A step-up ractopamine feeding program for finishing pigs / Niveles incrementales de ractopamina para cerdos en ceba / Plano de níveis aumentados de ractopamina para suínos em terminação

Abstract Background: Ractopamine (RAC) supplementation in the feed has been evaluated as a strategy to increase productive efficiency in finishing pigs. Objective: To evaluate the effects of different RAC dietary levels on performance, carcass traits, efficiency of lysine (ELU) and energy (EEU) utilization, and economic viability in finishing pig. Methods: A total of 40 barrows (74.75 ± 5.22 kg) were fed four RAC levels (0, 5, 10 and 5-10 mg/kg step-up program) from 0-14, 15-31 and 0-31 days. Performance, carcass characteristics, ELU, EEU, cost per unit of weight gain (CWG), payment and profit parameters were measured. The animals were distributed in a completely randomized design in four treatments, with ten replicates per treatment. The experimental unit was each animal. Results: Pigs fed RAC diets showed increased body weights at 14 and 31 days, average daily gain (ADG) at 0-14 and 0-31 days, ELU at 0-14 days, and hot carcass weight as compared with those fed the control diet. The step-up program as compared to the 10 mg/kg RAC concentration resulted in increased body weight, feed/gain ratio (FGR), ADG, ELU, EEU and CWG at 0-14 days. Payment by weight and bonus payment were better for treatments with RAC as compared to control. Conclusions: Pigs fed RAC improved performance, carcass weight, ELU, EEU and economic viability. The results were better for the step-up program compared with the intermittent use of 10 mg/kg RAC.

Resumen Antecedentes: La suplementación de cerdos con ractopamina (RAC) es una estrategia para aumentar la eficiencia productiva en ceba. Objetivo: Evaluar el efecto de diferentes planes de suplementación con RAC en dietas de cerdos en ceba sobre el rendimiento productivo, características de la canal, eficiencia de utilización de lisina (ELU) y energía (EEU), y viabilidad económica. Métodos: Un total de 40 machos castrados (74,75 ± 5,22 kg) fueron alimentados con cuatro niveles de RAC (0, 5, 10 y 5-10 mg/kg de plano escalonado) de 0-14, 15-31 y 0-31 días. Se evaluó el rendimiento, características de la canal, ELU, EEU, el costo por unidad de ganancia de peso (CWG), los tipos de pago y ganancias. Los animales se distribuyeron en un diseño completamente aleatorizado en cuatro tratamientos, con diez repeticiones por tratamiento. La unidad experimental fue cada animal. Resultados: Los animales suplementados con RAC tuvieron mayor peso corporal a los 14 y 31 días, ganancia de peso diaria (ADG) de 0-14 y 0-31 días, ELU de 0-14 días y peso de la canal caliente en comparación con el grupo control. En comparación con la concentración de 10 mg/kg de RAC, el plano escalonado resultó en un aumento de peso corporal, conversión alimenticia (FGR), ADG, ELU, EEU y CWG a los 0-14 días. El pago por peso y el pago por bonificación fueron mejores para los tratamientos con RAC en comparación con el control. Conclusiones: Los cerdos en ceba alimentados con RAC tienen mejor rendimiento, peso de la canal, ELU, EEU y viabilidad económica. Los resultados de los parámetros estudiados son mejores con el uso del plano escalonado en comparación con el uso continuo de 10 mg/kg de RAC.

