Effects of neonatal handling on central noradrenergic and nitric oxidergic systems and reproductive parameters in female rats

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NO x), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats. Copyright (c) 2007 S. Karger AG, Basel.

Lead Deposition in Bovine Enamel during a pH-Cycling Regimen Simulating the Caries Process

Like fluoride, lead (Pb) accumulates on the enamel surface pre-eruptively, but it is not yet known whether it also deposits on enamel while dental caries is developing. This study evaluates Pb distribution in bovine enamel slabs submitted to a pH-cycling regimen simulating the caries process. The slabs were subjected to 8 cycles of de- and remineralizing conditions, and Pb (as acetate salt) was added to the de- and remineralized solutions at concentrations of 30 mu g/l (experimental group, E1) and 300 mu g/l (experimental group, E2). The control group (C) consisted of solutions to which Pb was not added. After the pH cycling, 100-mu m sections of the slabs were analyzed by polarizing microscopy, to observe the extent of caries-like lesions, and these sections were used for Pb estimation by Synchrotron radiation X-ray microfluorescence. Caries lesions were observed along all superficial enamel surfaces to an extent of 120 mu m. A Pb concentration gradient was observed in enamel, which decreased toward dentine. The highest Pb signals were observed for group E2, and the differences were statistically significant at enamel depths of 0 (C vs. E2; p = 0.029) and 50 mu m (C vs. E2 and E1 vs. E2; p = 0.029). In conclusion, this study suggests that if Pb is present in the oral environment, it may deposit in enamel during the caries process. Copyright (C) 2011 S. Karger AG, Basel

Evaluation of the Frequency of Micronuclei in Exfoliated Cells from Oral Lesions Previously Identified by Toluidine Blue

Objective: Patients using a removable prosthesis are susceptible to a variety of oral lesions that may progress to cancer. Toluidine blue (TB) staining is used to identify premalignant lesions, but the results are still controversial. Since micronuclei (MN) are a biomarker of genetic instability, the objective of this study was to determine the frequency of MN in white lesions of the oral mucosa and to compare the results with those of the TB test. Study Design: The study included 20 removable prosthesis users with white lesions that were previously classified as toluidine positive or negative. The frequency of MN was evaluated in exfoliated cells from lesions and normal mucosa. Nuclear anomalies were also registered. Results: A significant increase (p < 0.05) in the frequency of MN was observed in exfoliated cells from lesions compared to normal mucosal cells, and no relationship was seen with TB staining. Lifestyle factors or gender did not influence the results. Conclusions: The frequency of MN is a sensitive biomarker and can be used to predict genomic instability in white oral lesions. The MN assay may serve as a good parameter in the battery of tests used to identify high-risk individuals, contributing to the identification of the biological conditions of oral lesions. Copyright (C) 2011 S. Karger AG, Basel

Mesenchymal Stem Cells and Bovine Bone Mineral in Sinus Lift Procedures-An Experimental Study in Sheep

Introduction: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. Purpose: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. Material and Methods: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. Results: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. Conclusion: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Prevalence of Vitamin D Insufficiency in Brazilian Adolescents

Background/Aims: Cutaneous sun exposure and dietary vitamin D intake are important determinants of vitamin D status. The objective of the present study was to evaluate the vitamin D status of a group of healthy adolescent students living in Brazil. Methods: One hundred and thirty-six adolescents, 64 boys and 72 girls, aged 16-20 years old, living in a rural town in the state of Sao Paulo, Brazil, participated in this study. Results: The mean dietary vitamin D intake was 140 (120-156) IU/day [3.5 (3.0-3.9) mu g/day]. Only 14.9% of the students met the daily adequate intake recommendation of vitamin D. Only 27.9% practice physical activity outdoors and 17.6% of the adolescents apply sunscreen daily. The mean 25(OH)D concentration was 73.0 (22.0) nmol/l [29.2 (8.8) ng/ml]. Vitamin D insufficiency was observed in 60% of adolescents. Conclusions: The present study suggests that even in a sunny climate like Brazil the prevalence of vitamin D insufficiency in adolescents is high. Most likely this is due to low intakes of vitamin D in this group. Due to the limited extent of natural dietary sources of vitamin D, a policy of vitamin D food fortification should be considered in the future, and in the meantime greater use of vitamin D supplements in this population group should be encouraged to provide the increased amounts of this essential nutrient for optimal health. Copyright (C) 2009 S. Karger AG, Basel

Constitutional Haploinsufficiency of Tumor Suppressor Genes in Mentally Retarded Patients With Microdeletions in 17p13.1

Chromosome microdeletions or duplications are detected in 10-20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of similar to 180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (similar to 1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer. Copyright (C) 2009 S. Karger AG, Basel

Non-homologous sex chromosomes in two species of the genus Eigenmannia (Teleostei : Gymnotiformes)

The Neotropical genus Eigenmannia is a fish group with unknown species diversity where representatives possess a broad range of chromosomal sex determining systems namely XY/XX, X1X2Y/X1X1X2X2, ZZ/ZW as well as homomorphic sex chromosomes. To test the homology of two heteromorphic XY sex chromosome systems present in two sympatric populations, reciprocal cross-species FISH experiments were performed using probes derived by microdissection of X and Y chromosomes present in analyzed specimens of Eigenmannia virescens and Eigenmannia sp.2, respectively. While X and Y paint probes hybridized to species-specific sex chromosomes, in reciprocal cross-FISH both probes hybridized exclusively to autosomes. The result suggests multiple independent origins of the XY systems in the analyzed populations. Copyright (C) 2008 S. Karger AG, Basel.

