Influence of Different Dye Substances on the Effectiveness of Bleaching and H2O2 Diffusion.

This study aimed to evaluate the influence of different dye substances on the effectiveness of bleaching and hydrogen peroxide diffusion (HO). From 300 central bovine incisors, 160 enamel/dentin disks with similar E* values were selected. The specimens were distributed according to the pigment treatment. Aiming to standardize the chromatic change provided by the different pigments, the specimens from each group remained immersed in the pigment solutions for different times (32 specimens per group): DW - distilled water (Control group); BT - black tea; CO - coffee; SD - cola-based soft drink; and RW - red wine. After pigmentation and chromatic change value analysis, only 10 specimens from each group (n=10) were selected, so the chromatic alteration of all groups was similar (ΔE=8.36±0.5). The samples were subjected to bleaching treatment and diffused peroxide was quantified in a visible ultraviolet light spectrophotometer. Two more bleaching sessions were conducted to evaluate ΔE and the Whiteness Index for Dentistry (ΔWID). Concurrently, solutions were prepared with dye agents, and the same ΔE value was obtained in the teeth (ΔE=8.49±0.5). The solutions received a standardized amount of H2O2, being analyzed by a visible ultraviolet light spectrophotometer. Data analysis comprised variance and Tukey's tests (α=0.05). Higher H2O2 diffusion was observed in pigmented groups when compared with DW (p<0.05). The CO and RW groups had the highest ΔE values (p>0.05), meaning greater difficulty in responding to treatment. In relation to ΔWID, RW bleached less than the other groups after the third bleaching session (p<0.05), resembling only the SD group (p=0.467). However, 21 days after ending the bleaching treatment, only RW and CO had the lowest values (p=0.481). Analysis of the solutions revealed that only RW was altered by the peroxide (p<0.05). In conclusion, teeth pigmented with coffee and, mainly, red wine were more resistant to bleaching treatment, although all pigmentations favored increases in transenamel and transdentinal H2O2 penetration.

Does the Bleaching Gel Application Site Interfere With the Whitening Result? A Randomized Clinical Trial.

This study aimed to evaluate the effect of the bleaching gel application site on chromatic changes and postoperative sensitivity in teeth. Thirty patients were selected and allocated to three groups (n=10 per group), according to the location of the gel: GI, cervical application; GII, incisal application; and GIII, total facial. The amount and time of application of the 35% hydrogen peroxide (H2O2) gel were standardized. Color changes were analyzed by ΔE and Wid (bleaching index), using the values obtained in the readings conducted on a digital spectrophotometer in the cervical (CRs) and incisal regions (IRs) of the teeth. Spontaneous sensitivity was assessed using the questionnaire, and the stimulated sensitivity caused by the thermosensory analysis (TSA). The analysis occurred in five stages: baseline, after the first, second, and third whitening sessions (S), and 14 days after the end of the whitening, using the linear regression statistical model with mixed effects and post-test by orthogonal contrasts (p<0.05). Although the IR was momentarily favored, at the end of the treatment, the restriction of the application site provided results similar to those obtained when the gel was applied over the entire facial surface. Regarding sensitivity, only the GI showed spontaneous sensitivity. In the TSA, GIII had less influence on the threshold of the thermal sensation. It was concluded that the chromatic alteration does not depend on the gel application site. Spontaneous sensitivity is greater when the gel is concentrated in the cervical region (CR), and the teeth remain sensitized by thermal stimuli even after 14 days.

Descrição anatômica da artéria carótida externa e seus ramos na preguiça-comum / Anatomical description of external carotid artery and its branches in sloth

