Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium bergheistrain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

Pattern of cytokine and chemokine production by THP-1 derived macrophages in response to live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin Moreau strain

Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.

Protein content of leaf-cutting ant queens before the nuptial flight and during the post-claustral phase

Protein content of leaf-cutting ant queens before the nuptial flight and during the post-claustral phase. This study evaluated the crude protein content of queens of Atta sexdens before the nuptial flight and after the claustral phase in laboratory and field colonies. The hypothesis was that protein is used for survival of the queen and for early colony growth during the claustral phase. Additionally, the nest morphology, live biomass and adult population of field colonies were evaluated. Crude protein was determined by digestion of the organic material with sulfuric acid at high temperatures. The mean crude protein content was 123.23 ± 11.20 mg for females before the nuptial flight and 70.44 ± 12.21 mg for laboratory-reared queens after the claustral phase. The post-claustral crude protein content of field-collected queen was 55.90 ± 9.18 mg. With respect to the loss of crude protein as a function of duration of the claustral phase, laboratory-reared queens lost 52.79 mg and field-collected queens lost 67.33 mg compared to females before the nuptial flight. A positive linear correlation was observed between the weight of field-collected queens (256.4 ± 36.3 mg) and colony biomass (13.02 ± 9.12 g), but there was no correlation between biomass and nest depth (13.11 ± 3.82 cm). As expected, the present results support the hypothesis that protein is used for survival of the queen and for early colony growth, as demonstrated by the reduction in crude protein content as a function of duration of the claustral phase. To our knowledge, this is the first study to provide data of the dynamics of protein reserves in leaf-cutting ant queens during the claustral phase.

Production of fungal protein by solid substrate fermentation of cactus Cereus peruvianus and Opuntia ficus indica

Agricultural wastes from cactus Cereus peruvianus and Opuntia ficus indica were investigated for protein production by solid substrate fermentation. Firstly, the polyelectrolytes were extracted and used in water cleaning as auxiliary of flocculation and coagulation. The remaining fibrous material and peels were used as substrate for fermentation with Aspergillus niger. Glucoamylase and cellulase were the main enzymes produced. Amino acids were determined by HPLC and protein by Lowry's method. After 120 hours of fermentation the protein increased by 12.8%. Aspartic acid (1.27%), threonine (0.97%), glutamic acid (0.88%), valine (0.70%), serine (0.68%), arginine (0.82%), and phenylalanine (0.51%) were the principal amino acids produced.

Association between peripheral arterial disease and creactive protein in the japanese-brazilian population

OBJECTIVE: To evaluate the relationship between peripheral arterial disease and elevated levels of C-reactive protein in the Japanese-Brazilian population of high cardiovascular risk.METHODS: We conducted a cross-sectional study derived from a population-based study on the prevalence of diabetes and associated diseases in the Japanese-Brazilian population. One thousand, three hundred and thirty individuals aged e" 30 underwent clinical and laboratory examination, including measurement of ultrasensitive C-reactive protein. The diagnosis of peripheral arterial disease was performed by calculating the ankle-brachial index. We considered with peripheral arterial disease patients who had ankle-brachial index d" 0.9. After applying the exclusion criteria, 1,038 subjects completed the study.RESULTS: The mean age of the population was 56.8 years; 46% were male. The prevalence of peripheral arterial disease was 21%, with no difference between genders. Data analysis showed no association between peripheral arterial disease and ultrasensitive C-reactive protein. Patients with ankle-brachial index d" 0.70 showed higher values of ultrasensitive C-reactive protein and worse cardiometabolic profile. We found a positive independent association of peripheral arterial disease with hypertension and smoking.CONCLUSION: The association between low levels of ankle-brachial index and elevated levels of ultrasensitive C-reactive protein may suggest a relationship of gravity, aiding in the mapping of high-risk patients.

Involvement of hippocampal AMPA glutamate receptor changes and the cAMP/protein kinase A/CREB-P signalling pathway in memory consolidation of an avoidance task in rats

Training in step-down inhibitory avoidance (0.3-mA footshock) is followed by biochemical changes in rat hippocampus that strongly suggest an involvement of quantitative changes in glutamate AMPA receptors, followed by changes in the dopamine D1 receptor/cAMP/protein kinase A (PKA)/CREB-P signalling pathway in memory consolidation. AMPA binding to its receptor and levels of the AMPA receptor-specific subunit GluR1 increase in the hippocampus within the first 3 h after training (20-70%). Binding of the specific D1 receptor ligand, SCH23390, and cAMP levels increase within 3 or 6 h after training (30-100%). PKA activity and CREB-P levels show two peaks: a 35-40% increase 0 h after training, and a second increase 3-6 h later (35-60%). The results correlate with pharmacological findings showing an early post-training involvement of AMPA receptors, and a late involvement of the D1/cAMP/PKA/CREB-P pathway in memory consolidation of this task

