Synthesis, characterization, electrochemical behavior and in vitro protein tyrosine kinase inhibitory activity of the cymene-halogenobenzohydroxamato [Ru(eta(6)-cymene)(bha)Cl] complexes

The ruthenium(II)-cymene complexes [Ru(eta(6)-cymene)(bha)Cl] with substituted halogenobenzohydroxamato (bha) ligands (substituents = 4-F, 4-Cl, 4-Br, 2,4-F-2, 3,4-F-2, 2,5-F-2, 2,6-F-2) have been synthesized and characterized by elemental analysis, IR, H-1 NMR, C-13 NMR, cyclic voltammetry and controlled-potential electrolysis, and density functional theory (DFT) studies. The compositions of their frontier molecular orbitals (MOs) were established by DFT calculations, and the oxidation and reduction potentials are shown to follow the orders of the estimated vertical ionization potential and electron affinity, respectively. The electrochemical E-L Lever parameter is estimated for the first time for the various bha ligands, which can thus be ordered according to their electron-donor character. All complexes exhibit very strong protein tyrosine kinase (PTK) inhibitory activity, even much higher than that of genistein, the clinically used PTK inhibitory drug. The complex containing the 2,4-difluorobenzohydroxamato ligand is the most active one, and the dependences of the PTK activity of the complexes and of their redox potentials on the ring substituents are discussed. (C) 2012 Elsevier B.V. All rights reserved.

Avaliação da cinetica bacteriana na biolixiviação de calcopirita

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Production, purification and characterisation of polysaccharides from pleurotus ostreatus with antitumor activity

Background: Mushroom polysaccharides play an important role in functional foods because they exhibit biological modulator properties such as antitumour, antiviral and antibacterial activities. The present study involved the production, purification and characterisation of intracellular and extracellular free and protein-bound polysaccharides from Pleurotus ostreatus and the investigation of their growth-inhibitory effect on human carcinoma cell lines. Results: Several fermentation parameters were obtained: batch polysaccharide productivities of 0.013 +/- 8.12 x 10-5 and 0.037 +/- 0.0005 g L-1 day-1 for intracellular and extracellular polysaccharides respectively, a maximum biomass concentration of 9.35 +/- 0.18 g L-1, Pmax = 0.935 +/- 0.018 g L-1 day-1, µmax = 0.218 +/- 0.02 day-1, YEP/X = 0.040 +/- 0.0015 g g-1 and YIP/X = 0.014 +/- 0.0003 g g-1. Some polysaccharides exhibited superoxide dismutase (SOD)-like activity of 50-200 units. Fourier transform infrared analysis of the polysaccharides revealed absorption bands characteristic of such biological macromolecules. Cytotoxicity assays showed that both intracellular and extracellular polysaccharides exhibited antitumour activity towards several tested human carcinoma cell lines in a dose-dependent manner. Conclusion - The polysaccharides of P. ostreatus exhibited high SOD-like activity, which strongly supports their biological effect on tumour cell lines. The extracellular polysaccharides presented the highest antitumour activity towards the RL95 carcinoma cell line and should be further investigated as an antitumour agent.

Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

Evaluation of standard reagents for radial-immunodiffusion assays. In vitro control of rabies vaccines

The RID assay is one of the in vitro methods used for in-process control in the production of rabies vaccines for veterinary use. It has been shown to be very useful for determining antigen concentration in the final bulk product. The work presented in this paper, including the production and standardization of candidate standard reagents for use in the Radial Immunodiffusion Assay (RID) was carried out at the Pan American Institute for Food Protection and Zoonoses (INPPAZ/PAHO/WHO). The study was completed with the cooperation of the Faculty of Veterinary Sciences, National University of La Plata (NULP), Argentina, where the validation of the proposed standards and the quality control of samples from 28 different batches of rabies vaccines produced with Pasteur strain rabies virus (PV) in BHK cells were performed. The activity of the vaccines was determined by in vivo (NIH) and in vitro (RID)assays. The results of the candidate reagents for the reagent standardization tests showed stability, sensitivity and reproducibility. The Relative Potency the 1.2 between the problem vaccines and the reference vaccine was estimated by variance and regression analysis. The results of our validation study show that the INPPAZ (PAHO/WHO) is capable of producing and distributing the above-mentioned standard reagents, as well as of providing support for the incorporation of the RID technique (sensitive, rapid and inexpensive) to the laboratories that manufacture rabies vaccines in Latin America and the Caribbean.

The effect of method, standard and sample components on the total antioxidant capacity of commercial waters assessed by optical conventional assays

The total antioxidant capacity (TAC) of 28 flavoured water samples was assessed by ferric reducing antioxidant potential (FRAP), oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity (TEAC) and total reactive antioxidant potential (TRAP) methods. It was observed that flavoured waters had higher antioxidant activity than the corresponding natural ones. The observed differences were attributed to flavours, juice and vitamins. Generally, higher TAC contents were obtained on lemon waters and lower values on guava and raspberry flavoured waters. Lower and higher TACs were obtained by TRAP and ORAC method, respectively. Statistical analysis suggested that vitamins and flavours increased the antioxidant content of the commercial waters.

