Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-g production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Microbicidal effect of medicinal plant extracts (Psidium guajava Linn. and Carica papaya Linn.) upon bacteria isolated from fish muscle and known to induce diarrhea in children

Out of the twenty-four samples of shrimp and fish muscle used for this study, twelve were collected near a large marine sewer for waste disposal, 3 km off the coast of Fortaleza (Brazil) and used for the isolation of E. coli. Other twelve were collected at the Mucuripe fresh fish market (Fortaleza, Brazil) and used for the isolation of Staphylococcus aureus. Ethanol, water and acetone-diluted extracts of guava and papaya leaf sprouts were tested on the bacteria in order to verify their microbicidal potential. The E. coli strains used in the trials were rated LT positive. The papaya leaf extracts (Carica papaya Linn) showed no microbicidal activity while the guava sprout extracts (Psidium guajava Linn) displayed halos exceeding 13 mm for both species, an effect considered to be inhibitory by the method employed. Guava sprout extracts by 50% diluted ethanol most effectively inhibited E. coli (EPEC), while those in 50% acetone were less effective. It may be concluded that guava sprout extracts constitute a feasible treatment option for diarrhea caused by E. coli or by S. aureus-produced toxins, due to their quick curative action, easy availability in tropical countries and low cost to the consumer.

Metals in proteins from sulphate-reducing bacteria: adenylate kinase and ATP sulfurylase. Proteins containing cobalt, zinc, iron (II) ions

A Thesis submitted at the Faculty Science and Technology of the New University of Lisbon for a degree in Doctor of Philosophy in Biochemistry with specialization in Physical Biochemistry

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Detection of cardiac biomarker proteins using a disposable based on a molecularly imprinted polymer grafted onto graphite

A low-cost disposable was developed for rapid detection of the protein biomarker myoglobin (Myo) as a model analyte. A screen printed electrode was modified with a molecularly imprinted material grafted on a graphite support and incorporated in a matrix composed of poly(vinyl chloride) and the plasticizer o-nitrophenyloctyl ether. The protein-imprinted material (PIM) was produced by growing a reticulated polymer around a protein template. This is followed by radical polymerization of 4-styrenesulfonic acid, 2-aminoethyl methacrylate hydrochloride, and ethylene glycol dimethacrylate. The polymeric layer was then covalently bound to the graphitic support, and Myo was added during the imprinting stage to act as a template. Non-imprinted control materials (CM) were also prepared by omitting the Myo template. Morphological and structural analysis of PIM and CM by FTIR, Raman, and SEM/EDC microscopies confirmed the modification of the graphite support. The analytical performance of the SPE was assessed by square wave voltammetry. The average limit of detection is 0.79 μg of Myo per mL, and the slope is −0.193 ± 0.006 μA per decade. The SPE-CM cannot detect such low levels of Myo but gives a linear response at above 7.2 μg · mL−1, with a slope of −0.719 ± 0.02 μA per decade. Interference studies with hemoglobin, bovine serum albumin, creatinine, and sodium chloride demonstrated good selectivity for Myo. The method was successfully applied to the determination of Myo urine and is conceived to be a promising tool for screening Myo in point-of-care patients with ischemia.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

Rapid automated method for on-site determination of sulfadiazine in fish farming: a stainless steel veterinary syringe coated with a selective membrane of PVC serving as a potentiometric detector in a flow-injection-analysis system

Sulfadiazine is an antibiotic of the sulfonamide group and is used as a veterinary drug in fish farming. Monitoring it in the tanks is fundamental to control the applied doses and avoid environmental dissemination. Pursuing this goal, we included a novel potentiometric design in a flow-injection assembly. The electrode body was a stainless steel needle veterinary syringe of 0.8-mm inner diameter. A selective membrane of PVC acted as a sensory surface. Its composition, the length of the electrode, and other flow variables were optimized. The best performance was obtained for sensors of 1.5-cm length and a membrane composition of 33% PVC, 66% onitrophenyloctyl ether, 1% ion exchanger, and a small amount of a cationic additive. It exhibited Nernstian slopes of 61.0 mV decade-1 down to 1.0×10-5 mol L-1, with a limit of detection of 3.1×10-6 mol L-1 in flowing media. All necessary pH/ionic strength adjustments were performed online by merging the sample plug with a buffer carrier of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 4.9. The sensor exhibited the advantages of a fast response time (less than 15 s), long operational lifetime (60 days), and good selectivity for chloride, nitrite, acetate, tartrate, citrate, and ascorbate. The flow setup was successfully applied to the analysis of aquaculture waters. The analytical results were validated against those obtained with liquid chromatography–tandem mass spectrometry procedures. The sampling rate was about 84 samples per hour and recoveries ranged from 95.9 to 106.9%.