Thalidomide protects mice against LPS-induced shock

Thalidomide has been shown to selectively inhibit TNF-a production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-a has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the production of TNF-a and other cytokines and on animal survival. After injection of 100-350 µg LPS into mice, cytokines including TNF-a, IL-6, IL-10, IL-1ß, GM-CSF and IFN-g were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-a levels were reduced by 93%, in a dose-dependent manner, and TNF-a mRNA expression in the spleens of mice was reduced by 70%. Serum IL-6 levels were also inhibited by 50%. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1ß or IFN-g. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 µg to 300 µg in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death

Acetylcholinesterase activity in the pons and medulla oblongata of rats after chronic electroconvulsive shock

An imbalance between cholinergic and noradrenergic neurotransmission has been proposed for the etiology of affective disorders. According to this hypothesis, depression would be the result of enhanced cholinergic and reduced noradrenergic neurotransmission. Repeated electroconvulsive shock (ECS) is an effective treatment for depression; moreover, in laboratory animals it induces changes in brain noradrenergic neurotransmission similar to those obtained by chronic treatment with antidepressant drugs (down-regulation of beta-adrenergic receptors). The aim of the present study was to determine whether repeated ECS in rats changes acetylcholinesterase (Achase) activity. Achase controls the level of acetylcholine (Ach) in the synaptic cleft and its levels seem to be regulated by the interaction between Ach and its receptor. Thus, a decrease in Achase activity would suggest decreased cholinergic activity. Adult male Wistar rats received one ECS (80 mA, 0.2 s, 60 Hz) daily for 7 days. Control rats were handled in the same way without receiving the shock. Rats were sacrificed 24 h after the last ECS and membrane-bound and soluble Achase activity was assayed in homogenates obtained from the pons and medulla oblongata. A statistically significant decrease in membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) (control 182.6 ± 14.8, ECS 162.2 ± 14.2, P<0.05) and an increase in soluble Achase activity in the medulla oblongata (control 133.6 ± 4.2, ECS 145.8 ± 12.3, P<0.05) were observed. No statistical differences were observed in Achase activity in the pons. Although repeated ECS induced a decrease in membrane-bound Achase activity, the lack of changes in the pons (control Achase activity: total 231.0 ± 34.5, membrane-bound 298.9 ± 18.5, soluble 203.9 ± 30.9), the region where the locus coeruleus, the main noradrenergic nucleus, is located, does not seem to favor the existence of an interaction between cholinergic and noradrenergic neurotransmission after ECS treatment

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells

Prostaglandins are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells is well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 µg/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87, 80, 70, 57, 29, 27 and 23 kDa in Aedes albopictus cells. When the proteins induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We also observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell

Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue

To investigate the role of nitric oxide in human sepsis, ten patients with severe septic shock requiring vasoactive drug therapy and mechanical ventilation were enrolled in a prospective, open, non-randomized clinical trial to study the acute effects of methylene blue, an inhibitor of guanylate cyclase. Hemodynamic and metabolic variables were measured before and 20, 40, 60, and 120 min after the start of a 1-h intravenous infusion of 4 mg/kg of methylene blue. Methylene blue administration caused a progressive increase in mean arterial pressure (60 [55-70] to 70 [65-100] mmHg, median [25-75th percentiles]; P<0.05), systemic vascular resistance index (649 [479-1084] to 1066 [585-1356] dyne s-1 cm-5 m-2; P<0.05) and the left ventricular stroke work index (35 [27-47] to 38 [32-56] g m-1 m-2; P<0.05) from baseline to 60 min. The pulmonary vascular resistance index increased from 150 [83-207] to 186 [121-367] dyne s-1 cm-5 m-2 after 20 min (P<0.05). Mixed venous saturation decreased from 65 [56-76] to 63 [55-69]% (P<0.05) after 60 min. The PaO2/FiO2 ratio decreased from 168 [131-215] to 132 [109-156] mmHg (P<0.05) after 40 min. Arterial lactate concentration decreased from 5.1 ± 2.9 to 4.5 ± 2.1 mmol/l, mean ± SD (P<0.05) after 60 min. Heart rate, cardiac filling pressures, cardiac output, oxygen delivery and consumption did not change. Methylene blue administration was safe and no adverse effect was observed. In severe human septic shock, a short infusion of methylene blue increases systemic vascular resistance and may improve myocardial function. Although there was a reduction in blood lactate concentration, this was not explained by an improvement in tissue oxygenation, since overall oxygen availability did not change. However, there was a significant increase in pulmonary vascular tone and a deterioration in gas exchange. Further studies are needed to demonstrate if nitric oxide blockade with methylene blue can be safe for patients with septic shock and, particularly, if it has an effect on pulmonary function.

Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

Cyanide-resistant alternative oxidase (AOX) is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP) is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX) and the proton electrochemical gradient energy-dissipating pathway (UCP) lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation). Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis

GLUT4 protein expression in white adipose tissue (WAT) and skeletal muscle (SM) was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, a ~70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as µg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as µg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.