Comparative Cytogenetics and Molecular Phylogeography in the Group Astyanax altiparanae - Astyanax aff. bimaculatus (Teleostei, Characidae)

The genus Astyanax comprises small characin fish of the neotropical region. The so-called `yellow-tailed characins` compose one of the most widely distributed Astyanax groups. A. altiparanae and A. aff. bimaculatus, are evolutionarily closely related and commonly found in several Brazilian hydrographic basins. In the present work, chromosomal data of specimens of A. altiparanae and A. aff. bimaculatus from 4 hydrographic basins in the states of Sao Paulo (Upper Tiete, Paranapanema, Ribeira de Iguape) and Rio de Janeiro (Guapimirim) are shown. All the populations showed 50 chromosomes, with different karyotypic formula. Although only a single Ag-NOR bearing chromosome pair was observed, all populations possess multiple cistrons of 18S rDNA. FISH with the 5S rDNA probe showed single signals at the interstitial position of one metacentric chromosome pair. C-bands are distributed in the terminal and interstitial regions of several chromosomes. However, the As-51 satDNA are frugally located in a few chromosomes of fishes from Upper Tiete, Paranapanema and Guapimirim Rivers, being absent in individuals of A. aff. bimaculatus from Ribeira de Iguape River basin. Beside these 4 populations, molecular phylogeography studies were also performed in individuals from Middle and Lower Tiete River basin and from 2 additional collection sites in the Paranapanema and Ribeira de Iguape River basins. The phylogeographic analysis using 2 mtDNA regions (totalizing 1.314 bp of ND2 and ATPase6/8 genes) of 8 populations of the group of `yellow-tailed characins` from 3 major hydrographic basins showed structuring of populations, suggesting a correlation between chromosomal (nuclear) and molecular (mitochondrial) data. Copyright (C) 2011 S. Karger AG, Basel

The Y Chromosome of the Atelidae Family (Platyrrhini): Study by Chromosome Microdissection

In order to study the intergeneric variability of the Y chromosome, we describe the hybridization of the Y chromosome of Brachyteles arachnoides, obtained by microdissection, to metaphases of Ateles belzebuth marginatus, Lagothrix lagothricha, and Alouatta male specimens. Brachyteles arachnoides (Atelinae) has 62 chromosomes and a very small Y chromosome. Our results showed that the Brachyteles arachnoides Y chromosome probe hybridized to Lagothrix lagothricha metaphases yielding one hybridization signal on only the tiny Y chromosome, and when hybridized with Ateles belzebuth marginatus metaphases it yielded one hybridization signal on two thirds of the small acrocentric Y chromosome. However, no hybridization signal was observed in Alouatta metaphases (subfamily Alouattinae), a closely related genus in the Atelidae family. Furthermore, our data support a close phylogenetic relationship among Brachyteles, Ateles, and Lagothrix and their placement in the Atelinae subfamily, but exclude Alouatta from this group indicating its placement as basal to this group. Copyright (C) 2009 S. Karger AG, Basel

Chromosomal Evolution in the Brazilian Geckos of the Genus Gymnodactylus (Squamata, Phyllodactylidae) from the Biomes of Cerrado, Caatinga and Atlantic Rain Forest: Evidence of Robertsonian Fusion Events and Supernumerary Chromosomes

Chromosomes of the South American geckos Gymnodactylus amarali and G. geckoides from open and dry areas of the Cerrado and Caatinga biomes in Brazil, respectively, were studied for the first time, after conventional and AgNOR staining, CBG- and RBG-banding, and FISH with telomeric sequences. Comparative analyses between the karyotypes of open areas and the previously studied Atlantic forest species G. darwinii were also performed. The chromosomal polymorphisms detected in populations of G. amarali from the states of Goias and Tocantins is the result of centric fusions (2n = 38, 39 and 40), suggesting a differentiation from a 2n = 40 ancestral karyotype and the presence of supernumerary chromosomes. The CBG- and RBG-banding patterns of the Bs are described. G. geckoides has 40 chromosomes with gradually decreasing sizes, but it is distinct from the 2n = 40 karyotypes of G. amarali and G. darwinii due to occurrence of pericentric inversions or centromere repositioning. NOR location seems to be a marker for Gymnodactylus, as G. amarali and G. geckoides share a medium-sized subtelocentric NOR-bearing pair, while G. darwinii has NORs at the secondary constriction of the long arm of pair 1. The comparative analyses indicate a non-random nature of the Robertsonian rearrangements in the genus Gymnodactylus. Copyright (C) 2010 S. Karger AG, Basel