Resumo Antecedentes: Suplementação de ractopamina (RAC) em dietas para suínos foi avaliada como uma estratégia para aumentar eficiência de produção de suínos em terminação. Objetivo: Avaliar os efeitos de diferentes planos de suplementação de RAC em dietas para suínos em terminação sobre o desempenho, características de carcaça, eficiência de utilização de lisina (ELU) e energia (EEU), e viabilidade econômica. Métodos: Um total de 40 machos castrados (74.75 ± 5.22 kg) foram alimentados com quatro níveis de RAC (0, 5, 10 e 5-10 mg/kg plano escalonado) em 0-14, 15-31 e 0-31 dias. Desempenho, características de carcaça, ELU, EEU, custo por unidade de ganho de peso (CWG), tipos de pagamento e lucro foram mensurados. Os animais foram distribuídos em um delineamento inteiramente casualizado em quatro tratamentos, dez repetições para cada tratamento. A unidade experimental foi cada animal. Resultados: Os animais alimentados com dietas contendo RAC mostraram aumento de peso corporal aos 14 e 31 dias, ganho de peso diário (ADG) de 0-14 e 0-31 dias, ELU de 0-14 dias e peso de carcaça quente comparado ao grupo controle. O plano escalonado comparado ao nível de 10 mg/kg de RAC mostrou maior peso corporal, conversão alimentar (FGR), ADG, ELU, EEU e CWG de 0-14 dias. Pagamento por peso e pagamento por bonificação foram melhor para tratamentos com RAC em comparação ao controle. Conclusões: Suínos alimentados com RAC mostram melhor desempenho, peso de carcaça, ELU, EEU e viabilidade econômica. Os resultados dos parâmetros estudados foram melhores com uso do plano escalonado quando comparado com uso constante de 10 mg/kg de RAC para suínos em terminação.

Bone characteristics of pigs fed different sources of phosphorus / Características óseas de cerdos alimentados con diferentes fuentes de fósforo / Características ósseas de suínos alimentados com diferentes fontes de fósforo

Summary Background: phosphorus supplementation should help to keep bone integrity and prevent fractures during the development and slaughter of animals. Objective: to evaluate the effect of different phosphorus sources on one characteristics of pigs. Methods: one-hundred and twelve piglets (28.65 ± 2.82 Kg body weight) were distributed into an 8×2 factorial arrangement (eight sources of phosphorus × two sexes) in blocks in a completely randomized design. The diets were formulated on a total-phosphorus basis, with 0.32 and 0.31% of P for the control diet and 0.56 and 0.42% of P for the other treatments in the growth and finishing phases, respectively. Phosphorus was supplemented as dicalcium phosphate (DCP); mono-dicalcium phosphate (MDCP); triple superphosphate (TSP); single superphosphate (SSP); Catalão-rock phosphate (ROCK); a mixture of sources (MIX); phosphoric acid (PPA); and the control diet (CTR). Results: there was no interaction between phosphorus sources and sex in any of the parameters. Thickness of the compact tissue was the lowest in the CTR, differing from the diets containing DCP, MDCP, and PPA, followed by diets SSP, TSP, and ROCK, with the greatest value for MDCP. Porosity of the compact tissue was higher for the CTR and SSP diets. Conclusion: the use of less elaborate sources of phosphorus, such as rock phosphate and single superphosphate, was less effective than the other sources to improve bone integrity of pigs.

Resumen Antecedentes: la suplementación dietaria con fósforo ayuda a mantener la integridad del hueso y prevenir fracturas durante el desarrollo y sacrificio de los animales. Objetivo: evaluar el efecto de diferentes fuentes de fósforo sobre las características óseas de los cerdos. Métodos: ciento doce lechones (peso corporal: 28,65 ± 2,82 Kg) se distribuyeron en un arreglo factorial 8×2 (ocho fuentes de fósforo × dos sexos) en bloques al azar. Las dietas se formularon con base en fósforo total, con 0,32 y 0,31% de P para la dieta control y 0,56 y 0,42% de P para los otros tratamientos en las fases de crecimiento y finalización, respectivamente. El fósforo se suplementó como fosfato dicálcico (DCP), monofosfato dicálcico (MDCP), superfosfato triple (TSP), superfosfato simple (SSP), fosfato de roca Catalão (ROCK), mezcla de fuentes (MIX), ácido fosfórico (PPA) y dieta control (CTR). Resultados: no se observó interacción entre las fuentes de fósforo y el sexo en ninguno de los parámetros estudiados. El espesor del tejido compacto fue más bajo en el CTR, y diferente a las dietas que contenían DCP, MDCP y PPA, seguido por las dietas SSP, TSP y ROCK; con el mayor valor para MDCP. La porosidad del tejido compacto fue mayor para las dietas CTR y SSP. Conclusión: el uso de fuentes menos elaboradas de fósforo, tales como el fosfato de roca y superfosfato simple, fue menos efectivo que las otras fuentes en mejorar la integridad ósea de los cerdos.

