Isolation, characterization and chromosome assignment of 341 newly isolated equine TKY microsatellite markers.
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Animals↗
Biomedical subjects
R P Oliveira
Publications and source records attributed to R P Oliveira.
A comparative histological evaluation of the biocompatibility of materials used in apical surgery.
AIM: To evaluate the biological properties of a variety of materials that could be used in apical surgery. METHODOLOGY: The intraosseous implant technique recommended by the FDI (1980) and ADA (1982) was used to test the following materials: zinc oxide-eugenol (ZOE), mineral trioxide aggregate (MTA), and Z-100 light-cured composite resin. Thirty guinea-pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks, received one implant on each side of the lower jaw symphysis. The connective tissue response alongside the lateral wall outside the cup served as a negative control for the technique. At the end of the observation periods, the animals were killed and the specimens prepared for routine histological examination to evaluate their biocompatibility. RESULTS: The reaction of the tissue to the materials diminished with time. The ZOE cement was highly toxic during the 4-week experimental period, but this profile changed significantly after 12 weeks, when it showed biocompatible characteristics. MTA and Z-100 showed biocompatibility in this test model at both time periods. CONCLUSIONS: MTA and Z-100 composite were biocompatible at 4 and 12 weeks in this experimental model.
Aluminum Compounds↗
Isolation and characterization of microsatellites from the tapeworm Echinococcus granulosus.
The Echinococcus granulosus genome was searched for microsatellites using 8 different repeated oligonucleotides as probes (GT15, CT15, AT15, CG15, CAT10, CAA10, CGG10 and CATA10). Southern blot experiments revealed that DNA regions containing GT, CAA, CATA and CT repeats are the most frequent in the E. granulosus genome. AT and CG probes showed no hybridization signal. Two loci containing CA/GT (Egmsca1 and Egmsca2) and 1 locus containing GA/CT (Egmsga1) repeats were cloned and sequenced. The locus Egmsca1 was analysed in 73 isolates from Brazil and Argentina whose strains were previously characterized. Brazilian isolates from cattle strain and Argentinean isolates from camel strain were monomorphic and shared the allele (CA)7. Argentinean isolates of sheep and Tasmanian sheep strains shared 2 alleles [(CA)8 and (CA)10] with Brazilian isolates of sheep strain. The allele (CA)11 was found only in Brazilian isolates of sheep strain at a low frequency. The Brazilian and the Argentinean sheep strain populations were tested for the Hardy-Weinberg equilibrium, and only the former was in agreement with the expectations. No polymorphism was found among individual protoscoleces from a single hydatid cyst, validating the utilization of pooled protoscoleces from 1 cyst, grouped as an isolate, in population studies. This work describes for the first time the isolation and characterization of microsatellites from E. granulosus.
Animals↗
Acute haemodynamic effects of a hypertonic saline/dextran solution in stable patients with severe sepsis.
OBJECTIVE: To study the haemodynamic effects of a hypertonic saline/dextran solution compared with a normal saline solution in patients with severe sepsis. DESIGN: Prospective double blind and control-randomised study. SETTING: Adult intensive care unit in a university hospital. PATIENTS: Twenty-nine patients with sepsis with a pulmonary artery occlusion pressure (PAOP) lower than 12 mmHg. INTERVENTIONS: Patients were randomised to receive 250 ml of blinded solutions of either normal saline (SS group, n=16) or hypertonic saline (NaCl 7.5%)/dextran 70 8% (HSS group, n=13) solutions. MEASUREMENTS AND RESULTS: Haemodynamic, blood gas, and sodium data were collected at the following time points: baseline, 30 min, 60 min, 120 min, and 180 min. PAOP was higher in the HSS group at 30 min (10.7+/-3.2 mmHg vs 6.8+/-3.2 mmHg) and 60 min (10.3+/-3 mmHg vs 7.4+/-2.9 mmHg); P<0.05. The cardiac index increased in the HSS group and it was greater than the SS group at 30 min (6.5+/-4.7 l min(-1) m(-2) vs 3.8+/-3.4 l min(-1) m(-2)), 60 min (4.9+/-4.5 l min(-1) m(-2) vs 3.7+/-3.3 l min(-1) m(-2)), and 120 min (5.0+/-4.3 l min(-1) m(-2) vs 4.1+/-3.4 l min(-1) m(-2)); P<0.05. The stroke volume index followed a comparable course and it was higher at 30 min [53.6(39.2-62.8) ml m(-2) vs 35.6(31.2-49.2) ml m(-2)] and 60 min [46.8(39.7-56.6) ml m(-2) vs 33.9(32.2-47.7) ml m(-2)]; P<0.05. Systemic vascular resistance decreased in the HSS group and became significantly lower at 30 min (824+/-277 dyne s(-1) cm(-5) m(-2) vs 1139+/-245 dyne s(-1) cm(-5) m(-2)), 60 min (921+/-256 dyne s(-1) cm(-5) m(-2) vs 1246+/-308 dyne s(-1) cm(-5) m(-2)), and 120 min (925+/-226 dyne s(-1) cm(-5) m(-2) vs 1269+/-494 dyne s(-1) cm(-5) m(-2)). Sodium levels increased in the HSS group (P=0.056) and were higher than in the SS group at 30 min (145+/-3 mEq l(-1)vs 137+/-7 mEq l(-1)), 60 min (143+/-4 mEq l(-1) vs 136+/-7 mEq l(-1)), 120 (142+/-5 mEq l(-1)vs 136+/-7 mEq l(-1)), and 180 min (142+/-5 mEq l(-1) vs 136+/-8 mEq l(-1)). CONCLUSION: Hypertonic saline/dextran solution may improve cardiovascular performance in severe sepsis without significant side effects. The haemodynamic effect appears related mainly to a volume effect.
