to check if mixed pollinations would lead to fruit set in LSI Adenocalymma peregrinum, and test for evidence of early-acting inbreeding depression in putative selfed seeds from mixed pollinations. Experimental pollinations were carried out in a natural population. Fruit and seed set from self-, cross and mixed pollinations were analysed. Further germination tests were carried out for the seeds obtained from treatments. Our results confirm self-incompatibility, and fruit set from cross-pollinations was three-fold that from mixed pollinations. This low fruit set in mixed pollinations is most likely due to a greater number of self- than cross-fertilized ovules, which promotes LSI action and pistil abortion. Likewise, higher percentage of empty seeds in surviving fruits from mixed pollinations compared with cross-pollinations is probably due to ovule discounting caused by self-fertilization. Moreover, germinability of seeds with developed embryos was lower in fruits from mixed than from cross-pollinations, and the non-viable seeds from mixed pollinations showed one-third of the mass of those from cross-pollinations. The great number of empty seeds, lower germinability, lower mass of non-viable seeds, and higher variation in seed mass distribution in mixed pollinations, strongly suggests early-acing inbreeding depression in putative selfed seeds. In this sense, LSI and inbreeding depression acting together probably constrain self-fertilized seedling establishment in A. peregrinum.

Pre-eruptive intracoronal resorption in a third upper molar: clinical, tomographic and histological analysis.

Radiolucent or hypodense lesions in the crown of unerupted teeth may be due to pre-eruptive intracoronal resorption. Clinicians must be aware of this risk so that they can diagnose and appropriately treat this condition. The purpose of this study is to present a well-documented clinical case of pre-eruptive intracoronal resorption in an impacted third upper left molar of a 63 year old female patient. This was an unexpected finding, which occurred after cone-beam computed tomography was used to investigate the first upper left molar, which had an acute periradicular abscess. A multidisciplinary team followed up the case to describe clinical, radiographic and histological findings. The available treatment options were discussed, and the tooth extraction was the option chosen. Previous case studies describing such resorption in third upper molars have not been reported. This case shows that all permanent teeth in a pre-eruptive stage must be analysed radiographically to detect early pre-eruptive intracoronal resorption.

Effects of oocytes exposure to bovine diarrhea viruses BVDV-1, BVDV-2 and Hobi-like virus on in vitro-produced bovine embryo development and viral infection.

As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 × 102 - 2.50 × 103 TCID50/100 µL), BVDV-2 (6.25 × 101 - 5.20 × 102 TCID50/100 µL) or Hobi-like virus (1.90 × 102 - 1.58 × 103 TCID50/100 µL) for 22-24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50-100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2-64.1% vs 49.8%; P = 0.003-0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20-50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3-38.0% vs 49.8% for negative controls; P ≤ 0.001-0.014). Blastocyst rates were only reduced in the 7.9 × 102 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P = 0.009), when calculated by oocyte number. 50-80% of degenerated embryos tested positive for Hobi-like virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and -2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity.

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Sex Hormones Promote Opposite Effects on ACE and ACE2 Activity, Hypertrophy and Cardiac Contractility in Spontaneously Hypertensive Rats.

BACKGROUND: There is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs). METHODOLOGY/PRINCIPAL FINDINGS: Twelve-week-old female (F) and male (M) SHRs were divided into 2 experimental groups (n = 7 in each group): sham (S) and gonadectomized (G). Fifty days after gonadectomy, we measured positive and negative first derivatives (dP/dt maximum left ventricle (LV) and dP/dt minimum LV, respectively), hypertrophy (morphometric analysis) and ACE and ACE2 catalytic activity (fluorimetrically). Expression of calcium handling proteins was measured by western blot. Male rats exhibited higher cardiac ACE and ACE2 activity as well as hypertrophy compared to female rats. Orchiectomy decreased the activity of these enzymes and hypertrophy, while ovariectomy increased hypertrophy and ACE2, but did not change ACE activity. For cardiac function, the male sham group had a lower +dP/dt than the female sham group. After gonadectomy, the +dP/dt increased in males and reduced in females. The male sham group had a lower -dP/dt than the female group. After gonadectomy, the -dP/dt increased in the male and decreased in the female groups when compared to the sham group. No difference was observed among the groups in SERCA2a protein expression. Gonadectomy increased protein expression of PLB (phospholamban) and the PLB to SERCA2a ratio in female rats, but did not change in male rats. CONCLUSION: Ovariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.

Internally quenched fluorogenic substrates for angiotensin I-converting enzyme.

OBJECTIVE: Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). DESIGN: We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids). METHODS: Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods. RESULTS: Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE. The best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively. This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril. CONCLUSIONS: We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group. They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum.

[Trial of labor after cesarean section. Two years' experience]. / Prova de trabalho de parto após cesariana. Dois anos de experiência.

A retrospective study was undertaken to evaluate the incidence of vaginal delivery in women that previously undergone a cesarean section; in addition, incidence of complications such as uterine rupture and scar dehiscence were evaluated. During the two years reviewed, 251 out of 324 women with a previous cesarean section were allowed to labor; 64.1% had a vaginal delivery. Uterine rupture occurred in 0.8% of the patients of the trial of labor group: no other significant maternal and/or fetal complications were seen; scar dehiscence occurred in 3.3%. Statistical analysis didn't show any increase in complications when oxytocin was used to stimulate uterine activity. It is concluded that trail of labor and delivery after cesarean section is a safe option in a large number of patients.