Bradypus variegatus, espécie pertencente à família Bradypodidae e à superordem Xenarthra, pode ser considerada modelo biológico de caráter multidisciplinar. Assim, realizou-se um trabalho de descrição anatômica da artéria carótida externa (ACE) e dos seus ramos no bicho-preguiça B. variegatus. Utilizaram-se 10 animais adultos, sendo todos fêmeas, que foram submetidos à dissecação, constatando-se que a artéria (a.) carótida comum se bifurca, em externa e interna, no nível do primeiro anel traqueal. A ACE, então, segue estendendo-se até a maxila, onde emite ramos para a região temporal e para o polo posterior do olho. Em todos os animais estudados, foram observados sete ramos principais da ACE, que, segundo a sua origem e localização, foram denominados como a. auricular, a. lingual, a. facial, a. alveolar, a. inferior, a. temporal, a. maxilar e a. oftálmica. Os ramos maxilar e oftálmico correspondem aos terminais e os demais são ramos colaterais. Em 50% dos animais analisados, foi verificada a presença de anastomoses arteriais e 40% deles apresentaram o acréscimo de um ramo aos principais. Desses, 30% demonstraram a presença de um ramo traqueal e 10% de um ramo sublingual, sendo esses ramos colaterais.(AU)

Bradypus variegatus is a species belonging to the family Bradypodidae and superorder Xenarthra, which should be considered as a multidisciplinary biological model. Thus, an anatomical description of the external carotid artery (ACE) and its branches in sloth B. variegatus was studied. Ten adult animals, all of them female, were submitted to dissection, and it was observed that the common carotid artery (a.) bifurcates in external and internal at the level of the first tracheal ring. Then, ACE extends through the maxilla where it launches branches to the temporal region and posterior eye side. For all sampled animals, seven principal branches of ACE were observed, and according to their origin and location were denominated as auricular, lingual, facial, bottom alveolar, temporal, maxillary and ophthalmic arteries. The maxillary and ophthalmic branches correspond to the terminals and the other branches are collateral. Presence of arterial anastomoses was observed in 50% of the sampled animals and 40% of them had increase of a branch on the principal. In these, 30% had presence of one tracheal branch and 10% of a sublingual branch, considering these branches as collateral.(AU)

Radiologist participation in multi-disciplinary teams in breast cancer improves reflective practice, decision making and isolation.

This study aims to explore Australian radiologists' experiences of participating in breast cancer multi-disciplinary team (MDT) meetings to identify enablers and barriers to participation as well their perception of confidence and patient care. Qualitative methods incorporating observation and interviews were used. Twenty-one breast cancer MDT meetings were observed across Sydney to study the dynamics of the meetings, the level of participation by radiologists and their most important interactions. Qualitative semi-structured interviews were conducted with 10 radiologists participating in these meetings regarding participation, educational opportunities and improvements to work practices. Radiologists' participation in breast cancer MDT meetings is influenced by the type of meeting they attend with higher levels of participation and a more dominant 'valued' role being evident in pre-interventional meetings. The key themes to emerge from the data include the importance of 'sharing experiences', the 'radiologist-pathologist relationship' and the value of 'continuing participation'. Radiologists believed their confidence in their clinical decision making increased when there was immediate feedback from pathologists. This study highlights the benefits of radiologists regularly participating in breast cancer MDT meetings in terms of continuing professional education resulting from collegial experiential learning. Radiologists' perceived patient care and workplace isolation were improved by sharing experiences with other cancer care colleagues.

Phenotypic integration in flowers of neotropical lianas: diversification of form with stasis of underlying patterns.

Phenotypic integration is essential to the understanding of organismal evolution as a whole. In this study, a phylogenetic framework is used to assess phenotypic integration among the floral parts of a group of Neotropical lianas. Flowers consist of plant reproductive organs (carpels and stamens), usually surrounded by attractive whorls (petals and sepals). Thus, flower parts might be involved in different functions and developmental constraints, leading to conflicting selective forces. We found that Bignonieae flowers have very similar patterns of variance/covariance among traits and that such patterns are uncorrelated with the phylogenetic relationships between species. However, in spite of pattern stasis, our results also indicate that diversification of floral morphology in this group has occurred throughout the evolution of magnitudes of correlation among traits. Thus, we suggest that stabilizing selection has played an important role in phenotypic integration, resulting in the long-term stasis of covariance patterns underlying flower diversification during the ca. 50 Myr of evolution of Bignonieae. This is the first report of long-term stasis in the phenotypic integration of angiosperms, suggesting that patterns of floral morphology can be recognizable as specific attributes of distinct botanical families.