Adult↗
Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi.
Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group (1/2). These results suggest different origins for these strains.
Animals↗
The population structure of Trypanosoma cruzi: expanded analysis of 54 strains using eight polymorphic CA-repeat microsatellites.
Recently we cloned and sequenced the first eight Trypanosoma cruzi polymorphic microsatellite loci and studied 31 clones and strains to obtain valuable information about the population structure of the parasite. We have now studied 23 further strains, increasing from 11 to 31 the number of strains obtained from patients with chronic Chagas disease. This expanded set of 54 strains and clones analyzed with the eight microsatellites markers confirmed the previously observed diploidy, clonal population organization and very high polymorphism of T. cruzi. Moreover, this new study disclosed two new features of the population genetic structure of T. cruzi. The first was the discovery that, similarly to what we had previously shown for strains isolated from insect vectors, mammals and humans with acute disease, isolates from patients in the chronic phase of Chagas disease could also be multiclonal, albeit at a reduced proportion. Second, when we used parsimony to display the genetic relationship among the clonal lineages in an unrooted Wagner network we observed, like before, a good correlation of the tree topography with the classification in three clusters on the basis of single locus analysis of the ribosomal RNA genes. However, a significant new finding was that now the strains belonging to cluster 2 split in two distant sub-clusters. This observation suggests that the evolutionary history of T. cruzi may be more complex than we previously thought.
Animals↗
Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue.
To investigate the role of nitric oxide in human sepsis, ten patients with severe septic shock requiring vasoactive drug therapy and mechanical ventilation were enrolled in a prospective, open, non-randomized clinical trial to study the acute effects of methylene blue, an inhibitor of guanylate cyclase. Hemodynamic and metabolic variables were measured before and 20, 40, 60, and 120 min after the start of a 1-h intravenous infusion of 4 mg/kg of methylene blue. Methylene blue administration caused a progressive increase in mean arterial pressure (60 [55-70] to 70 [65-100] mmHg, median [25-75th percentiles]; P<0.05), systemic vascular resistance index (649 [479-1084] to 1066 [585-1356] dyne s-1 cm-5 m-2; P<0.05) and the left ventricular stroke work index (35 [27-47] to 38 [32-56] g m-1 m-2; P<0.05) from baseline to 60 min. The pulmonary vascular resistance index increased from 150 [83-207] to 186 [121-367] dyne s-1 cm-5 m-2 after 20 min (P<0.05). Mixed venous saturation decreased from 65 [56-76] to 63 [55-69]% (P<0.05) after 60 min. The PaO2/FiO2 ratio decreased from 168 [131-215] to 132 [109-156] mmHg (P<0.05) after 40 min. Arterial lactate concentration decreased from 5.1 +/- 2.9 to 4.5 +/- 2.1 mmol/l, mean +/- SD (P<0.05) after 60 min. Heart rate, cardiac filling pressures, cardiac output, oxygen delivery and consumption did not change. Methylene blue administration was safe and no adverse effect was observed. In severe human septic shock, a short infusion of methylene blue increases systemic vascular resistance and may improve myocardial function. Although there was a reduction in blood lactate concentration, this was not explained by an improvement in tissue oxygenation, since overall oxygen availability did not change. However, there was a significant increase in pulmonary vascular tone and a deterioration in gas exchange. Further studies are needed to demonstrate if nitric oxide blockade with methylene blue can be safe for patients with septic shock and, particularly, if it has an effect on pulmonary function.
Adult↗
Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites.
We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.
Animals↗
Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.
Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.
Animals↗
An alternative approach to evaluating the intraspecific genetic variability of parasites.
Analysis of DNA polymorphisms provides important information for the molecular characterization of parasite strains and clones. Because we still know little about the genomes of parasites, such analysis has to rely on methods applicable to any eukaryotic genome, such as DNA fingerprinting with multilocal minisatellite probes and the polymerase chain reaction (PCR)-based random amplified polymorphic DNA technique (RAPD). However, DNA fingerprinting is cumbersome and needs large amounts of parasite DNA, and RAPD can exhibit low reproducibility and spurious bands, both of which appear to be related to the low stringency of the PCR procedure. Riva Oliveira, Andréa Macedo, Egler Chiari and Sérgio Pena here evaluate the applicability to parasites of a technique described two years ago called simple sequence repeat-anchored PCR amplification (SSR-PCR), in which a single primer is needed [the (CA)(8)RY primer] and highstringency conditions are applied.
