Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma

Actin-based motor protein requirements and nitric oxide (NO) production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma), with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS) expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity.

Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus

Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

Protection against neurotoxicity by an autophagic mechanism

The objective of the present study was to investigate the effects of 3-n-butylphthalide (NBP) on a 1-methyl-4-phenylpyridinium (MPP+)-induced cellular model of Parkinson’s disease (PD) and to illustrate the potential mechanism of autophagy in this process. For this purpose, rat PC12 pheochromocytoma cells were treated with MPP+ (1 mM) for 24 h following pretreatment with NBP (0.1 mM). Cell metabolic viability was determined by the MTT assay and cell ultrastructure was examined by transmission electron microscopy. The intracellular distribution and expression of α-synuclein and microtubule-associated protein light chain 3 (LC3) were detected by immunocytochemistry and Western blotting. Our results demonstrated that: 1) NBP prevented MPP+-induced cytotoxicity in PC12 cells by promoting metabolic viability. 2) NBP induced the accumulation of autophagosomes in MPP+-treated PC12 cells. 3) Further study of the molecular mechanism demonstrated that NBP enhanced the colocalization of α-synuclein and LC3 and up-regulated the protein level of LC3-II. These results demonstrate that NBP protects PC12 cells against MPP+-induced neurotoxicity by activating autophagy-mediated α-synuclein degradation, implying that it may be a potential effective therapeutic agent for the treatment of PD.

Protein defects in neuromuscular diseases

Muscular dystrophies are a heterogeneous group of genetically determined progressive disorders of the muscle with a primary or predominant involvement of the pelvic or shoulder girdle musculature. The clinical course is highly variable, ranging from severe congenital forms with rapid progression to milder forms with later onset and a slower course. In recent years, several proteins from the sarcolemmal muscle membrane (dystrophin, sarcoglycans, dysferlin, caveolin-3), from the extracellular matrix (alpha2-laminin, collagen VI), from the sarcomere (telethonin, myotilin, titin, nebulin), from the muscle cytosol (calpain 3, TRIM32), from the nucleus (emerin, lamin A/C, survival motor neuron protein), and from the glycosylation pathway (fukutin, fukutin-related protein) have been identified. Mutations in their respective genes are responsible for different forms of neuromuscular diseases. Protein analysis using Western blotting or immunohistochemistry with specific antibodies is of the utmost importance for the differential diagnosis and elucidation of the physiopathology of each genetic disorder involved. Recent molecular studies have shown clinical inter- and intra-familial variability in several genetic disorders highlighting the importance of other factors in determining phenotypic expression and the role of possible modifying genes and protein interactions. Developmental studies can help elucidate the mechanism of normal muscle formation and thus muscle regeneration. In the last fifteen years, our research has focused on muscle protein expression, localization and possible interactions in patients affected by different forms of muscular dystrophies. The main objective of this review is to summarize the most recent findings in the field and our own contribution.

High doses of riboflavin and the elimination of dietary red meat promote the recovery of some motor functions in Parkinson's disease patients

Abnormal riboflavin status in the absence of a dietary deficiency was detected in 31 consecutive outpatients with Parkinson's disease (PD), while the classical determinants of homocysteine levels (B6, folic acid, and B12) were usually within normal limits. In contrast, only 3 of 10 consecutive outpatients with dementia without previous stroke had abnormal riboflavin status. The data for 12 patients who did not complete 6 months of therapy or did not comply with the proposed treatment paradigm were excluded from analysis. Nineteen PD patients (8 males and 11 females, mean age ± SD = 66.2 ± 8.6 years; 3, 3, 2, 5, and 6 patients in Hoehn and Yahr stages I to V) received riboflavin orally (30 mg every 8 h) plus their usual symptomatic medications and all red meat was eliminated from their diet. After 1 month the riboflavin status of the patients was normalized from 106.4 ± 34.9 to 179.2 ± 23 ng/ml (N = 9). Motor capacity was measured by a modification of the scoring system of Hoehn and Yahr, which reports motor capacity as percent. All 19 patients who completed 6 months of treatment showed improved motor capacity during the first three months and most reached a plateau while 5/19 continued to improve in the 3- to 6-month interval. Their average motor capacity increased from 44 to 71% after 6 months, increasing significantly every month compared with their own pretreatment status (P < 0.001, Wilcoxon signed rank test). Discontinuation of riboflavin for several days did not impair motor capacity and yellowish urine was the only side effect observed. The data show that the proposed treatment improves the clinical condition of PD patients. Riboflavin-sensitive mechanisms involved in PD may include glutathione depletion, cumulative mitochondrial DNA mutations, disturbed mitochondrial protein complexes, and abnormal iron metabolism. More studies are required to identify the mechanisms involved.

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Alguns aspectos epidemiológicos da mortalidade por acidentes de trânsito de veículo a motor na Cidade de São Paulo, Brasil

Foram estudadas algumas características dos óbitos por acidentes de trânsito de veículos a motor, no município de São Paulo, ocorridos entre 1.° de janeiro a 31 de dezembro de 1970. Todas as características do falecido e do acidente foram coletadas a partir dos dados registrados nos laudos de necrópsias existentes no Instituto Médico Legal. O estudo evidenciou que a mortalidade por acidentes de veículo a motor é alta, maior no sexo masculino, aumenta com a idade, sendo que o maior coeficiente foi para maiores de 60 anos. A zona da cidade com maior número de acidentes é a zona Sul, existindo áreas (distritos policiais) e vias públicas preferenciais quanto a ocorrência, em todas as 4 zonas do município; a maior ocorrência de acidentes foi aos sábados e domingos; os pedestres compreendem a grande maioria dos falecidos; proporção apreciável dos falecidos recebeu atendimento hospitalar após o acidente. Foram relacionados também o número total de acidentes, vítimas e mortes mostrando que para cada 100 acidentes ocorreram 62,50 vítimas e 5,13 mortes, e para cada 100 vítimas, 18, 22 mortes.

Aspectos epidemiológicos dos acidentes fatais a veículo a motor na cidade do Salvador (Bahia), Brasil

Foi realizado um estudo com o objetivo de descrever certos aspectos epidemiológicos dos acidentes de trânsito de veículo a motor, na cidade do Salvador, Bahia, Brasil, o qual envolveu coleta de informações no Departamento de Trânsito (DETRAN) e no Instituto de Medicina Legal. Ficou demonstrado que os acidentes de trânsito de veículo a motor são mais freqüentes ao fim da tarde e concentram-se, sobretudo, no fim de semana, isto é, sábado e domingo. Motoristas do sexo masculino foram muito mais freqüentemente envolvidos em acidentes de trânsito do que os do sexo feminino. Foi concluído que os acidentes fatais envolvendo pedestres constituem-se importante problema urbano necessitando cuidadosa atenção por parte de educadores e dos serviços públicos responsáveis pelo setor do trânsito. Foi sugerida a necessidade de melhorar e tornar mais uniforme o sistema de notificação e classificação dos acidentes de veículo a motor pelo DETRAN da cidade do Salvador.

Traumatismo crânio-encefálico: diferenças das vítimas pedestres e ocupantes de veículos a motor

OBJETIVO: Caracterizar as diferenças das vítimas com diagnóstico de traumatismo crânio-encefálico envolvidas em diferentes condições, em acidentes de trânsito de veículo a motor e evidenciar indicadores para prevenção e seu atendimento. MÉTODO: Foram analisados os prontuários de todas as vítimas com diagnóstico de trauma crânio-encefálico, assistidas em hospital de referência para atendimento do trauma, entre março e junho de 1993. A população foi de 156 vítimas, sendo 80 pedestres, 50 ocupantes de veículos a motor, exceto de moto e, 26 motociclistas ou passageiros de motos. RESULTADOS E CONCLUSÕES: Os resultados mostraram que a mortalidade entre pedestres foi a mais alta entre os três grupos (25,0%). Os óbitos ocorrem em 19,2% dos ocupantes de motocicleta e 8,0% dos ocupantes de demais veículos a motor. Foram observadas diferenças estatísticas entre os grupos quando a variável gravidade do trauma crânio-encefálico foi analisado. Análise de certas variáveis mostraram importantes diferenças na distribuição dos três grupos.

Oportunidades do ambiente domiciliar para o desenvolvimento motor

OBJETIVO: Avaliar as oportunidades presentes no ambiente domiciliar para o desenvolvimento motor de lactentes. MÉTODOS: Estudo epidemiológico de base populacional, transversal, realizado com 239 lactentes com idade entre três e 18 meses, residentes no município de Juiz de Fora, MG, em 2010. Os participantes foram selecionados por amostragem aleatória estratificada, conglomerada, em múltiplos estágios. Para avaliar a qualidade e quantidade de estímulo motor no ambiente domiciliar foi utilizado o instrumento Affordance in the Home Environment for Motor Development - Infant Scale. Procedeu-se a análise bivariada com aplicação do teste qui-quadrado, seguida de regressão logística multinomial para verificar a associação entre as oportunidades presentes no domicílio e fatores biológicos, comportamentais, demográficos e socioeconômicos. RESULTADOS: As oportunidades de estimulação ambiental foram relativamente baixas. Na análise bivariada, para a faixa etária de três a nove meses, foi encontrada associação com os fatores: ordem de nascimento (p = 0,06), classificação socioeconômica (p = 0,08), renda mensal (p = 0,06) e renda per capita (p = 0,03). No modelo de regressão, prevaleceu a classificação socioeconômica (RC = 7,46; p = 0,03). Para a faixa etária de dez a 18 meses, os fatores associados, na análise bivariada, foram: estado civil materno (p < 0,01), convívio da criança com o pai (p = 0,08), chefe da família (p = 0,04), número de pessoas no domicílio (p = 0,05), escolaridade materna (p < 0,01) e paterna (p < 0,01), classificação socioeconômica (p < 0,01) e renda per capita (p = 0,03). No modelo de regressão, o estado civil materno (RC = 4,83; p = 0,02), escolaridade materna (RC = 0,29; p = 0,03) e paterna (RC = 0,33; p = 0,04) permaneceram associados às oportunidades de estimulação ambiental. CONCLUSÕES: A união estável dos pais, maior escolaridade materna e paterna e maior nível econômico foram os fatores associados às melhores oportunidades de estimulação motora no lar.

Content validity and reliability of test of gross motor development in Chilean children

ABSTRACT OBJECTIVE To validate a Spanish version of the Test of Gross Motor Development (TGMD-2) for the Chilean population. METHODS Descriptive, transversal, non-experimental validity and reliability study. Four translators, three experts and 92 Chilean children, from five to 10 years, students from a primary school in Santiago, Chile, have participated. The Committee of Experts has carried out translation, back-translation and revision processes to determine the translinguistic equivalence and content validity of the test, using the content validity index in 2013. In addition, a pilot implementation was achieved to determine test reliability in Spanish, by using the intraclass correlation coefficient and Bland-Altman method. We evaluated whether the results presented significant differences by replacing the bat with a racket, using T-test. RESULTS We obtained a content validity index higher than 0.80 for language clarity and relevance of the TGMD-2 for children. There were significant differences in the object control subtest when comparing the results with bat and racket. The intraclass correlation coefficient for reliability inter-rater, intra-rater and test-retest reliability was greater than 0.80 in all cases. CONCLUSIONS The TGMD-2 has appropriate content validity to be applied in the Chilean population. The reliability of this test is within the appropriate parameters and its use could be recommended in this population after the establishment of normative data, setting a further precedent for the validation in other Latin American countries.

Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment

Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In general, the levels and/or positivity rate of circulating antigens and specific IgG antibodies were significantly higher in patients with hepatointestinal (HI) and hepatosplenic (HS) forms than in mild intestinal (I) forms. An association between Po antigens and clinical features of the disease was observed, as the level of these antigens was low (137 ng/ml) as well as the positivity rate (7.9%) in patients with I forms; values that were intermediate (593 ng/ml and 33.3%) in those with HI forms, and high (1.563 ng/ml and 50.0%) in more severe HS forms. The Pt antigens were detected in the studied clinical forms not differing statistically but, the positivity rate was significantly higher in HS forms comparatively to I forms. The antisera studied revealed distinct circulating antigen profiles, and the prognostic value of Po and Pt antigens was suggested.