[Impact of quality improvement process upon the state of nutritional support in a critical care unit]. / Impacto de un proceso de mejora de la calidad en el estado del soporte nutricional en una unidad de cuidados intensivos.

INTRODUCTION: In a preceding article the state of Nutritional support (NS) in an Intensive Care Unit (ICU) was documented [Martinuzzi A et al. Estado del soporte nutricional en una unidad de Cuidados críticos. RNC 2011; 20: 5-17]. In this follow-up work we set to assess the impact of several organizational, recording and educational interventions upon the current state of NS processes. MATERIALS AND METHODS: Interventions comprised presentation of the results of the audit conducted at the ICU before the institution's medical as well as paramedical personnel; their publication in a periodical, peer-reviewed journal; drafting and implementation of a protocol regulating NS schemes to be carried out at the ICU; and conduction of continuous education activities on Nutrition (such as "experts talks", interactive courses, and training in the implementation of the NS protocol). The state of NS processes documented after the interventions was compared with the results annotated in the preceding article. Study observation window ran between March the 1st, 2011 and May 31th, 2011, both included. RESULTS: Study series differed only regarding overall-mortality: Phase 1: 40.0% vs. Phase 2: 20.5%; Difference: 19.5%; Z = 1.927; two-tailed-p = 0.054. Interventions resulted in a higher fulfillment rate of the prescribed NS indication; an increase in the number of patients receiving ≥ 80% of prescribed energy; and a reduction in the number of NS lost days. Mortality was (numerically) lower in patients in which the prescribed NS scheme was fulfilled, NS was early initiated, and whom received ≥ 80% of prescribed energy. Adopted interventions had no effect upon average energy intakes: Phase 1: 574.7 ± 395.3 kcal/24 h⁻¹ vs. Phase 2: 591.1 ± 315.3 kcal/24 h⁻¹; two-tailed-p > 0.05. CONCLUSIONS: Educational, recording and organizational interventions might result in a better conduction of NS processes, and thus, in a lower mortality. Hemodynamic instability is still the most formidable obstacle for initiating and completing NS.

Weight loss induced by 6-month lifestyle intervention improves early endothelial activation and fibrinolysis in obese adolescents.

BACKGROUND: Adolescent obesity is associated with an increased risk of adult obesity and subsequent cardiovascular diseases. The present study aimed to assess the effect of weight loss after 6-month lifestyle intervention in obese adolescents on biomarkers of endothelial activation and fibrinolytic system. METHODS: Eighty-five obese adolescents aged 10 to 16 years were assigned to a 6-month lifestyle intervention and 61 completed the programme. We examined the effect of the intervention on adhesion molecules (selectin E, soluble intercellular adhesion molecule 1 and soluble vascular adhesion molecule 1) and fibrinolytic parameters [plasminogen activator inhibitor-1 (PAI-1) and fibrinogen]. Thirty-six lean adolescents were studied only at baseline as a comparison group. RESULTS: Compared with lean participants, obese adolescents at baseline demonstrated significantly higher levels of triglycerides, glucose, insulin, homeostasis model assessment, soluble intercellular adhesion molecule 1, PAI-1 and fibrinogen. After 6-month lifestyle intervention, those obese adolescents with decreased standard deviation score-body mass index (SDS-BMI) displayed significant decreases in insulin (19.2 ± 11.2 vs. 26.8 ± 13.2 mU/L, P≤ 0.01), homeostasis model assessment (4.24 ± 3.19 vs. 6.58 ± 4.08, P≤ 0.01), selectin E (100.2 ± 60.9 vs. 116.0 ± 69.0 ng/mL, P≤ 0.01) and PAI-1 (39.6 ± 38.0 vs. 51.8 ± 25.6 ng/mL, P≤ 0.05) with respect to the baseline levels. No changes in these parameters were observed in the obese adolescents with stable or increased SDS-BMI. The changes of triglycerides after intervention in subgroup with decreased SDS-BMI were significantly greater than those in subgroup with stable SDS-BMI. CONCLUSIONS: The present study demonstrated increased endothelial activation and impairment of the fibrinolytic system in early life, which is in part reversible by a 6-month lifestyle intervention.