Journal Article↗
Isolation and characterisation of mutants from the halotolerant yeast Pichia sorbitophila defective in H+/glycerol symport activity.
Pichia sorbitophila, a yeast species that is highly resistant to osmotic stress in general and to salt stress in particular, was subjected to a mutagenesis strategy in order to obtain mutants deficient in the glycerol active uptake previously described. Density centrifugation was used for enrichment of NaCl sensitive mutants in either glucose or glycerol media. Several phenotypic classes of mutants were identified, to which physiological tests were applied concerning the activity of the symporter, its accumulation capacity and the detection of the activity of glycerol pathway specific enzymes. From these, two mutant strains were selected, presenting a clearly deficient phenotype on H+/glycerol symport activity.
Biological Transport, Active↗
Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly from infected tissues.
We report here a polymerase chain reaction (PCR)-based DNA profiling technique that permits Trypanosoma cruzi strain characterization by direct study of infected tissues. This is based on application of a recently developed method of DNA fragment identification, called low-stringency single specific primer PCR (LSSP-PCR), to the study of the variable region of kinetoplast DNA (kDNA) minicircles from T. cruzi Thus, we can translate the intraspecific polymorphism in the nucleotide sequence of kDNA minicircles into a specific and highly reproducible kDNA signature. Comparison with the phenogram obtained by DNA fingerprinting analysis of a set of T. cruzi strains showed good qualitative correlation between the degree of divergence of the LSSP-PCR profiles and the genetic distance between the strains. kDNA signatures of heart tissue from acutely or chronically infected animals revealed perfect concordance with the patterns obtained from cultured parasites for the CL and Colombiana strains but not for the Y strain, which is known to be multiclonal. However, the match was perfect for studies with two clones of the Y strain. We take this as evidence that in some multiclonal strains there is heterogeneity among the clones in the degree of tropism for the heart tissue. Finally, we showed that it is possible to obtain a T. cruzi kDNA signature from the heart of a human patient with chronic Chagasic myocardiopathy. kDNA signatures obtained by LSSP-PCR of sequences amplified from infected tissues constitute a new tool to study the molecular epidemiology of Chagas' disease.
Animals↗
Hereditary multiple exostoses and cervical ventral protuberance causing dysphagia. A case report.
STUDY DESIGN: A case report of a patient with hereditary multiple exostosis and who presented with cervical ventral protuberance causing dysphagia. OBJECTIVES: To present this rare situation and to discuss the treatment and the result obtained. SUMMARY OF BACKGROUND DATA: We found in the literature only one case of exostosis of the cervical spine causing dysphagia. METHODS: The patient, a 16-year-old girl, was affected by hereditary multiple exostosis, as was her father. The diagnosis was confirmed by radiograph, computed tomography, and magnetic resonance imaging, which showed a tumor in the anterior arch of the atlas. The patient was submitted to a transoral approach, and the tumor was excised. RESULTS: The patient had a good evolution 2 years after the surgery without sign of recurrence. CONCLUSIONS: This was a very rare situation, and the result validated the treatment used.
Adolescent↗
Hemisection of the cervical spinal cord caused by a stab wound: MR findings.
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Adult↗
Biological parameters and molecular markers of clone CL Brener--the reference organism of the Trypanosoma cruzi genome project.
Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28 degrees C is 58 +/- 13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace's medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37 degrees C; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24S alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.
Animals↗
Atlanto-axial dislocation in children.
The authors present 14 cases of C1 C2 dislocations in children (8 boys and 6 girls; mean age, 6.9 years) seen at the Orthopedics and Traumatology Institute of the University Hospital, Faculty of Medicine, University of São Paulo from 1978 to 1990. In view of the disappointing results obtained by conservative treatment in 6 of the 14 patients, the authors recommend early atlanto-axial arthrodesis in cases of C1 C2 dislocation in children.
Atlanto-Axial Joint↗
[Traumatic disk hernia of the cervical spine].
The authors present 26 cases of traumatic disc herniation in the cervical spine submitted to anterior discectomy. All of the patient presented neurologic deficit, being 18 classified as Frankel A, three as Frankel B, 2 Frankel C and 3 Frankel D. There were observed three deaths in the immediate post-operative period due to respiratory insufficiency in patients classified as Frankel A. The other 23 cases have a follow-up of 14 months (6-84 months), presenting improvement of the neurologic deficit in six patients classified as Frankel B, C and D and only one as Frankel A. The authors emphasize the importance of the image methods in the diagnosis of this lesion.
Cervical Vertebrae↗
[Femoral fracture in patients with spinal cord injury].
Femoral fractures during the physical therapy in two patients with spinal cord injury and consequent osteoporosis in the paralyzed limbs are reported. The fractures were caused by a minor trauma and the spasticity was considered to be an additional factor in the accident. During the physical therapy the patients were seated with hip abduction and flexion of the knee. It is important that the patients participate in a program of physiotherapy. This, however, should be performed under strict control.
Adolescent↗