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 2-Chromosomal location of a satellite DNA

Studies about composition of repetitive sequences and their chromosomal location have been helpful to evolutionary studies in many distinct organisms. In order to keep on assessing the possible relationships among different cytotypes of Astyanax fasciatus (Teleostei, Characiformes) in the Mogi-Guacu River (Sao Paulo State, Brazil), C-banding, chromomycin A 3 staining, and fluorescent in situ hybridization with a repetitive DNA sequence (As51) isolated from Astyanax scabripinnis were performed in the present work. The constitutive heterochromatin was distributed in terminal regions on long arms of submetacentric, subtelocentric, and acrocentric chromosomes and in the terminal region on short arms of a pair of submetacentric chromosomes in both standard cytotypes. This latter heterochromatic site was also GC-rich, as revealed by chromomycin A(3) staining, corresponding to the nucleolar organizer region (NOR), as shown by previous studies. The sites of the satellite As51 DNA were located in terminal regions on long arms of several chromosomes. Some variant karyotypic forms, which diverge from the two standard cytotypes, also presented distinctive chromosomes carrying As51 satellite DNA. It is possible that the standard 2n = 46 cytotype represents an invader population in the Mogi-Guacu River able to interbreed with the resident standard 2n = 48 cytotype. Therefore, the variant karyotypes would be related to a possible viable offspring, where complementary chromosomal rearrangements could favor new locations of the satellite DNA analyzed. Copyright (C) 2008 S. Karger AG, Basel

The Antiapoptotic Effect of Granulocyte Colony-stimulating Factor Reduces Infarct Size and Prevents Heart Failure Development in Rats

Background/Aim. Granulocyte colony-stimulating factor (G-CSF) reduces myocardial injury and improves cardiac function after myocardial infarction (MI). We investigated the early alterations provided by G-CSF and the chronic repercussions in infarcted rats. Methods. Male Wistar rats (200-250g) received vehicle (MI) or G-CSF (MI-GCSF) (50 mu g/kg, sc) at 7, 3 and 1 days before MI surgery. Afterwards MI was produced and infarct size was measured 1 and 15 days after surgery. Expression of anti-and proapoptotic proteins was evaluated immediately before surgery. 24 hours after surgery, apoptotic nuclei were evaluated. Two weeks after MI, left ventricular (LV) function was evaluated, followed by in situ LV diastolic pressure-volume evaluation. Results. Infarct size was decreased by 1 day pretreatment before occlusion (36 +/- 2.8 vs. 44 +/- 2.1% in MI; P<0.05) and remained reduced at 15 days after infarction (28 +/- 2.2 vs. 36 +/- 1.4% in MI; P<0.05). G-CSF pretreatment increased Bcl-2 and Bcl-xL protein expression, but did not alter Bax in LV. Apoptotic nuclei were reduced by treatment (Sham: 0.46 +/- 0.42, MI: 15.5 +/- 2.43, MI-GCSF: 5.34 +/- 3.34%; P<0.05). Fifteen days after MI, cardiac function remained preserved in G-CSF pretreated rats. The LV dilation was reduced in MI-G-CSF group as compared to MI rats, being closely associated with infarct size. Conclusion. The early beneficial effects of G-CSF were essentials to preserve cardiac function at a chronic stage of myocardial infarction. Copyright (C) 2011 S. Karger AG, Basel

Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries

Background/Aims: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. Methods: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 mu g.day(-1)) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-alpha, and IL-6 protein expression were investigated. Results: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-alpha protein expression increased in MRA in this group. Conclusion: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro-inflammatory cytokines and reduced NO bioavailability. Copyright (C) 2011 S. Karger AG, Basel

Regulation of Na(+)/H(+) Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca(2+)/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA(1K3R/4E) and SB(1K3R/4E). Wild type and mutants were transfected into PS120 cells and their activity was examined by H(+) flux (J(H+)). The basal J(H+) of wild type was 4.71 +/- 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10(-12) and 10(-9) M) increased the J(H+) in wild type and SB. Ang II (10(-6) M) increased this parameter only in SA. Ang II (10(-9) M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10(-6) M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10(-7) M), a calcium chelator, suppressed the effect of Ang II (10(-9) M) in wild type. With Ang II (10(-6) M), Bapta failed to affect wild type or SA, but it increased the J(H+) in SB. W13 or calmidazolium chloride (10(-5) M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10(-9) M) in wild type or SB. With Ang II (10(-6) M), W13 or calmidazolium chloride decreased the J(H+) in wild type or SA and increased it in SB. Thus, with Ang II (10(-12) and 10(-9) M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10(-6) M), site B is important to maintain its basal activity. Copyright (C) 2010 S. Karger AG, Basel