Análise das metodologias diretas e indiretas para a contagem de células somáticas no leite de cabras hígidas

A particularidade da secreção láctea caprina, do tipo apócrina, diferente da secreção merócrina da vaca, leva a erros de interpretação durante a realização de técnicas de avaliação da celularidade do leite de fêmeas desta espécie. Portanto, o presente trabalho teve o objetivo de determinar a contagem de células somáticas pelo método indireto California Mastitis Test (CMT), e por métodos diretos, incluindo a contagem por citometria de fluxo e a contagem microscópica direta, através da coloração de verde de metil e pironina-Y, além de comparar os métodos de contagem celular. Foram analisadas 102 amostras de 51 fêmeas caprinas, das raças Saanen, Parda Alpina e Toggenburg, criadas no Estado de São Paulo. Os animais foram categorizados segundo a fase da lactação, exame físico da glândula mamária e exame do leite. As amostras foram colhidas, após a realização do exame Califórnia Mastitis Test, em duas alíquotas, uma destinada à contagem celular automática e a outra, a contagem microscópica direta, utilizando-se o corante verde de metil e pironina- Y. De acordo com os diferentes escores do CMT, observou- se 74,5% de amostras negativas, 8,8% de amostras com escore traços, 8,8% de amostras ligeiramente positivas (+), 6,8% de amostras fracamente positivas (++) e 0,9% de amostras fortemente positivas (+++). Os valores medianos das contagens de células somáticas presentes no leite de cabras, avaliadas através de contador automático e microscopia direta, e analisadas de acordo com os diferentes escores do CMT, foram, respectivamente, 181.000, 578.000, 628.000, 1.421.500 e 5.542.000 células/mL de leite e 74.991, 271.396, 71.420, 640.995 e 5.049.394 células/ mL de leite, nos escores negativo, traços, +, ++ e +++. Os valores medianos obtidos através da contagem de células somáticas pelo método automático e microscópico direto, de acordo com as fases de lactação foram de 159.500, 508.000 e 277.500 células/mL de leite, e 62.493, 89.275 e 146.411. A correlação obtida entre a contagem celular automática e microscópica direta foi de 88%. A partir dos resultados observados pode-se concluir que existe diferença na contagem celular determinada através do método automático e microscópico sendo este último o mais adequado para a determinação da celularidade no leite de cabras.

Identification and control of occupational risks in a Sao Paulo metropolitan region quarry

Crushed stone mining is the third largest mining economy in Brazil, where almost half is produced in the Sao Paulo metropolitan region. The segment registers the highest number of accidents among the extractive industries, which justifies the concern with workers` health and safety, and the importance of controlling occupational hazards. Since 2002, the NR-22 Standard (NR-22: Occupational Health and Safety in Mining) makes compulsory the elaboration of a Risk Management Program that identifies risks and establishes control measures. Considering the crushed stone mining industry importance to the state, this paper evaluates and discusses the risks identified in unit operations during the production process of crushed stone in an open pit mine in order to propose control measures for the development of the Risk Management Program. Although this study refers to a specific quarry, it can be applied to other mines from the same sector since some considerations are made regarding differences in manufacturing processes. The research was based on the identification of the main risks associated with drilling, blasting, load & haulage, crushing and screening through field measurements of some hazardous agents, together with company reports. The results contributed to the choice of the appropriate control measures for the improvement Of workers` health and safety conditions.

Efeitos da alteração do limite de exposição ocupacional à sílica cristalina no processo de seleção de respiradores.

The purpose of this study was to investigate the respiratory protective device selection process and to identify changes in this process when an exposure limit value is updated. Two previous studies conducted in mining industries in the metropolitan area of Sao Paulo were put through the respiratory protective device selection process. The protection factors of the equipment provided by the companies were compared with the required protection factors and with the FUNDACENTRO`s respiratory protection program. The results showed that until 2005, some companies were providing inadequate protection, and after the change in crystalline silica exposure limit value in 2006, all the analyzed companies were providing inadequate respirators. This study suggests that there is an opportunity to create a web portal, where the selection process can be done by the companies with updated information.

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl(2). The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants. (C) 2009 Elsevier B.V. All rights reserved.

Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl(2). Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H(2)O(2) production, reduced and enhanced APX activity in NaCl and CdCl(2), respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure. (C) 2011 Elsevier B.V. All rights reserved.

