Interaction of bovine serum albumin (BSA) with ionic surfactants evaluated by electron paramagnetic resonance (EPR) spectroscopy

EPR spectra of 5- and 16-doxyl stearic acid nitroxide probes (5-DSA and 16-DSA, respectively) bound to bovine serum albumin (BSA) revealed that in the presence of ionic surfactants, at least, two label populations coexist in equilibrium. The rotational correlation times (tau) indicated that component I displays a more restricted mobility state, associated to the spin labels bound to the protein; the less immobilized component 2 is due to label localization in the surfactant aggregates. For both probes, the increase of surfactant concentration leads to higher motional levels of component 1 followed by a simultaneous decrease of this fraction of nitroxides and its conversion into component 2. For 10 mM cethyltrimethylammonium chloride (CTAC), the nitroxides are 100% bound to the protein, whereas at 10mM N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and sodium dodecyl sulfate (SDS) the fractions of bound nitroxides are reduced to 18% and 86%, respectively. No significant polarity changes were observed in the whole surfactant concentration range for component 1. Moreover, at higher surfactant concentration, component 2 exhibited a similar polarity as in the pure surfactant micelles. For 16-DSA the surfactant effect is different: at 10mM of HPS and CTAC the fractions of bound nitroxides are 76% and 49%, respectively, while at 10 mM SDS they are present exclusively in a micellar environment, consistent with 100% of component 2. Overall, both SDS and HPS are able to effectively displace the nitroxide probes from the protein binding sites. while CTAC seems to affect the nitroxide binding to a significantly smaller extent. (C) 2008 Elsevier B.V. All rights reserved.

A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Macronutrientes em cultivares de gérbera sob dois níveis de fertirrigação

A gérbera de vaso é uma planta ornamental conhecida mundialmente; entretanto, dispõem-se de reduzido volume de informações sobre suas técnicas de cultivo, em especial a nutrição e adubação, fatores essenciais para ganhos na qualidade produtiva. O presente trabalho teve por objetivo avaliar o teor e o acúmulo de macronutrientes em quatro cultivares de gérbera sob dois níveis de fertirrigação. O experimento foi conduzido em casa de vegetação, de maio a julho de 2006, na UNESP em Botucatu. O mesmo foi instalado em blocos casualizados e constituído de um fatorial 4x2, sendo 4 cultivares de gérbera (Cherry, Golden Yellow, Salmon Rose e Orange) e 2 concentrações de solução nutritiva (50% e 100%), correspondendo respectivamente às condutividades elétricas de 0,92 e 1,76 dS m-1, no período vegetativo e 1,07 e 2,04 dS m-1, no reprodutivo. Os nutrientes foram aplicados via fertirrigação, realizada manualmente, uma vez ao dia. Ao final dos períodos vegetativo e reprodutivo foram quantificados no tecido vegetal, os teores e os acúmulos dos macronutrientes. As demandas destes nutrientes foram diferenciadas entre as cultivares e a concentração da solução apresentou um efeito marcante no teor e acúmulo de N, P, Ca, Mg e S na planta. Os maiores acúmulos foram registrados no terço final do ciclo de produção, apresentando a seguinte ordem de absorção: K>N>Ca>Mg>P>S (415, 327, 33, 32, 20 mg planta-1).

