Effects of protein-calorie restriction on mechanical function of hypertrophied cardiac muscle

OBJECTIVE: To assess the effect of food restriction (FR) on hypertrophied cardiac muscle in spontaneously hypertensive rats (SHR). METHODS: Isolated papillary muscle preparations of the left ventricle (LV) of 60-day-old SHR and of normotensive Wistar-Kyoto (WKY) rats were studied. The rats were fed either an unrestricted diet or FR diet (50% of the intake of the control diet) for 30 days. The mechanical function of the muscles was evaluated through monitoring isometric and isotonic contractions. RESULTS: FR caused: 1) reduction in the body weight and LV weight of SHR and WKY rats; 2) increase in the time to peak shortening and the time to peak developed tension (DT) in the hypertrophied myocardium of the SHR; 3) diverging changes in the mechanical function of the normal cardiac muscles of WKY rats with reduction in maximum velocity of isotonic shortening and of the time for DT to decrease 50% of its maximum value, and increase of the resting tension and of the rate of tension decline. CONCLUSION: Short-term FR causes prolongation of the contraction time of hypertrophied muscles and paradoxal changes in mechanical performance of normal cardiac fibers, with worsening of the shortening indices and of the resting tension, and improvement of the isometric relaxation.

High-Sensitivity C-Reactive Protein as a Predictor of Cardiovascular Events after ST-Elevation Myocardial Infarction

Background: The association between high-sensitivity C-reactive protein and recurrent major adverse cardiovascular events (MACE) in patients with ST-elevation myocardial infarction who undergo primary percutaneous coronary intervention remains controversial. Objective: To investigate the potential association between high-sensitivity C-reactive protein and an increased risk of MACE such as death, heart failure, reinfarction, and new revascularization in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention. Methods: This prospective cohort study included 300 individuals aged >18 years who were diagnosed with ST-elevation myocardial infarction and underwent primary percutaneous coronary intervention at a tertiary health center. An instrument evaluating clinical variables and the Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) risk scores was used. High-sensitivity C-reactive protein was determined by nephelometry. The patients were followed-up during hospitalization and up to 30 days after infarction for the occurrence of MACE. Student's t, Mann-Whitney, chi-square, and logistic regression tests were used for statistical analyses. P values of ≤0.05 were considered statistically significant. Results: The mean age was 59.76 years, and 69.3% of patients were male. No statistically significant association was observed between high-sensitivity C-reactive protein and recurrent MACE (p = 0.11). However, high-sensitivity C-reactive protein was independently associated with 30-day mortality when adjusted for TIMI [odds ratio (OR), 1.27; 95% confidence interval (CI), 1.07-1.51; p = 0.005] and GRACE (OR, 1.26; 95% CI, 1.06-1.49; p = 0.007) risk scores. Conclusion: Although high-sensitivity C-reactive protein was not predictive of combined major cardiovascular events within 30 days after ST-elevation myocardial infarction in patients who underwent primary angioplasty and stent implantation, it was an independent predictor of 30-day mortality.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Longevity of adults of Peckia chrysostoma and Adiscochaeta ingens (Diptera: Sarcophagidae) reared with and without protein

With access to a proteic source in the diet the mean longevity and lethal time (MLT) of Peckia chrysostoma was 52.6 ± 5.5 and 30.3 ± 5.9 days, respectively. With an isolated protein source, the mean longevity was 49.1 ± 2.6 days and the MLT was 28.5 ± 0.8 days. Without a proteic source the mean longevity and the MLT lowered to 37.4 ± 4.0 and 18.1 ± 1.3 days, respectively. For Adiscochaeta ingens the mean longevity with access to a proteic source in the diet was 29.0 ± 6.0 days and the MLT was 16.7 ± 2.7 days. The figures with an isolated proteic source were 26.9 ± 4.8 and 14.9 ± 2.0 days, and without a proteic source were 24.7 ± 4.2 and 13.3 ± 1.4 days, respectively. These results show that in P. chrysostoma the longevity is higher than in A. ingens and that the access to the proteic source increase the longevity in both species.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Detection of antibodies to the 97 kDa component of Toxoplasma gondii in samples of human serum

This study was carried out to investigate the immune response against 97 kDa (p97) molecular marker of Toxoplasma gondii that has been characterized as a cytosolic protein and a component of the excreted-secreted antigens from this parasite. A total of 60 serum samples from patients were analyzed by enzime-linked immunosorbent assay and Western blot for toxoplasmosis. These samples were organized in three groups, based on clinical symptoms and results of serological tests. Group I: 20 samples reactive to IgG and IgM (acute phase); group II: 20 non-reactive samples (control group); and group III: 20 samples reactive only to IgG (chronic phase). Western blot was performed with total antigenic extracts or with excreted and secreted antigen from T. gondii to identify the fraction correspondent to p97. It was observed that this cytosolic component from T. gondii stimulates the immunologic system to produce both IgM and IgG antibodies in the beginning of the acute infection and IgG throughout the chronic stage of the asymptomatic toxoplasmosis.

Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole). This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Naturally acquired antibodies to merozoite surface protein (MSP)-1(19) and cumulative exposure to Plasmodium falciparum and Plasmodium vivax in remote populations of the Amazon Basin of Brazil

To infer recent patterns of malaria transmission, we measured naturally acquired IgG antibodies to the conserved 19-kDa C-terminal region of the merozoite surface protein (MSP)-1 of both Plasmodium vivax (PvMSP-1(19)) and Plasmodium falciparum (PfMSP-1(19)) in remote malaria-exposed populations of the Amazon Basin. Community-based cross-sectional surveys were carried out between 2002 and 2003 in subjects of all age groups living along the margins of the Unini and Jaú rivers, Northwestern Brazil. We found high prevalence rates of IgG antibodies to PvMSP-1(19) (64.0 - 69.6%) and PfMSP-1(19) (51.6 - 52.0%), with significant differences in the proportion of subjects with antibodies to PvMSP-1(19) according to age, place of residence and habitual involvement in high-risk activities, defining some groups of highly exposed people who might be preferential targets of malaria control measures. In contrast, no risk factor other than age was significantly associated with seropositivity to PfMSP-1(19). Only 14.1% and 19.3% of the subjects tested for antibodies to PvMSP-1(19) and PfMSP-1(19) in consecutive surveys (142 - 203 days apart) seroconverted or had a three fold or higher increase in the levels of antibodies to these antigens. We discuss the extent to which serological data correlated with the classical malariometric indices and morbidity indicators measured in the studied population at the time of the seroprevalence surveys and highlight some limitations of serological data for epidemiological inference.

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Rhodnius prolixus is the main Trypanosoma rangeli vector in several Latin-American countries and is susceptible to infection with KP1(+) strains; however, it presents an invasion-resistant response to KP1(-) strains. The present work has identified a trypanolytic protein against T. rangeli KP1(-) in the R. prolixus hemolymph which was fractioned with ammonium sulfate (following dialysis). The results revealed a protein component which did not depend on divalent cations for its biological function whilst keeping its trypanolytic activity at temperatures ranging from -20ºC to 37ºC, at 7.0 to 10.5 pH. The protein was partially purified by gel filtration chromatography and ionic exchange chromatography. The major component presented a molecular weight of around 79 kDa and an isoelectric point between 4.9 and 6.3 and may be directly related to hemolymph trypanolytic activity against T. rangeli KP1(-) populations.

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.

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30%) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61%) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis.