Standardization and comparison of nonautomated assays to measure the collagen binding activity of von Willebrand factor.

INTRODUCTION: Diagnosis of von Willebrand disease (VWD) is difficult due to the heterogenic phenotype of patients and to the complex tests that are required for an adequate investigation. The collagen binding assay (VWF:CB) reveals the adhesion capacity of von Willebrand factor (VWF) to collagen and can be useful to reduce the misleading diagnosis of VWD. This study aimed the standardization of 2 nonautomated VWF:CB assays based on ELISA and flow cytometry. METHODS: Plasma samples from 87 patients previously diagnosed with VWD and 22 healthy controls were analyzed. Measurement of the VWF-collagen binding activity was performed using a commercial assay and the 2 tests proposed. VWF:CB/VWF:Ag ratio was calculated for samples and the differentiation between types 1, 2A, and 2M was analyzed. RESULTS: ELISA and flow cytometry tests presented strong correlation with the gold standard test (r2  = .8976 and r2  = .8143, respectively). Tests based on ELISA and flow cytometry presented a bias of +7.2% and -3.6%, respectively. CONCLUSION: The ELISA test demonstrated better performance to detect VWF-collagen binding activity in healthy individuals and VWD patients. This test could differentiate 2A and 2M subtypes using a feasible protocol that can be easily implemented.

Characterization of non-dialyzable constituents from cranberry juice that inhibit adhesion, co-aggregation and biofilm formation by oral bacteria.

An extract prepared from cranberry juice by dialysis known as nondialyzable material (NDM) has been shown previously to possess anti-adhesion activity toward microbial species including oral bacteria, uropathogenic Escherichia coli and Helicobacter pylori. Bioassay-guided fractionation of cranberry NDM was therefore undertaken to identify the anti-adhesive constituents. An aqueous acetone-soluble fraction (NDMac) obtained from Sephadex LH-20 inhibited adhesion-linked activities by oral bacteria, including co-aggregation of oral bacteria Fusobacterium nucleatum with Streptococcus sanguinis or Porphyromonas gingivalis, and biofilm formation by Streptococcus mutans. Analysis of NDMac and subsequent subfractions by MALDI-TOF MS and 1H NMR revealed the presence of A-type proanthocyanidin oligomers (PACs) of 3-6 degrees of polymerization composed of (epi)catechin units, with some (epi)gallocatechin and anthocyanin units also present, as well as quercetin derivatives. Subfractions containing putative xyloglucans in addition to the mixed polyphenols also inhibit biofilm formation by S. mutans (MIC = 125-250 µg mL-1). These studies suggest that the anti-adhesion activities of cranberry NDM on oral bacteria may arise from a combination of mixed polyphenol and non-polyphenol constituents.

Increased incidence of femoral fractures in small femurs and women undergoing uncemented total hip arthroplasty - why?

The aim of this study was to investigate the relationship between the geometry of the proximal femur and the incidence of intra-operative fracture during uncemented total hip arthroplasty (THA). We studied the pre-operative CT scans of 100 patients undergoing THA with an uncemented femoral component. We measured the anteroposterior and mediolateral dimensions at the level of division of the femoral neck to calculate the aspect ratio of the femur. Wide variations in the shape of the femur were observed, from round, to very narrow elliptic. The femurs of women were narrower than those of men (p < 0.0001) and small femurs were also narrower than large ones. Patients with an intra-operative fracture of the calcar had smaller and narrower femurs than those without a fracture (p < 0.05) and the implanted Corail stems were smaller in those with a fracture (mean size 9 vs 12, p < 0.0001). The variability of the shape of the femoral neck at the level of division contributes to the understanding of the causation of intra-operative fractures in uncemented THA.

Karyoevolutionary aspects of Atlantic hogfishes (Labridae-Bodianinae), with evidence of an atypical decondensed argentophilic heterochromatin.

Fish from the family Labridae elicit considerable ecological interest, especially due to their complex interactions with the reef environment. Different karyoevolutionary tendencies have been identified in the subfamilies Bodianinae, Corinae and Cheilinae. Chromosomal analyses conducted in the Atlantic species Bodianus rufus (2n=48; 6m+12sm+14st+16a, FN=80), Bodianus pulchellus (2n=48; 4m+12sm+14st+18a, FN=78) and Bodianus insularis (2n=48; 4m+12sm+14st+18a, FN=78) identified Ag-NOR/18SrDNA sites located only in the terminal region of the short arm (p) of the largest subtelocentric pair. The 5S rDNA genes were mapped in the terminal region of the long arm (q) of the largest acrocentric pair and the p arm of chromosome 19 in B. insularis. The karyotype of the three species shows an extensive heterochromatic and argentophilic region, exceptionally decondensed, located in the p arm of the second subtelocentric pair. This region does not correspond to a NOR site, since it is not hybridized with 18S rDNA probes, and is not GC-rich, as generally occurs with nucleolus organizer regions of lower invertebrates. Heterochromatin in the three species is reduced and distributed over the centromeric and pericentromeric regions of chromosomes. The elevated number of two-armed chromosomes in species of Bodianus, in relation to other Labridae, shows karyotype diversification based on pericentric inversions, differentiating them markedly in terms of evolutionary tendencies that occur in subfamilies Corinae and Cheilininae. Structural cytogenetic similarities between B. pulchellus and B. insularis, in addition to the conserved chromosomal location pattern of ribosomal multigenic families, indicate phylogenetic proximity of these species.

Isolation and experimental infection with Vibrio alginolyticus in the sea horse, Hippocampus reidi Ginsburg, 1933 (Osteichthyes: Syngnathidae) in Brazil.

The aim of this study was to evaluate the pathogenicity of Vibrio alginolyticus isolated from an outbreak of sea horse Hippocampus reidi reared in the State of Santa Catarina, Brazil, by experimental infection. Sea horses with necrosis on the mouth epithelium were collected from aquaria at the Aquaculture Department, UFSC and the bacterium isolated from the mouth, liver, heart and blood in thiosulphate citrate bilesalt sucrose agar broth. The strains were identified by API 20E kit with 99.1% probability as Vibrio alginolyticus. Twelve adult sea horses (9.63 +/- 2.42 g and 15.12 +/- 0.87 cm) were distributed in six aquaria of 10 L capacity with aerated sea water. Fish from three aquaria were submitted to an immersion bath in a solution containing 1.0 x 10(7) CFU of V. alginolyticus/mL for 15 minutes. Fish from the other three aquaria received the same procedure without bacteria. Twenty four hours after this challenge, 100% mortality was observed in the animals infected with V. alginolyticus. No mortality was observed in non-infected fish. Hyperplasia, displacement and fusion of secondary lamellae of the gills; leukocyte infiltration and necrotic foci in the kidney; hyperplasia, sinusoidal deformation and necrotic foci in the liver were observed in histopathological analysis. The V. alginolyticus isolated in this study was pathogenic to H. reidi and constitutes an important sanitary problem to its culture.

Effects of retinoids on the in vitro development of Capra hircus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 µg/ml RT and 0.5 µm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

Haematological changes in Nile tilapia experimentally infected with Enterococcus sp.

This study evaluated the haematological changes in Nile tilapia experimentally infected with 1 x 10(3) and 1 x 10(6) colony-forming units (CFU)/mL of Enterococcus sp. in the swim bladder. The experiment consisted of four treatments in triplicates: non-injected fish (NI); fish injected with 1 mL of sterile saline solution 0.65% (SAL); fish injected with 1 x 10(3) and 1 x 10(6) CFU/mL of Enterococcus diluted in 1 mL sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected. The haematological tests included red blood cell (RBC) and white blood cell (WBC) counts, hematocrit, number of total thrombocytes, and differential counting of WBC. Fish injected with 1 x 10(6) CFU/mL of Enterococcus showed a higher number of thrombocytes than the other treatments. White blood cell and lymphocyte numbers increased significantly in fish injected with 1 x 10(6) CFU/mL of Enterococcus when compared to non-injected control. There was significant increase in the number of neutrophils in saline injected fish and reduced number of monocytes after injections with 1 x 10(6) CFU/mL of Enterococcus. Hematocrit increased in fish injected with 1 x 10(3) and 1 x 10(6) CFU/mL of Enterococcus.

Haematological changes in Nile tilapia experimentally infected with Enterococcus sp[period] / Alterações hematológicas em tilápia do Nilo infectada experimentalmente com Enterococcus sp[ponto final]

This study evaluated the haematological changes in Nile tilapia experimentally infected with 1 x 10³ and 1 x 10(6) colony-forming units (CFU)/mL of Enterococcus sp. in the swim bladder. The experiment consisted of four treatments in triplicates: non-injected fish (NI); fish injected with 1 mL of sterile saline solution 0.65 percent (SAL); fish injected with 1 x 10³ and 1 x 10(6) CFU/mL of Enterococcus diluted in 1 mL sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected. The haematological tests included red blood cell (RBC) and white blood cell (WBC) counts, hematocrit, number of total thrombocytes, and differential counting of WBC. Fish injected with 1 x 10(6) CFU/mL of Enterococcus showed a higher number of thrombocytes than the other treatments. White blood cell and lymphocyte numbers increased significantly in fish injected with 1 x 10(6) CFU/mL of Enterococcus when compared to non-injected control. There was significant increase in the number of neutrophils in saline injected fish and reduced number of monocytes after injections with 1 x 10(6) CFU/mL of Enterococcus. Hematocrit increased in fish injected with 1 x 10³ and 1 x 10(6) CFU/mL of Enterococcus.