Resumo Antecedentes: a suplementação de fósforo deve manter a integridade do tecido ósseo e prevenir fraturas durante o desenvolvimento e abate dos animais. Objetivo: avaliar o efeito de diferentes fontes de fósforo sobre as características ósseas dos suínos. Métodos: cento e doze leitões com peso médio inicial de 28,65 ± 2,82 Kg foram distribuídos em esquema fatorial 8×2 (oito fontes de fósforo × dois sexos) em blocos casualizados. As dietas foram formuladas baseadas em fósforo total com 0,32 e 0,31% de P para a dieta controle e com 0,56 e 0,42% de P para os outros tratamentos nas fases de crescimento e terminação, respectivamente. O fósforo nas dietas foi suplementado com fosfato bicálcico (DCP); mono-fosfato bicálcico (MDCP); superfosfato triplo (TSP); superfosfato simples (SSP); fosfato de rocha Catalão (ROCK); uma mistura de fontes (MIX); ácido fosfórico (PPA); e a dieta controle (CTR). Resultados: não houve interação entre as fontes de fósforo e sexo dos animais para qualquer um dos parâmetros estudados. A espessura do tecido compacto é menor na CTR, diferenciando-se das dietas DCP, MDCP e PPA, seguido pelas dietas SSP, TSP e ROCK, sendo que o maior valor foi observado na dieta com MDCP. A porosidade do tecido compacto foi maior com as dietas CTR e SSP. Conclusão: o uso de fontes menos elaboradas de fósforo como superfosfato simples e fosfato de rocha foram menos eficientes do que os outros tratamentos para melhorar a integridade óssea de suínos.

Frequency of Equine Monocytic Ehrlichiosis (EME) in Brazil / Relato epidemiológico da Erlichiose monocítica equina (EME) no Brasil

From a cross-sectional observational study with convenience samples, 347 blood samples from horses were collected from different physiographic regions, as follows: Santa Catarina Plateau (Santa Catarina State - SC), Médio Paraíba do Sul (São Paulo State - SP and Rio de Janeiro State RJ), Mountainous and Metropolitan regions (Rio de Janeiro State - RJ). Samples were tested for the presence of antibodies (IgG) anti Neorickettsia risticii by indirect immunofluorescence assay (IFA). The frequency obtained in this study corroborates with the ones obtained in the U.S.A., which refers to endemic regions. Fisher's exact test showed significant differences in the number of positive animals between regions, indicating that the probability of an animal becoming infected varies depending on the area. The CI 95% revealed no association between infection and geopolitical space. Moreover, Odds ratio test showed differences of an animal getting infected in different regions. This event could be influenced by the type of treatment used in each area, as the seasonal frequency of injury or even potential vectors. Therefore, there are seropositive animals for N. risticii in the studied areas, suggesting that this agent may be circulating in those regions. Future studies mainly based on molecular analyzes are needed to confirm these serological findings.

A partir de um delineamento observacional transversal com amostras de conveniência, 347 amostras de sangue foram coletadas de diferentes regiões fisiográficas: Planalto de Santa Catarina (Estado de Santa Catarina - SC), Região do Médio Paraíba do Sul (Estados de São Paulo - SP e Rio de Janeiro - RJ), Região Serrana e Metropolitana (ambas do Estado do Rio de Janeiro - RJ). As amostras foram testadas para a presença de anticorpos (IgG) anti-Neorickettsia risticii por imunofluorescência indireta (IFI). A prevalência obtida no presente estudo corrobora com demais resultados obtidos nos Estados Unidos da América. O Teste Exato de Fisher demonstrou diferença significativa no número de animais positivos entre as regiões, indicando assim que a probabilidade de um animal se infectar varia dependendo da região. O intervalo de confiança (IC 95%) revelou não haver associação entre a infecção e o espaço geopolítico, este evento pode ser influenciado pelo tipo de tratamento em cada área, como sazonalidade do agravo ou frequência de potenciais vetores. Assim, a soropositividade ora encontrada sugere a circulação de N. risticii nas áreas estudadas. Estudos futuros baseados, principalmente, em análises moleculares serão importantes para a confirmação dos achados sorológicos no presente trabalho.

Genotoxicity of polar and apolar extracts obtained from Qualea multiflora and Qualea grandiflora.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Qualea grandiflora and Qualea multiflora, which belong to the Vochysiaceae family, are common in the Brazilian savannah (Cerrado biome), and the local inhabitants use these species to treat external ulcers and gastric diseases and as an anti-inflammatory agent. Studies have demonstrated that these plants contain compounds that exhibit pharmacological activities; however, the risks associated with their consumption are not known. MATERIAL AND METHODS: In the present study, the mutagenicity of polar and apolar extracts from Qualea grandiflora and Qualea multiflora were assessed by employing the Ames assay with and without metabolic activation. Additionally, phytochemical analyses (HPLC-ESI-IT-MS, HPLC-UV-PDA and GC-IT-MS) were performed to identify the chemical constituents present in these species, including the evaluation of physico-chemical properties, such as polarity or apolarity of the organic compounds, which are related to each fraction obtained. These studies provide important information regarding the biochemical behaviour of these compounds. RESULTS: All extracts exhibited mutagenicity, inducing frameshift mutations and base substitutions in DNA. Phytochemical analysis identified terpenes, ellagic acid derivatives and phytosteroids. CONCLUSIONS: The mutagenicity observed might be due to the presence of pentacyclic triterpenes and polyphenols, which are able to generate reactive oxygen species (ROS) and result in the potential to cause DNA damage. The genetic risk identified in this present work shows that special attention should be considered for the use of compounds obtained from these plant species in medicinal treatments. Further studies must be conducted to identify safe therapeutic doses.

Assessment of the genetic risks of a metallic alloy used in medical implants.

The use of artificial implants provides a palliative or permanent solution for individuals who have lost some bodily function through disease, an accident or natural wear. This functional loss can be compensated for by the use of medical devices produced from special biomaterials. Titanium alloy (Ti-6Al-4V) is a well-established primary metallic biomaterial for orthopedic implants, but the toxicity of the chemical components of this alloy has become an issue of concern. In this work, we used the MTT assay and micronucleus assay to examine the cytotoxicity and genotoxicity, respectively, of an extract obtained from this alloy. The MTT assay indicated that the mitochondrial activity and cell viability of CHO-K1 cells were unaffected by exposure to the extract. However, the micronucleus assay revealed DNA damage and an increase in micronucleus frequency at all of the concentrations tested. These results show that ions released from Ti-6Al-4V alloy can cause DNA and nuclear damage and reinforce the importance of assessing the safety of metallic medical devices constructed from biomaterials.

Low-level laser therapy (LLLT) acts as cAMP-elevating agent in acute respiratory distress syndrome.

The aim of this work was to investigate if the low-level laser therapy (LLLT) on acute lung inflammation (ALI) induced by lipopolysaccharide (LPS) is linked to tumor necrosis factor (TNF) in alveolar macrophages (AM) from bronchoalveolar lavage fluid (BALF) of mice. LLLT has been reported to actuate positively for relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased TNF mRNA expression and dysfunction of cAMP generation observed in ALI can be influenced by LLLT. For in vivo studies, Balb/c mice (n = 5 for group) received LPS inhalation or TNF intra nasal instillation and 3 h after LPS or TNF-α, leukocytes in BALF were analyzed. LLLT administered perpendicularly to a point in the middle of the dissected bronchi with a wavelength of 660 nm and a dose of 4.5 J/cm(2). The mice were irradiated 15 min after ALI induction. In vitro AM from mice were cultured for analyses of TNF mRNA expression and protein and adenosine3':5'-cyclic monophosphate (cAMP) levels. One hour after LPS, the TNF and cAMP levels in AM were measured by ELISA. RT-PCR was used to measure TNF mRNA in AM. The LLLT was inefficient in potentiating the rolipram effect in presence of a TNF synthesis inhibitor. LLLT attenuated the neutrophil influx and TNF in BALF. In AM, the laser increased the cAMP and reduced the TNF-α mRNA. LLLT increases indirectly the cAMP in AM by a TNF-dependent mechanism.

Assessment of the genetic risks of a metallic alloy used in medical implants

The use of artificial implants provides a palliative or permanent solution for individuals who have lost some bodily function through disease, an accident or natural wear. This functional loss can be compensated for by the use of medical devices produced from special biomaterials. Titanium alloy (Ti-6Al-4V) is a well-established primary metallic biomaterial for orthopedic implants, but the toxicity of the chemical components of this alloy has become an issue of concern. In this work, we used the MTT assay and micronucleus assay to examine the cytotoxicity and genotoxicity, respectively, of an extract obtained from this alloy. The MTT assay indicated that the mitochondrial activity and cell viability of CHO-K1 cells were unaffected by exposure to the extract. However, the micronucleus assay revealed DNA damage and an increase in micronucleus frequency at all of the concentrations tested. These results show that ions released from Ti-6Al-4V alloy can cause DNA and nuclear damage and reinforce the importance of assessing the safety of metallic medical devices constructed from biomaterials.

Candida albicans: genotyping methods and clade related phenotypic characteristics

Several molecular methods, such as Southern blotting hybridization, Multilocus Sequence Typing, and DNA microsatellite analysis, have been employed to genotype Candida albicans. The genotype analysis allows to group strains in clades, that is, a group composed of one ancestor and its descendants. These genotype studies demonstrate that clades distribution is influenced by geographic area as well as that antifungal resistance is associated with particular clades. These findings suggested that C. albicans reproduces mainly in a clonal manner, with certain degree of DNA microevolution. Additionally, virulence factors and site of isolation have also been associated with clade specificity. The present article is a brief review about the methods used for Candida genotyping and the correlated clade systems established. Special emphasis is given to Ca3 hybridization, MLST, and Microsatellites. The present work is also focused on the phenotypic and physiological traits associated with Candida clades.

Antifungal susceptibility profile of trichosporon isolates: correlation between CLSI and etest methodologies

The aim of the present study was to evaluate the antifungal susceptibility profile of Trichosporon species isolated from different sources employing the Clinical and Laboratory Standards Institute (CLSI) method and E-test method. Thirty-four isolates of Trichosporon spp. and six CBS reference samples were tested for their susceptibility to Amphotericin B, 5-flucytosine, Fluconazole, Itraconazole, Voriconazole and Terbinafine. All species showed high Minimun Inhibitory Concentrations (MIC) for Itraconazole and susceptibility to Fluconazole, The comparison among the results obtained by the CLSI method and E-test revealed larger discrepancies among 5-flucytosine and Itraconazole. The present work provides epidemiological data that could influence therapeutic choices. Furthermore, the comparison between different methodologies could help to analyze results obtained by different laboratories.

Antifungal susceptibility profile of Trichosporon isolates: correlation between CLSI and etest methodologies.

The aim of the present study was to evaluate the antifungal susceptibility profile of Trichosporon species isolated from different sources employing the Clinical and Laboratory Standards Institute (CLSI) method and E-test method. Thirty-four isolates of Trichosporon spp. and six CBS reference samples were tested for their susceptibility to Amphotericin B, 5-flucytosine, Fluconazole, Itraconazole, Voriconazole and Terbinafine. All species showed high Minimun Inhibitory Concentrations (MIC) for Itraconazole and susceptibility to Fluconazole, The comparison among the results obtained by the CLSI method and E-test revealed larger discrepancies among 5-flucytosine and Itraconazole. The present work provides epidemiological data that could influence therapeutic choices. Furthermore, the comparison between different methodologies could help to analyze results obtained by different laboratories.