Crescimento e produção de gérbera fertirrigada com solução nutritiva

Este trabalho foi conduzido com o objetivo de avaliar as características de crescimento e produção de quatro cultivares de gérbera de vaso fertirrigadas com duas soluções nutritivas. O experimento foi realizado em casa-de-vegetação, durante o período de maio a julho de 2006, na UNESP-FCA em Botucatu-SP. O delineamento experimental foi de blocos casualizados e constituído em esquema fatorial 4x2, sendo 4 cultivares de gérbera (Cherry, Golden Yellow, Salmon Rose e Orange) e 2 soluções nutritivas. As plantas foram conduzidas em vasos com volume de 1,3 L, preenchidos com substrato composto de 70% de casca de pinus fina e 30% de terra de subsolo. Avaliou-se o número de folhas e diâmetro de planta semanalmente durante o ciclo da cultura; área foliar e fitomassa seca ao final dos períodos vegetativo e reprodutivo; tempo necessário para a produção, número de inflorescências, diâmetro das inflorescências e hastes e altura de planta, no ponto de comercialização. As cultivares responderam de maneira diferenciada quanto às características de crescimento, Salmon Rose e Cherry apresentaram maior formação de parte aérea; Golden Yellow e Orange foram mais compactas. No ponto de comercialização, somente altura, diâmetro de inflorescência e haste apresentaram variação entre as cultivares. As duas soluções nutritivas foram eficientes para a produção de plantas de qualidade.

Surveillance of canine visceral leishmaniasis in a disease-free area

A leishmaniose é uma importante parasitose re-emergente observada no mundo, particularmente em países tropicais. Não há ainda relatos de casos autóctones no estado do Paraná. Não há até o momento referência de vigilância no reservatório canino, tais como Curitiba e região metropolitana do estado. O objetivo do estudo foi determinar a soroprevalência da leishmaniose visceral em cães entregues ao Centro de Controle de Zoonoses de São José dos Pinhais, Paraná para eutanásia. A detecção sorológica da presença de anticorpos contra Leishmania sp. foi realizada por (ELISA) indireto e pela Reação de Imunofluorescência Indireta (RIFI). Além disso, impressão de linfonodo poplíteo coletadas ao acaso de 50 cães com sinais clínicos suspeitos para leishmaniose visceral e analisados sob microscopia óptica para detecção de formas amastigotas, foram negativas. Amostras de soro de 364 animais foram testadas, e os resultados mostraram somente uma amostra positiva (0,0027%), reagente ao ELISA e negativa à RIFI, entretanto, o cão não apresentava sinais clínicos. A vigilância ao acaso em uma população de vários locais de uma área metropolitana pode ser uma forma de prevenção da disseminação da doença. Com base nos resultados observados, Curitiba e região metropolitana foram consideradas de baixo risco para a leishmaniose visceral.

The effect of elevated blood cortisol levels on the extinction of a conditioned stress response in rainbow trout

Following previously published observations that a conditioned response (CR) was lost more quickly by rainbow trout (Oncorhynchus mykiss) exhibiting a high responsiveness to stressors than by low responding individuals this study was designed to investigate the effects of exogenous cortisol on the retention of a CR in unselected rainbow trout. Fish held in isolation were conditioned over a 10-day period by pairing an innocuous signal (conditioned stimulus, CS: a water jet played on the surface of the tank water) with a mild stressor (unconditioned stimulus, US: 30 min of confinement). This resulted in a brief elevation of plasma cortisol levels (the CR) when the fish was exposed to the CS only. The effect of exogenous cortisol on the retention of the CR was evaluated by comparing the performance of fish that received cortisol-containing slow-release intraperitoneal implants, with fish receiving vehicle-only implants. Retention of the CR was assessed at intervals up to 35 days after conditioning ceased. The CR was considered to be evident when 30 min following presentation of the CS, mean plasma cortisol levels were significantly higher in conditioned than untrained fish. on day 1 both cortisol-implanted and vehicle-implanted conditioned fish exhibited a CR. However, from day 5 onwards the CR was observed only in the vehicle-implanted and conditioned group. This finding indicates that administration of cortisol accelerated the extinction of the CR in the cortisol-implanted fish, suggesting that elevated plasma cortisol levels can impair memory processes in rainbow trout. (c) 2006 Elsevier B.V. All rights reserved.

Chronic social stress in rainbow trout: Does it promote physiological habituation?

The effect of chronic social stress on growth, energetic substrates and hormones was tested in rainbow trout, Oncorhynchus mykiss. After a 14-day isolation period, the fish were paired for 8 days. In order to expose fish to chronic intermittent social contact during pairing, they were maintained in direct contact with each other during the first day. After that, a black plastic screen partition was introduced in each tank, preventing direct contact between animals. Every day the partition was removed for 30 min, allowing physical interaction between fish. At the end of pairing period, they were isolated again for 13 days. Fish were weighed and blood was sampled frequently during the experiment. Plasma levels of cortisol, growth hormone, glucose, total protein and free amino acids were quantified. Both dominants and subordinates had specific growth rate decreased during the pairing period, but only subordinates increased when the stressor was abolished (dominants: 0.32 +/- 0.21 and 0.24 +/- 0.41, subordinates: -0.77 +/- 0.29 and 0.37 +/- 0.31, respectively). Dominants showed a higher cortisol level one week after pairing condition had been abolished than subordinates (dominants: 56.76 +/- 13.26, subordinates: 31.89 +/- 13.36). We conclude that chronic condition of intermittent social stress represents a stressful condition for animals of both hierarchical ranks and a treatment of one daily short direct contact between conspecifics does not promote habituation in fish, as mentioned for other stressors. (C) 2007 Elsevier B.V. All rights reserved.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Calcitonin in bone-guided regeneration of mandibles in ovariectomized rats: densitometric, histologic and histomorphometric analysis

The aim of the present study was to investigate bone promotion in surgical defects created in the mandible of normal and ovariectomized female rats using calcitonin associated with a polytetrafluoroethylene barrier. The 100 female rats were divided into four groups: control (C), control treated with calcitonin (CM), ovariectomized control (OV) and ovariectomized treated with calcitonin (OVM). A circumscribed bone defect 4 mm in diameter was created in the region of the mandibular angle, and covered with the barrier. Groups CM and OVM received 2 IU/kg of synthetic salmon calcitonin intramuscularly three times a week. The animals were killed 3, 7, 14, 21 and 28 days after surgery. The bone defects were submitted to densitometric, histologic and histomorphometric analysis. Groups C and CM showed higher levels of bone formation after 7 days compared to the OV and OVM groups. A significant difference was observed between groups C and OV at 3-14 days. The OV group presented slower bone regeneration of the surgical bone defect created in the mandibular angle than group C. Synthetic salmon calcitonin accelerated regeneration of the bone defect in the mandibles of OVM animals similarly to group C, and also increased the formation of new bone during the regeneration process in CM.

Effect of Low-Level Laser Therapy and Calcitonin on Bone Repair in Castrated Rats: A Densitometric Study

Objective: To investigate the healing of bone defects in male rats treated with salmon calcitonin, low-level laser therapy (LLLT), or both. Background: Healing of bone defects still represents a challenge to health professionals in several areas. In this article, the effect of calcitonin in combination with LLLT on bone repair was studied. Densitometry was used as a valuable tool for the measurement of bone regeneration. Methods: Sixty male Wistar rats underwent bilateral castration surgery before the creation of a surgical bone defect. The animals were randomly divided into four groups: control, treated with calcitonin (Ca), treated with LLLT (La), and treated with calcitonin and LLLT (CaLa). Groups Ca and CaLa received 2 IU/kg of synthetic salmon calcitonin intra-muscularly three times a week. Groups La and CaLa received laser therapy using a gallium-aluminum-arsenide laser (10mW, 20 J/cm(2), wavelength 830 nm). Control animals were submitted to sham irradiation. The animals were sacrificed 7, 14, and 21 days after surgery, and bone defects were analyzed using densitometry. Results: The CaLa group had a higher degree of bone regeneration 14 and 21 days after surgery. Conclusions: The La and CaLa had significantly higher bone mineral density than the control and Ca groups.