Behavioral and synaptic circuit analysis in models of neuropsychiatric disorders: Dissecting the in vivo role of the postsynaptic density proteins nArgBP2 and Shank3 using genetic engineered mice

Dissertation presented to obtain the Ph.D degree in Biology

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

New approach for vitamin E extraction in rainbow trout flesh: Application in fish fed commercial and red seaweed-supplemented diets

A vitamin E extraction method for rainbow trout flesh was optimized, validated, and applied in fish fed commercial and Gracilaria vermiculophylla-supplemented diets. Five extraction methods were compared. Vitamers were analyzed by HPLC/DAD/fluorescence. A solid-liquid extraction with n-hexane, which showed the best performance, was optimized and validated. Among the eight vitamers, only α- and γ-tocopherol were detected in muscle samples. The final method showed good linearity (>0.999), intra- (<3.1%) and inter-day precision (<2.6%), and recoveries (>96%). Detection and quantification limits were 39.9 and 121.0 ng/g of muscle, for α-tocopherol, and 111.4 ng/g and 337.6 ng/g, for γ-tocopherol, respectively. Compared to the control group, the dietary inclusion of 5% G. vermiculophylla resulted in a slight reduction of lipids in muscle and, consequently, of α- and γ-tocopherol. Nevertheless, vitamin E profile in lipids was maintained. In general, the results may be explained by the lower vitamin E level in seaweed-containing diet. Practical Applications: Based on the validation results and the low solvent consumption, the developed method can be used to analyze vitamin E in rainbow trout. The results of this work are also a valuable information source for fish feed industries and aquaculture producers, which can focus on improving seaweed inclusion in feeds as a source of vitamin E in fish muscle and, therefore, take full advantage of all bioactive components with an important role in fish health and flesh quality.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Significance of Biogenic Amines in Cold-Smoked Fish and Their Relation to Microbiological Characteristics of Products Available in Portuguese Retail Markets

Studies on microbial characterization of cold-smoked salmon and salmon trout during cold storage were performed on samples available in the Portuguese market. Samples were also classified microbiologically according to guidelines for ready-to-eat (RTE) products. Further investigations on sample variability and microbial abilities to produce tyramine and histamine were also performed. The coefficient of variation for viable counts of different groups of microorganisms of samples collected at retail market point was high in the first 2 wk of storage, mainly in the Enterobacteriaceae group and aerobic plate count (APC), suggesting that microbiological characteristics of samples were different in numbers, even within the same batch from the same producer. This variation seemed to be decreased when storage and temperature were controlled under lab conditions. The numbers of Enterobacteriaceae were influenced by storage temperature, as indicated by low microbial numbers in samples from controlled refrigeration. Lactic acid bacteria (LAB) and Enterobacteriaceae were predominant in commercial products, a significant percentage of which were tyramine and less histamine producers. These results might be influenced by (1) the technological processes in the early stages of production, (2) contamination during the smoking process, and (3) conditions and temperature fluctuations during cold storage at retail market point of sale.

Clinical and epidemiological study of 27 poisonings caused by ingesting puffer fish (Tetrodontidae) in the states of Santa Catarina and Bahia, Brazil

Puffer fish can be poisonous due to the presence of the potent neurotoxins such as Tetrodotoxin (TTX) and Saxitoxin (STX) found in its tissues. The authors report 27 human poisonings from ingestion of puffer fish in patients treated at Toxicology Centers in the states of Santa Catarina and Bahia, Brazil, between 1984 and January 2009. Poisonings were classified as moderate (52%) and severe (33%), two deaths were observed. Early diagnosis is very important to ensure respiratory support.

Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission

Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.