Biochemical responses of glyphosate resistant and susceptible soybean plants exposed to glyphosate

Glyphosate is a wide spectrum, non-selective, post-emergence herbicide. It acts on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products, leading to plant death. Transgenic glyphosate-resistant (GR) soybean [Glycine max (L.)] expressing an glyphosate-insensitive EPSPS enzyme has provided new opportunities for weed control in soybean production. The effect of glyphosate application on chlorophyll level, lipid peroxidation, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GOPX) and superoxide dismutase (SOD) activities, soluble amino acid levels and protein profile, in leaves and roots, was examined in two conventional (non-GR) and two transgenic (GR) soybean. Glyphosate treatment had no significant impact on lipid peroxidation, whilst the chlorophyll content decreased in only one non-GR cultivar. However, there was a significant increase in the levels of soluble amino acid in roots and leaves, more so in non-GR than in GR soybean cultivars. Root CAT activity increased in non-GR cultivars and was not altered in GR cultivars. In leaves, CAT activity was inhibited in one non-GR and one GR cultivar. GOPX activity increased in one GR cultivar and in both non-GR cultivars. Root APX activity increased in one GR cultivar. The soluble protein profiles as assessed by 1-D gel electrophoresis of selected non-GR and GR soybean lines were unaffected by glyphosate treatment. Neither was formation of new isoenzymes of SOD and CAT observed when these lines were treated by glyphosate. The slight oxidative stress generated by glyphosate has no relevance to plant mortality. The potential antioxidant action of soluble amino acids may be responsible for the lack of lipid peroxidation observed. CAT activity in the roots and soluble amino acids in the leaves can be used as indicators of glyphosate resistance.

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): Study of enzymes and nitrogen-containing compounds

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-L-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine, synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound. (c) 2007 Elsevier Masson SAS. All rights reserved.

Nutritional Quality of Sorghum Seeds: Storage Proteins and Amino Acids

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.

Differences in both matrix metalloproteinase 9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence of amorphous silica or silicate salts in blood collection devices

Matrix metalloproteinases (MMPs) are promising diagnostic tools, and blood sampling/handling alters MMP concentrations between plasma and serum and between serum with and without clot activators. To explain the higher MMP-9 expression in serum collected with clot accelerators relative to serum with no additives and to plasma, we analyzed the effects of increasing amounts of silica and silicates (components of clot activators) in,citrate plasma, serum, and huffy coats collected in both plastic and glass tubes from 50 healthy donors, and we analyzed the effects of silica and silicate on cultured leukemia cells. The levels of MMP-2 did not show significant changes between glass and plastic tubes, between serum and plasma, between serum with and without clot accelerators, or between silica and silicate treatments. No modification of MMP-9 expression was obtained by the addition of silica or silicate to previously separated plasma and serum. Increasing the amounts of nonsoluble silica and soluble silicate added to citrate and empty tubes prior to blood collection resulted in increasing levels of MMP-9 relative to citrate plasma and serum. Silica and silicate added to buffy coats and leukemia cells significantly induced MMP-9 release/secretion, demonstrating that both silica and silicate induce the release of pro- and complexed MMP-9 forms. We recommend limiting the misuse of serum and avoiding the interfering effects of clot activators. (c) 2007 Elsevier Inc. All rights reserved.

HLA-DRB1*allele-associated genetic susceptibility and protection against multiple sclerosis in Brazilian patients

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system that causes neurological disorders in young adults. Previous studies in various populations highlighted an association between the HLA-DRB1*1.5 allele and MS. This study investigated the association between HLA-DRB1*15 and other HLA-DRB1 alleles and MS in a Brazilian Caucasian population sample from Londrina, Southern Brazil. HLA-DRB1 alleles were analyzed by polymerase chain reaction with specific sequence oligonucleotide primers in 119 MS patients and in 305 healthy blood donors as a control. Among the MS patients, 89 (75.0%) presented with relapsing remitting MS, 24 (20.0%) with secondary progressive MS and 6 (5.0%) with primary progressive MS. The frequency of the HLA-DRB1*15 allele observed in the MS Brazilian patients was similar to findings reported in previous studies carried out in populations worldwide. However, the results showed a higher frequency of the HLA-DRB1*15 allele in the MS patients compared to the controls, with a relative frequency of 0.1050 (10.50%) and 0.0443 (4.4%), respectively (OR=2.53; 95% CI 1.43-4.46; p=0.0009). A protector allele was also detected. The frequency of the HLA-DRB1*11 allele was reduced in the MS patients compared to the controls, with a relative frequency of 0.1345 (13.4%) and 0.1869 (18.7%), respectively (OR=0.67; 95% CI 0.44-1.03; p=0.0692). The results demonstrated that the HLA-DRB1*15 allele in heterozygosity is positively associated with MS (p=0.0079), and may be considered a genetic marker of susceptibility to the disease. A negative association between the HLA-DRB1*11 allele in homozygosity and MS was also verified (p=0.0418); this allele may be considered a genetic marker of resistance to MS in the Brazilian population.