Este estudo avaliou alterações hematológicas em tilápia do Nilo infectada experimentalmente com 1 x 10³ e 1 x 10(6) unidades formadoras de colônia (CFU)/mL de Enterococcus sp. na bexiga natatória. O experimento consistiu de quatro tratamentos com três repetições cada: peixes não injetados (NI); peixes injetados com 1 mL de solução salina a 0,65 por cento esterilizada (SAL); peixes injetados com 1 x 10³ e 1 x 10(6) CFU/mL de Enterococcus diluída em 1 mL de solução salina esterilizada. Vinte e quatro horas após as injeções, os peixes foram anestesiados com benzocaína e o sangue coletado. Os testes hematológicos incluíram as contagens totais de eritrócitos (RBC), de leucócitos (WBC) e de trombócitos, o percentual do hematócrito e a contagem diferencial de leucócitos. Peixes injetados com 1 x 10(6) CFU/mL de Enterococcus mostraram maior número de trombócitos no sangue do que os dos outros tratamentos. O número de leucócitos totais e o número de linfócitos foram significativamente mais altos após injeção de 1 x 10(6) CFU/mL de Enterococcus, quando comparados ao controle não injetado. Houve aumento no número de neutrófilos nos injetados com salina e redução no número de monócitos nos injetados com 1 x 10(6) CFU/mL de Enterococcus. O hematócrito aumentou nos animais injetados com 1 x 10³ e 1 x 10(6) CFU/mL de Enterococcus.

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions.

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.

Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.

A bioactive spirolactone iridoid and triterpenoids from Himatanthus sucuuba.

Himatanthus sucuuba is an Amazonian tree with abundant, yet conflicting ethnobotanical information. Investigation of the polar and non-polar constituents led to the isolation of plumericin, a bioactive spirolactone iridoid, and four known pentacylic triterpenes: lupeol acetate, lupeol cinnamate, lupeol beta-phenyl propionate, and alpha-amyrin cinnamate.

Cytotoxic triterpene acids from the Peruvian medicinal plant Polylepis racemosa.

Cytotoxicity-guided fractionation of the bark and stem extract of Polylepis racemosa led to the identification of ursolic acid, pomolic acid, 3-O-acetylpomolic acid, and 2-oxopomolic acid. Pomolic acid was the most cytotoxic component, and was specific for M-14 melanoma and ME180 cervical carcinoma, with GI50 values of 6.9 and 8.3 micrograms/mL respectively.

Brazilian bioethanol program.

Brazil is the largest producer of bioethanol, and sugarcane is the main raw material. Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used. This article analyzes the Brazilian Ethanol Program. For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane. These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol. The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

Primary coronary angioplasty and stent implantation in acute myocardial infarction. Comparative analysis of the in-hospital results in the CENIC/SBHCI registry. National Center of Cardiovascular Interventions members.

OBJECTIVE: Comparative analysis of the in-hospital results after primary implantation of stents or coronary balloon angioplasty in patients with acute myocardial infarction (MI). METHODS: CENIC (National Center of Cardiovascular Interventions) gathered data on 3, 924 patients undergoing coronary angioplasty (in the primary form, without the previous use of thrombolytic agents) in the first 24 hours after a MI, during the period of 1996-1998. From these 3,924 patients, 1,337 (34%) underwent stent implantation. We analyzed the success of the procedure and the occurrence of adverse cardiac events. RESULTS: In patients undergoing stent implantation there were more males (77% vs 69%, p=0.001), previous by pass surgery (6. 3% vs. 4.5%, p=0.01), anterior MI and stent implantation in left descending artery (55% vs. 48% vs. p=0.009), and saphenous vein bypass grafts (3.3% vs. 1.9%). the procedure was more succesful in the group of stents (97% vs. 84%, p=0.001) and reinfarction rate (2. 5 vs. 4%, p=0.002). The need for emergency revascularization was similar (1% vs. 1.1%, NS). Total in-hospital mortality was lower in stent group (3.4% vs. 7. 2%, p=0.0001) and this effect was in patients Killip class III/V (19.5% vs. 32.5%, p= 0.002) because there was no difference in patients class I/II (1.7% vs. 2.8%, p=0. 9). CONCLUSION: Primary stent implantation in acute myocardial infarction showed better early results than balloon angioplasty alome.

The A3B phenotype: a further contribution to family studies

Estudaram-se os grupos e sub-grupos sanguíneos ABO em vinte e nove componentes de tres famílias caucasoides e de uma negroide. Exames sorológicos complementares permitiram caracterizar a variante A3B. Entre os probandos, dois eram doadores de sangue, uma era portadora de um mioma uterino e a outra criança com anemia falciforme. Os padröes de herança parecem apoiar nossas sugestöes anteriores de que o fenótipo A3B deve corresponder aos genótipos A2B* ou AintB*, sendo B* um alelo repressor. A repressäo parece ser de grau variável em uma mesma família porque: a) o genótipo AintB* pode condicionar um fenótipo AintB sem campo misto aparente ou um fenótipo A3B com um campo misto típico; b) os indivíduos A3B podem diferir significantemente no conteúdo de antígeno A de suas hemácias. Numa família informativa pôde-se presumir, pelo padräo de herança, a ausência do alelo A3 e do heterozigoto A3B. Devido a casos deste tipo, foi salientada a importância de estudos detalhados em células e soros, em casos de exclusäo de paternidade pelo sistema ABO