Composted municipal waste effects on chemical properties of a Brazilian soil.

The spread of composted municipal waste (CMW) on land can be used for sustainable crop production. Nevertheless, heavy metals availability may be a problem. Therefore, the main objective of this study was to assess the impact of CMW disposal on heavy metal accumulation in soil and plants. The treatments consisted of an untreated plot (control) and four rates of CMW application. All plots were cultivated in succession of carrot, cauliflower, sweet corn, and radish. Cu and Pb significantly accumulated in the topsoil (0-5 cm) with a similar pattern in the depths of 5-10 cm and 10-20 cm. Cauliflower, for Fe and Cu, and radish, for Pb and Cu, had their tissue analysis significantly affected due to the increasing rates of application of CMW. Nevertheless, the levels of accumulation in both, soil and plant, are within permissible limits. The evidences provided by this experiment indicated that heavy metals are less likely to cause problems for the estimation of CMW loadings to Brazilian agricultural land.

Pleiotrophin disrupts calcium-dependent homophilic cell-cell adhesion and initiates an epithelial-mesenchymal transition.

Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)beta/zeta is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell-cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPbeta/zeta; PTN inactivates RPTPbeta/zeta, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPbeta/zeta at sites dephosphorylated by RPTPbeta/zeta in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPbeta/zeta, the PTN/RPTPbeta/zeta signaling pathway coordinately regulates the levels of tyrosine phosphorylation of proteins in many cellular systems. We now demonstrate that PTN disrupts cytoskeletal protein complexes, ablates calcium-dependent homophilic cell-cell adhesion, stimulates ubiquitination and degradation of N-cadherin, reorganizes the actin cytoskeleton, and induces a morphological epithelial-mesenchymal transition (EMT) in PTN-stimulated U373 cells. The data suggest that increased tyrosine phosphorylation of the different substrates of RPTPbeta/zeta in PTN-stimulated cells alone is sufficient to coordinately stimulate the different functions needed for an EMT; it is possible that PTN initiates an EMT in cells at sites where PTN is expressed in development and in malignant cells that inappropriately express Ptn.

Oxygen transfer and consumption in a thiosulfate oxidizing bioreactor with sulfur production.

AIMS: To evaluate the contribution of oxygen transfer and consumption in a sulfoxidizing system to increase the elemental sulfur yield from thiosulfate oxidation. METHODS AND RESULTS: A 10 l thiosulfate oxidizing bioreactor with suspended cells operating under microaerophilic conditions and a separated aerator with a variable volume of 0.8--1.7 l were operated with a consortium containing mainly Thiobacillus sp. that oxidizes several sulfide species to elemental sulfur and sulfate. From the gas-liquid oxygen balance, the k(L)a was estimated under different operation conditions. A k(L)a of around 200 h(-1) favoured elemental sulfur production and can serve as scale-up criterion. It was further shown that more than 50% of the oxygen fed to the system was consumed in the aerator. CONCLUSIONS: The performance of the sulfoxidizing system can be improved by controlling oxygen transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method for the k(L)a determination was based on the oxygen balance, which incorporates the oxygen concentrations measured in the liquid in steady state, reducing the interference of the response time in the traditional non-steady state methods. This approach can be used to optimize reactors where microaerophilic conditions are desirable.

Positive control wells: a potential answer to remote-area quality assurance of malaria rapid diagnostic tests.

Malaria rapid diagnostic tests (RDTs) are a potential breakthrough in the provision of accurate diagnosis in remote areas, but widescale use is hampered by uncertainty over accuracy under field conditions. Positive control wells, which contain recombinant malaria parasite antigen, are a novel method for addressing this need for quality assurance. The potential of a commercially available positive control well, reconstituted with blood, was assessed for use in routine monitoring of RDT sensitivity in a remote malaria-endemic region. When maintained at 4 degrees C, the wells produced a consistent level of parasite lactate dehydrogenase (pLDH) antigen activity, as detected by pLDH-detecting RDTs, but activity reduced after cumulative exposure to temperatures likely to be encountered over a few months in a malaria-endemic area. This limitation was successfully overcome in the field through centralized, controlled storage. Monitoring of RDT sensitivity was successfully incorporated into routine supervisory visits to remote clinics. However, improved temperature stability of the wells would enhance their potential. The threshold at which the wells' signal reduced RDT sensitivity requires further investigation. The wells show potential to overcome an important obstacle to the wide implementation of accurate parasite-based diagnosis and appropriate treatment. Further assessment of their place in malaria management is warranted.