Low-Level Laser Therapy and Calcitonin in Bone Repair: Densitometric Analysis

The aim of this work was to evaluate the association of low-level laser therapy (LLLT, 830 nm) and calcitonin in bone repair considering that bone healing remains a challenge to health professionals. Calcitonin has antiosteoclastic action and LLLT is a treatment that uses low-level lasers or light-emitting diodes to alter cellular function. Both are used to improve bone healing. Densitometry is a clinical noninvasive valuable tool used to evaluate bone mineral density (BMD). Sixty male rats were submitted to bone defect with a trephine bur, randomly divided into four groups of 15 animals each: control (C); synthetic salmon calcitonin (Ca); LLLT (La); LLLT combined with calcitonin (LaCa). Animals from Ca and LaCa received 2UI/Kg synthetic salmon calcitonin intramuscularly on alternate days after surgery. Animals from groups La and LaCa were treated with infrared LLLT (830 nm, 10mW, 20 J/cm(2), 6 s, contact mode). Five animals from each group were euthanized 7, 14, and 21 days after surgery and bone defects were analyzed by densitometry. Statistical analysis showed a significant difference in BMD values in LaCa group at 7 and 21 days (P = 0, 005). The results of the densitometric study showed that LLLT (830 nm) combined with calcitonin improved bone repair.

Extinction of a conditioned response in rainbow trout selected for high or low responsiveness to stress

Two lines of rainbow trout (Oncorhynchus mykiss) that exhibit divergent endocrine responsiveness to stressors also display disparate behavioral traits. To investigate whether the high-responding (HR) and low-responding (LR) fish also differ in cognitive function, the rate of extinction of a conditioned response was compared between the two lines. Groups of HR and LR fish were exposed to a paired conditioned stimulus (CS- water off) and unconditioned stimulus (US; confinement stressor). After exposure to 18 CS-US pairings, at least 70% of individuals of both lines acquired a conditioned response (CR) manifested as an elevation of blood cortisol levels on presentation of the CS only. Post-conditioning, the fish were tested by presentation of the CS at weekly intervals, for 4 weeks, with no further reinforcement, and the extinction of the CR in the two lines was compared. The decline in mean plasma cortisol levels after exposure to the CS over successive tests suggested that the CR was retained for a shorter period among the HR (<14 days) than LR fish (<21 days). The frequency of individuals within each line whose plasma cortisol levels indicated a stress response when exposed to the CS was significantly greater among the LR than HR fish at 14 and 21 days with no HR fish falling into this category at 21 days. At 28 days post-conditioning, there were no HR fish and only three LR fish were categorized as stressed. These results suggest that there are differences in cognitive function between the two lines. Possible mechanisms underlying these differences are discussed. (C) 2004 Elsevier B.V. All rights reserved.

Screening and HPLC methods for carotenoids in sweetpotato, cassava and maize for plant breeding trials

Analytical methods for sweetpotato, cassava and maize were developed. In orange and salmon-fleshed sweetpotatoes, (all-E)-beta-carotene predominated and results of spectrophotometric screening and HPLC quantification did not differ significantly. In yellow-fleshed sweetpotato and cassava, however, spectrophotometric screening overestimated the HPLC values because of the presence of several minor carotenoids. Aside from (all-E)-beta-carotene, Z-isomers were present in cassava in appreciable amounts. For both crops, extraction with acetone or tetrahydrofuran: methanol. (1: 1), using a mortar and pestle or a Polytron homogenizer, gave equivalent results. Rehydration of dry maize at room temperature for 30 min or at 85 degrees C for 5, 10 or 15 min gave equivalent results. Concentrations obtained with the C18 and C30 columns did not differ significantly for zeaxanthin, lutein, beta-cryptoxanthin and beta-carotene in the all-E-configuration, but their Z-isomers were difficult to locate in the chromatogram obtained with the C30 column. Extraction with tetrahydrofuran:methanol (1:1) gave significantly lower results for zeaxanthin and lutein. (c) 2005 Elsevier Ltd. All rights reserved.