Functional deficiencies of sulfite oxidase: Differential diagnoses in neonates presenting with intractable seizures and cystic encephalomalacia

Sulfite oxidase is a mitochondrial enzyme encoded by the SUOX gene and essential for the detoxification of sulfite which results mainly from the catabolism of sulfur-containing amino acids. Decreased activity of this enzyme can either be due to mutations in the SUOX gene or secondary to defects in the synthesis of its cofactor, the molybdenum cofactor. Defects in the synthesis of the molybdenum cofactor are caused by mutations in one of the genes MOCS1, MOCS2, MOCS3 and GEPH and result in combined deficiencies of the enzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Although present in many ethnic groups, isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are rare inborn errors of metabolism, which makes awareness of key clinical and laboratory features of affected individuals crucial for early diagnosis. We report clinical, radiologic, biochemical and genetic data on a Brazilian and on a Turkish child with sulfite oxidase deficiency due to the isolated defect and impaired synthesis of the molybdenum cofactor, respectively. Both patients presented with early onset seizures and neurological deterioration. They showed no sulfite oxidase activity in fibroblasts and were homozygous for the mutations c.1136A>G in the SUOX gene and c.667insCGA in the MOCS1 gene, respectively. Widely available routine laboratory tests such as assessment of total homocysteine and uric acid are indicated in children with a clinical presentation resembling that of hypoxic ischemic encephalopathy and may help in obtaining a tentative diagnosis locally, which requires confirmation by specialized laboratories. (C) 2009 Published by Elsevier B.V.

Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer`s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia

Background: The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self-renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets. Methods: The relative expression of the drug-efflux pumps P-gp, MRP, LRP, and BCRP was evaluated by mean fluorescence index (MFI) and the Kolmogorov-Smirnov analysis (D values) in five leukemic subpopulations: CD34(+)CD38(-)CD123(+) (LSCs), CD34(+)CD38(+)CD123(-), CD34(+)CD38(+)CD123(+), CD34(+)CD38(+)CD123(-), and CD34(-) mature cells in 26 bone marrow samples of CD34(+) AML cases. Results: The comparison between the two more immature subsets (LSC versus CD34(+)CD38(-)CD123(-) cells) revealed a higher P-gp, MRP, and LRP expression in LSCs. The comparative analysis between LSCs and subsets of intermediate maturation (CD34(+)CD38(+)) demonstrated the higher BCRP expression in the LSCs. In addition, P-gp expression was also significantly higher in the LSC compared to CD34(+)CD38(+)CD123(-) subpopulation. Finally, the comparative analysis between LSC and the most mature subset (CD34(-)) revealed higher MRP and LRP and lower P-gp expression in the LSCs. Conclusions: Considering the cellular heterogeneity of AML, the higher MDR transporters expression at the most immature, self-renewable, and quiescent LSC population reinforces that MDR is one of the mechanisms responsible for treatment failure. (C) 2008 Clinical Cytometry Society.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Analysis of the Ontogenetic Variation in the Venom Proteome/Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Infection by Spotted Fever Rickettsiae in People, Dogs, Horses and Ticks in Londrina, Parana State, Brazil

Spotted fever is a disease caused by bacteria from the genus Rickettsia of the spotted fever group (SFG). Rickettsia rickettsii is likely the main agent of Brazilian spotted fever (BSF). With the objective of gathering information on the circulation of SFG rickettsiae in Londrina, Parana state, ticks from dogs and horses and also blood from dogs, horses and humans were collected in a neighbourhood of the city which presented potential for circulation of rickettsiae between hosts and vectors. Amblyomma cajennense, Dermacentor nitens, and Rhipicephalus sanguineus ticks were subjected to Polymerase Chain Reaction targeting a fragment of the Rickettsia gltA gene. This specific gene encodes the enzyme citrate synthase of Rickettsia spp., and results on all ticks were negative. Human and animal sera were tested by Indirect Immunofluorescence Assay in which R. rickettsii and R. parkeri were used as antigens. Sera from 4.7% human, 2.7% canine and 38.5% equine were positive for R. rickettsii. For R. parkeri, 0.9% human, 2.7% canine and 11.5% equine samples were positive. All samples reactive to R. parkeri also reacted to R. rickettsii. An epidemiological questionnaire was applied, but there were no statistically significant results. Comparison of our serological results with previous studies in Brazil, among BSF endemic and non-endemic areas, indicates that there is no established rickettsial infection in the study area, a statement corroborated with our molecular analysis. Nonetheless, as humans of the present study are highly exposed to tick infestations, health education within the population is needed to obtain efficient tick control. Zoonoses and Public Health 416 (C) 2011 Blackwell Verlag GmbH . Zoonoses Public Health. 58 (2011) 416-423

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

Preventive Effect of Commercial Desensitizing Toothpastes on Bovine Enamel Erosion in vitro