Measuring the cytochrome c nitrite reductase activity—practical considerations on the enzyme assays

Hindawi Publishing Corporation Bioinorganic Chemistry and Applications Volume 2010, Article ID 634597, 8 pages

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5%) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9%) and HTLV-2 in 21 sera (2.8%). Negative and indeterminate results were detected in four (0.5%) and 16 (2.1%) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas. A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

A contribuição das bombas de efluxo QacA e Smr para a multirresistência em Staphylococcus aureus

RESUMO: O efluxo de compostos antimicrobianos é um mecanismo importante na multirresistência em bactérias. Bombas de efluxo codificadas em plasmídeos, como a QacA e a Smr, estão implicadas na susceptibilidade reduzida a biocidas, geralmente utilizados na prevenção e controlo de infecções nosocomiais, incluindo as causadas por estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). Neste trabalho pretendeu-se avaliar a relevância de QacA e Smr no perfil de susceptibilidade dos isolados clínicos MRSA SM39 e SM52, que transportam os plasmídeos pSM39 e pSM52 com os determinantes qacA e smr, respectivamente. A actividade de efluxo das estirpes SM39 e SM39 curada (sem pSM39) e das estirpes SM52 e RN4220:pSM52 (estirpe susceptível RN4220 transformada com pSM52) foi caracterizada por: (1) determinação da concentração mínima inibitória (CMI) de biocidas, corantes e antibióticos, na ausência e presença dos inibidores de efluxo tioridazina, clorpromazina, verapamil e reserpina; e (2) fluorometria em tempo-real. A determinação de CMIs demonstrou que a actividade de efluxo mediada por QacA e Smr está envolvida na susceptibilidade reduzida aos biocidas e corantes testados, que incluíram o brometo de hexadeciltrimetilamónio, a cetrimida, o cloreto de benzalcónio, a berberina, o cloreto de dequalínio, a pentamidina e o brometo de etídeo. Os ensaios fluorométricos confirmaram a elevada actividade de efluxo presente nas estirpes com os genes qacA ou smr. A determinação de CMIs para antibióticos β-lactâmicos em conjunto com o teste da nitrocefina revelou a presença simultânea do gene qacA e de uma β-lactamase no plasmídeo pSM39. Este trabalho evidencia a importância das bombas de efluxo QacA e Smr na resistência a biocidas em estirpes MRSA e na sobrevivência destas estirpes em ambiente hospitalar e na comunidade, para além de destacar a questão da potencial co-resistência entre biocidas e antibióticos.--------------- ABSTRACT: Drug efflux has become an important cause of multidrug resistance (MDR) in bacteria. Plasmid-encoded MDR efflux pumps, such as QacA and Smr, are implicated in reduced susceptibility to biocides, generally used in the prevention and control of nosocomial infections, including the ones caused by methicillin-resistant Staphylococcus aureus (MRSA). In this work, we aimed to evaluate the relevance of QacA and Smr to the susceptibility profile of the clinical MRSA isolates SM39 and SM52, which harbor the plasmids pSM39 and pSM52 that carry the determinants qacA and smr, respectively. Efflux activity of strain SM39 and its plasmid-free counterpart, SM39 cured, SM52 and RN4220:pSM52 (susceptible strain RN4220 transformed with pSM52) was characterized by: (1) determination of minimum inhibitory concentration (MIC) of biocides, dyes and antibiotics, in the absence and presence of the efflux inhibitors thioridazine, chlorpromazine, verapamil and reserpine; and (2) real-time fluorometry. MIC determination showed that QacA and Smr mediated efflux was involved in the reduced susceptibility profile to the biocides and dyes tested, which included hexadecyltrymethylammonium bromide, cetrimide, benzalkonium chloride, berberine, dequalinium chloride, pentamidine and ethidium bromide. Fluorometric assays confirmed the higher efflux activity present in strains harboring qacA or smr genes. Moreover, MIC determination for β-lactam antibiotics together with the nitrocefin test confirmed the presence of a β-lactamase in the plasmid carried by SM39 strain, pSM39. This work highlights the relevance of QacA and Smr to the biocide resistance in MRSA strains, and consequently to their survival and maintenance in the hospital environment and in the community. Furthermore, the presence of a β-lactamase and qacA determinants in the the same plasmid reinforces the question of the potencial biocide/antibiotic co-resistance in MRSA strains.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

DETERMINATION OF VIRAL TROPISM BY GENOTYPING AND PHENOTYPING ASSAYS IN BRAZILIAN HIV-1-INFECTED PATIENTS

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.