Sulfur formation by steady-state continuous cultures of a sulfoxidizing consortium and Thiobacillus thioparus ATCC 23645.

The elemental sulfur formation by the partial oxidation of thiosulfate by both a sulfoxidizing consortium and by Thiobacillus thioparus ATCC 23645 was studied under aerobic conditions in chemostat. Steady state was attained with essentially total conversion to sulfate when the dissolved oxygen concentration was 5 mgO2 l(-1) and below a dilution rate (D) of 3.0 d(-1)for the consortium and 0.9 d(-1) for T thioparus. The consortium formed elemental sulfur in steady state under oxygen limitation. Fifty percent of the theoretical elemental sulfur yield was obtained with a dissolved oxygen concentration of 0.2 mgO2 l(-1). Growth of T thioparus was negatively affected with a concentration below 1.9 mgO2 l(-1). Consortium yield from batch cultures was 2.1 g(-1) (protein) mol(-1) (thiosulfate), which was comparable with the values obtained in the chemostat at dilution rates of 0.4 d(-1) and 1.2 d(-1). The consortium showed a maximum degradation rate of 0.105 g(thiosulfate) g(-1) (protein) min(-1) and a saturation rate for S2O3(2-) of 1.9 mM.

Use of OM-85 BV in children suffering from recurrent respiratory tract infections and subnormal IgG subclass levels.

BACKGROUND: Recurrent acute respiratory tract infections (RARTIs) in children are related to IgG subclass deficiencies. The aim of the trial was to evaluate the effect of OM-85 BV in the number of RARTIs as well as in the IgG subclass levels. METHODS: This was a randomized, double-blind, placebo-controlled clinical trial. Patients of ages three to six years, having three or more documented ARTIs during the last six months with subnormal IgG subclass levels were included. Patients took either one capsule of OM-85 BV (3.5 mg) or placebo orally every day for ten consecutive days per month during three consecutive months. Patients were followed three further months without drug intake. IgG subclass levels were determined before and after treatment. RESULTS: IgG4 levels diminished after the OM-85 BV treatment (-3 [-8.0, -1.0] median difference [95 % CI] p < 0.05 by Wilcoxon test). No other significant changes in IgG subclasses were observed. After six months the patients in the OM-85 BV group (n = 20) experienced 2.8 1.4 (mean SD) ARTIs, while the patients in the placebo group (n = 20) suffered 5.2 1.5 ARTIs (-2.4 [3.3, -1.5] mean difference [95 % CI] p < 0.001 by Student's t test). Three patients with OM-85 BV had gastrointestinal events related to drug administration, as well as three placebo patients. CONCLUSION: This study demonstrated the clinical benefit of OM-85 BV in patients suffering from RARTIs and subnormal levels of IgG subclasses. This trial opens new perspectives in the research of the mechanism of action of OM-85 BV.

Effect of salmeterol and salmeterol plus beclomethasone on saliva flow and IgA in patients with moderate-persistent chronic asthma.