Candida albicans: genotyping methods and clade related phenotypic characteristics.

Several molecular methods, such as Southern blotting hybridization, Multilocus Sequence Typing, and DNA microsatellite analysis, have been employed to genotype Candida albicans. The genotype analysis allows to group strains in clades, that is, a group composed of one ancestor and its descendants. These genotype studies demonstrate that clades distribution is influenced by geographic area as well as that antifungal resistance is associated with particular clades. These findings suggested that C. albicans reproduces mainly in a clonal manner, with certain degree of DNA microevolution. Additionally, virulence factors and site of isolation have also been associated with clade specificity. The present article is a brief review about the methods used for Candida genotyping and the correlated clade systems established. Special emphasis is given to Ca3 hybridization, MLST, and Microsatellites. The present work is also focused on the phenotypic and physiological traits associated with Candida clades.

Catheters: instrumental advancements in biomedical applications of optical fibers.

This review is focused on the advancements in biomedical engineering regarding the elaboration of new prototypes of optical fiber catheters to be applied in spectroscopic analysis, such as Raman and fluorescence spectroscopy. Our group has contributed to the development of new prototypes with interesting properties, such as side-viewing signal excitation and collection, distal tip with bending control, and Raman scattering minimization from the optical fiber. In addition, several groups have contributed to other new catheter-improving properties of this spectroscopic device. However, a relatively small number of studies has been published in the literature, due to industrial interest in this interdisciplinary and multidisciplinary area. To our knowledge, no review that has focused on the applications of catheters to several modes of spectroscopy has been published. In this work we revised this topic, analyzing the advancements and limitations of the recent biomedical catheters.

Dynamic light scattering and optical absorption spectroscopy study of pH and temperature stabilities of the extracellular hemoglobin of Glossoscolex paulistus.

The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 x 10(6) Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D(h)) of 27 +/- 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D(h) of 10 +/- 1 nm. The decrease in D(h) suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DLS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25 degrees C and 38 degrees C. At 25 degrees C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38 degrees C is faster than that at 25 degrees C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25 degrees C. Autoxidation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS): effects of oligomeric dissociation.

The present work focuses on the interaction between the zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp). Electronic optical absorption, fluorescence emission and circular dichroism spectroscopy techniques, together with Gel-filtration chromatography, were used in order to evaluate the oligomeric dissociation as well as the autoxidation of HbGp as a function of the interaction with HPS. A peculiar behavior was observed for the HPS-HbGp interaction: a complex ferric species formation equilibrium was promoted, as a consequence of the autoxidation and oligomeric dissociation processes. At pH 7.0, HPS is more effective up to 1mM while at pH 9.0 the surfactant effect is more intense above 1mM. Furthermore, the interaction of HPS with HbGp was clearly less intense than the interaction of this hemoglobin with cationic (CTAC) and anionic (SDS) surfactants. Probably, this lower interaction with HPS is due to two factors: (i) the lower electrostatic attraction between the HPS surfactant and the protein surface ionic sites when compared to the electrostatic interaction between HbGp and cationic and anionic surfactants, and (ii) the low cmc of HPS, which probably reduces the interaction of the surfactant in the monomeric form with the protein. The present work emphasizes the importance of the electrostatic contribution in the interaction between ionic surfactants and HbGp. Furthermore, in the whole HPS concentration range used in this study, no folding and autoxidation decrease induced by this surfactant were observed. This is quite different from the literature data on the interaction between surfactants and tetrameric hemoglobins, that supports the occurrence of this behavior for the intracellular hemoglobins at low surfactant concentration range. Spectroscopic data are discussed and compared with the literature in order to improve the understanding of hemoglobin-surfactant interaction as well as the acid isoelectric point (pI) influence of the giant extracellular hemoglobins on their structure-activity relationship.