This study evaluated in vitro commercial desensitizing toothpastes with respect to the prevention of erosion and explored the effect of their agents alone or in combination with fluoride. Bovine enamel blocks were randomly allocated to five groups of 20 and exposed to: Sensodyne ProNamel (1,425 ppm F as NaF, 5% KNO(3)), Sensodyne Original (no fluoride, 10% SrCl(2)), Colgate Sensitive (1,450 ppm F as sodium monofluorophosphate, 5% K citrate), Crest (fluoride-only toothpaste, 1,100 ppm F as NaF) and water (negative control). A second experiment was conducted with experimental dentifrices containing fluoride (NaF, 1,100 ppm F), 10% SrCl(2), 5% KNO(3) or 5% K citrate alone or the latter three combined with F. The samples were submitted to four cycles, alternating demineralization (cola, 10 min) and remineralization (artificial saliva, 1 h). Before and between cyclic de- and remineralization, blocks were treated with slurries of the respective toothpastes or water (1 min). Erosive tissue loss was analyzed by profilometry. Data were analyzed by Kruskal-Wallis and Dunn`s tests (p < 0.05). The mean erosion depth (+/- SE, mu m) was significantly less for Colgate Sensitive (0.04 +/- 0.00), Sensodyne Original (0.06 +/- 0.01) and Crest (0.07 +/- 0.01) than for Sensodyne ProNamel (2.36 +/- 0.25) or water (2.92 +/- 0.24), which did not significantly differ from each other. Both F and the desensitizing agents alone reduced erosion, but no additive effect was found. In addition, the combination of F and KNO(3) did not reduce erosion. These in vitro results suggest that the presence of fluoride or desensitizing substances in toothpastes, alone or in combination, can reduce erosion of enamel, but this is not valid for all the formulations. Copyright (C) 2010 S. Karger AG, Basel

In Vitro Proliferation and Osteoblastic Phenotype Expression of Cells Derived From Human Vertebral Lamina and Iliac Crest

Study Design. Osteoblastic cells derived from vertebral lamina and iliac crest were isolated and cultured under the same conditions (osteogenic medium, pH, temperature, and CO(2) levels). Objective. To compare proliferation and expression of osteoblastic phenotype of cells derived from vertebral lamina and iliac grafting. Summary of Background Data. Many factors play a role in the success of bone graft in spinal fusion including osteoblastic cell population. Two common sources of graft are vertebral lamina and iliac crest, however, differences in proliferation and osteoblastic phenotype expression between cells from these sites have not been investigated. Methods. Cells obtained from cancellous bone of both vertebral lamina and iliac crest were cultured and proliferation was evaluated by direct cell counting and viability detected by Trypan blue. Alkaline phosphatase (ALP) activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red S. Gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, osteoprotegerin, and receptor activator of NF-kB ligand was analyzed by real-time PCR. All comparisons were donor-matched. Results. Proliferation was greater at days 7 and 10 in cells from vertebral lamina compared with ones from iliac crest without difference in cell viability. ALP activity was higher in cells from vertebral lamina compared with cells from iliac crest at days 7 and 10. At 21 days, mineralized matrix was higher in cells derived from vertebral lamina than from iliac crest. At day 7, gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, receptor activator of NF-kB ligand, and osteoprotegerin was higher in cells derived from vertebral lamina compared with iliac crest. Conclusion. Cell proliferation and osteoblastic phenotype development in cells derived from cancellous bone were more exuberant in cultures of vertebral lamina than of iliac crest.

Atomic Absorption Spectrometry and Scanning Electron Microscopy Evaluation of Concentration of Calcium Ions and Smear Layer Removal With Root Canal Chelators

Aim. The aim of this study was to evaluate the concentration of calcium ions and smear layer removal by using root canal chelators according to flame atomic absorption spectrophotometry and scanning electron microscopy. Forty-two human maxillary central incisors were irrigated with 15% ethylenediaminetetraacetic acid (EDTA), 10% citric acid, 10% sodium citrate, apple vinegar, 5% acetic acid, 5% malic acid, and sodium hypochlorite. The concentration of calcium ions was measured by using flame atomic absorption spectrometry, and smear layer removal was determined by scanning electron microscopy. Mean +/- standard deviation, one-way analysis of variance, Tukey-Kramer, Kruskal-Wallis, Dunn, and kappa tests were used for statistical analysis. The use of 15% EDTA resulted in the greatest concentration of calcium ions followed by 10% citric acid; 15% EDTA and 10% citric acid were the most efficient solutions for removal of smear layer. (J Endod 2009;35:727-730)

Effect of Chelating Solutions on the Microhardness of Root Canal Lumen Dentin

Introduction: The greatest reduction in microhardness of the most superficial layer of dentin of the root canal lumen is desired. The use of chelating agents during biomechanical preparation of root canals removes smear layer, increasing the access of the irrigant into the dentin tubules to allow adequate disinfection, and also reduces dentin microhardness, facilitating the action of endodontic instruments. This study evaluated the effect of different chelating solutions on the microhardness of the most superficial dentin layer from the root canal lumen. Methods: Thirty-five recently extracted single-rooted maxillary central incisors were instrumented, and the roots were longitudinally sectioned in a mesiodistal direction to expose the entire canal extension. The specimens were distributed in seven groups according to the final irrigation: 15% EDTA, 10% citric acid, 5% malic acid, 5% acetic acid, apple vinegar, 10% sodium citrate, and control (no irrigation). A standardized volume of 50 mu L of each chelating solution was used for 5 minutes. Dentin microhardness was measured with a Knoop indenter under a 10-g load and a 15-second dwell time. Data were analyzed statistically by one-way analysis of variance and Tukey-Kramer multiple-comparison test at 5% significance level. Results: EDTA and citric acid had the greatest overall effect, causing a sharp decrease in dentin microhardness without a significant difference (p > .05) from each other. However, both chelators differed significantly from the other solutions (p < .001). Sodium citrate and deionized water were similar to each other (p > .05) and did not affect dentin microhardness. Apple vinegar, acetic acid, and malic acid were similar to each other (p > .05) and presented intermediate results. Conclusion: Except for sodium citrate, all tested chelating solutions reduced microhardness of the most superficial root canal dentin layer. EDTA and citric acid were the most efficient. (J Endod 2011;37:358-362)