BACKGROUND: The use of short-acting beta2-agonists is associated with oral mucosa injuries that are probably provoked by decreased saliva flow and decreased concentrations of immunoglobulin (Ig)A in saliva. OBJECTIVES: To explore the effect of salmeterol, alone or combined with beclomethasone, on the health of oral mucosa, as well as its effect on saliva flow and IgA concentration in saliva. METHODS: Patients ranging in age from 6 to 15 years with moderate-persistent chronic asthma were enrolled. Patients received two 6-week treatments, one with salmeterol plus beclomethasone and the other with only salmeterol, with a 1-week washout period between treatments. Patients had oral cavity examinations and assessments of saliva flow, IgA in saliva, and total protein in saliva before the beginning and at the end of each treatment RESULTS: The results of the baseline oral examinations were normal in all patients. The postsalmetrol (PS) examinations detected 13 patients with gingivitis and the postbeclomethasone-salmeterol (PBS) examinations disclosed 10 patients with gingivitis and 1 with lower-lip ulceration. Baseline saliva flow was 16.25 +/- 7.04 mm/minute (confidence interval [CI] 95% 13.67; 18.89), PS was 13.53 +/- 5.93 mm/minute (CI 95% 11.33; 15.73), and PBS was 16.57 +/- 5.54 mm/minute (CI 95% 14.51; 18.62). No statistical differences between the different assessments were found. Mean saliva IgA at baseline was 4.99 +/- 1.96 mg/dL (CI 95% 4.26; 5.71), PS IgA was 6.53 +/- 3.02 mg/dL (CI 95% 5.41; 7.65), and PBS IgA was 4.82 +/- 1.98 mg/dL (CI 95% 4.08; 5.56). PS IgA was significantly higher than the other two determinations (P < 0.05 by Bonferroni and Tukey tests). Baseline saliva IgA-to-protein ratio was 0.72 +/- 0.24 (95% CI 0.64; 0.80), PS IgA:protein ratio was 1.02 +/- 0.38 (95% CI 0.88; 1.16), and PBS IgA:protein ratio was 0.72 +/- 0.25 (95% CI 0.62; 0.82). PS IgA:protein ratio was significantly higher than the other two determinations (P < 0.05 by Bonferroni and Tukey tests). CONCLUSIONS: In the present study it was demonstrated that salmeterol alone or in combination with beclomethasone induced injuries in the oral mucosa, but only salmeterol alone induced increases in the total and protein-adjusted IgA in saliva.

Dscr1, a novel endogenous inhibitor of calcineurin signaling, is expressed in the primitive ventricle of the heart and during neurogenesis.

We have demonstrated that DSCR1 acts as a negative regulator of calcineurin-mediated signaling and that its transcript is overexpressed in the Down syndrome (DS) fetal brain. To evaluate the possible involvement of DSCR1 in DS, we have cloned the mouse gene and analyzed its expression pattern in the central nervous system (CNS). Early expression of Dscr1 is detected mainly in the heart tube and in the CNS in rhombomere 4 and the pretectum. From embryonic day 14.5 onwards, Dscr1 is widely distributed in the CNS but becomes more restricted as the brain matures. We confirmed its neuronal expression pattern in the adult, preferentially in Purkinje and pyramidal cells, by double labeling with glial fibrillary acidic protein. We also show that although Dscr1 is present in trisomy in the Ts65Dn mouse, the adult brain expression pattern is not significantly altered. This expression pattern indicated that Dscr1 is a developmentally regulated gene involved in neurogenesis and cardiogenesis and suggests that it may contribute to the alterations observed in these organ systems in DS patients.

Aberrant development of hippocampal circuits and altered neural activity in netrin 1-deficient mice.

Diffusible factors, including netrins and semaphorins, are believed to be important cues for the formation of neural circuits in the forebrain. Here we have examined the role of netrin 1 in the development of hippocampal connections. We show that netrin 1 and its receptor, Dcc, are expressed in the developing fimbria and in projection neurons, respectively, and that netrin 1 promotes the outgrowth of hippocampal axons in vitro via DCC receptors. We also show that the hippocampus of netrin 1-deficient mice shows a misorientation of fiber tracts and pathfinding errors, as detected with antibodies against the surface proteins TAG-1, L1 and DCC. DiI injections show that hippocampal commissural axons do not cross the midline in these mutants. Instead, when axons approach the midline, they turn ventrally and form a massive aberrant projection to the ipsilateral septum. In addition, both the ipsilateral entorhino-hippocampal and the CA3-to-CA1 associational projections show an altered pattern of layer-specific termination in netrin 1-deficient mice. Finally, optical recordings with the Ca(2+) indicator Fura 2-AM show that spontaneous neuronal activity is reduced in the septum of netrin 1-mutant mice. We conclude that netrin 1 is required not only for the formation of crossed connections in the forebrain, but also for the appropriate layer-specific targeting of ipsilateral projections and for the control of normal levels of spontaneous neural activity.

Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system.

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Here we used tissue explants to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. We show that netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14, which give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, did not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibres and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration.

Alu-splice cloning of human Intersectin (ITSN), a putative multivalent binding protein expressed in proliferating and differentiating neurons and overexpressed in Down syndrome.

By Alu-splice PCR we have trapped two exons and subsequently identified the full length cDNA of a human gene, Intersectin (ITSN), which maps to chromosome 21q22.1 between markers D21S320 and D21S325. The gene has the potential to code for at least two different protein isoforms by alternative splicing (ITSN-L and ITSN-S). Intersectin exists with a high degree of similarity in flies, frogs and mammals, suggesting a conserved role in higher eukaryotes. Analysis of the expression pattern of human and mouse Intersectin detected mRNAs in all adult and foetal tissues tested, with the longer isoform present in brain. In situ hybridisation studies in the developing mouse brain showed ITSN expression in both proliferating and differentiating neurons. The genomic structure of ITSN was determined using the chromosome 21 sequences deposited in the public databases. The protein contains several known motifs which implicate ITSN in clathrin mediated endocytosis and synaptic vesicle recycling. The expression pattern of Intersectin in mouse brain, its presumed function and its overexpression in brains from Down syndrome patients, suggest that Intersectin may contribute in a gene dosage-dependent manner to some of the abnormalities of Down syndrome.

Thyroid hormone regulates reelin and dab1 expression during brain development.

The reelin and dab1 genes are necessary for appropriate neuronal migration and lamination during brain development. Since these processes are controlled by thyroid hormone, we studied the effect of thyroid hormone deprivation and administration on the expression of reelin and dab1. As shown by Northern analysis, in situ hybridization, and immunohistochemistry studies, hypothyroid rats expressed decreased levels of reelin RNA and protein during the perinatal period [embryonic day 18 (E18) and postnatal day 0 (P0)]. The effect was evident in Cajal-Retzius cells of cortex layer I, as well as in layers V/VI, hippocampus, and granular neurons of the cerebellum. At later ages, however, Reelin was more abundant in the cortex, hippocampus, cerebellum, and olfactory bulb of hypothyroid rats (P5), and no differences were detected at P15. Conversely, Dab1 levels were higher at P0, and lower at P5 in hypothyroid animals. In line with these results, reelin RNA and protein levels were higher in cultured hippocampal slices from P0 control rats compared to those from hypothyroid animals. Significantly, thyroid-dependent regulation of reelin and dab1 was confirmed in vivo and in vitro by hormone treatment of hypothyroid rats and organotypic cultures, respectively. In both cases, thyroid hormone led to an increase in reelin expression. Our data suggest that the effects of thyroid hormone on neuronal migration may be in part mediated through the control of reelin and dab1 expression during brain ontogenesis.

Influence of carbon and nitrogen sources on Flavobacteriumgrowth and zeaxanthin biosynthesis.

Growth and production of zeaxanthin by Flavobacterium sp were studied using different carbon and nitrogen sources in a chemically defined medium. The best growth was supported by sucrose, but glucose yielded similar carotenoid concentrations. Both asparagine and glutamine stimulated growth and pigment formation. Carotenoid production and glucose consumption increased as a function of asparagine concentration. In the presence of asparagine, high glucose concentrations decreased pigment production without affecting biomass formation. In the absence of glucose, asparagine could not support growth and zeaxanthin production. When compared to the effect of 55 mM glucose, 10 mM oxaloacetate increased growth and carotenoid production. Pyruvate and other intermediates of the citric acid cycle showed a similar stimulatory effect. The intermediates of glycolysis: glucose 6-phosphate and fructose 1,6-diphosphate did not support growth. These results suggest that Flavobacterium sp utilizes asparagine primarily as a nitrogen source for growth and production of zeaxanthin.