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation.

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.

Phosphate group effects upon the equilibrium of iron(III) meso-tetrakis (4-N-methylpyridiniumyl) porphyrin in aqueous solution.

Iron(III) meso-tetrakis (4-N-methylpyridiniumyl) porphyrin (FeTMPyP) undergoes a complex equilibrium in aqueous solution as a function of pH. Use of phosphate buffers, a common practice in biomedical applications of porphyrins, suggests the complexation of phosphate anion at the sixth coordination position to the iron, which contributes to the complexity of the equilibrium in the pH range from 1 to 4. In the absence of phosphate the equilibrium is simplified in a similar way as in the presence of high salt concentrations. Combined use of optical absorption, (1)H NMR and infrared spectroscopies, together with the literature data, suggest the formation of hexacoordinated monoaqueous-phosphate FeTMPyP complex in a limited acidic pH range. Discussion of the behavior of cationic FeTMPyP as compared to anionic iron(III) meso-tetrakis (4-sulfonatophenyl) porphyrin (FeTPPS(4)) is presented in regard to equilibrium of different species to explain the observed complex equilibrium.

SDS (sodium dodecyl sulfate) effect on the autoxidation of the Glossoscolex paulistus giant extracellular hemoglobin: kinetic studies at pH 7.0 and 9.0.

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on the autoxidation process of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is addressed in the present work. The complex oligomeric assembly of hemoglobin subunits may influence the autoxidation rate and the exponential decay behavior. Kinetic studies were developed using UV-vis measurements at 415 nm. These spectroscopic measurements are analyzed at two pH values, 7.0 and 9.0, where the hemoglobin presents different oligomeric assembly. At pH 7.0 a high stability of the native form of the oxy-hemoglobin is observed, while at pH 9.0 an intense dissociation of the oligomer is promoted by alkalization. This difference is evident by comparison of the rate constants in the absence of surfactant: at pH 7.0 the kinetics presents a mono-exponential behavior with a rate constant of 0.27 x 10(-4)s(-1) while at pH 9.0 a bi-exponential behavior was observed with rate constant increase to 7 x 10(-4)s(-1) (fast process) and 1 x 10(-4)s(-1) (slow process). In the autoxidation induced by SDS two factors affect significantly the process rate, namely, the oligomeric arrangement of the hemoglobin and the strength of the interaction between SDS and HbGp. At pH 7.0, for SDS concentrations up to 0.3mM, a mono-exponential behavior was observed, showing rate constants around 0.4 x 10(-4)s(-1), which suggest that the hemoglobin still maintains the more compact structure observed at this pH for the native protein. In the SDS concentration range 0.75-1.0mM, the mono-exponential process changes into a bi-exponential behavior with rate constants varying from 48 x 10(-4) up to 99 x 10(-4)s(-1) for the fast process and from 1.7 x 10(-4) up to 3.7 x 10(-4)s(-1) for the slow process, suggesting hemoglobin dissociation. At pH 9.0, a bi-exponential decay is observed for all studied SDS concentration range, presenting rate constants from 11.0 x 10(-4) up to 179 x 10(-4)s(-1) for the fast process and from 1.0 x 10(-4) up to 8 x 10(4)s(-1) for the slow process probably due to hemoglobin dissociation, which is already present in the absence of surfactant. At pH 7.0, the highly packed native protein structure should inhibit the autoxidation process, but the SDS/HbGp interaction is more intense as compared to pH 9.0, due to the acid pI value, promoting oligomeric dissociation. So, the autoxidation process is regulated at pH 7.0 by the interaction with SDS, which triggers oligomeric dissociation and increase of autoxidation rate. At pH 9.0, the autoxidation process should be very fast, probably due to the oligomeric dissociation, which is already present in the absence of surfactant. At alkaline pH, the interaction with SDS seems be weaker than at pH 7.0. This behavior at pH 7.0 can be observed through the higher autoxidation rate for the faster chains and it is associated to the acid pI of the giant extracellular hemoglobins.