Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil

INTRODUCTION: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.

Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

Spectrophotometric study of the chromium(III)/azide system in the ultraviolet region and its analytical application

A sensitive and alternative method for the spectrophotometric determination of chromium(III) based on the formation of chromium(III)/azide complexes was established by investigating a new band in the ultraviolet region. The best experimental conditions for the analytical determination of this metallic ion were: ligand and perchloric acid analytical concentration = 493 and 12.0 mmol L-1, respectively; aqueous medium; T = 25.0 ºC; contact time = 1 hour. The maximum molar absorptivity coefficient occurred at 287 nm (average 1.481 ± 0.008 ´ 10(4) L mol-1 cm-1), leading to the determination of metal ion concentrations one hundred times lower than the ones formerly determined in the visible region. The system obeys Beer's Law and is suitable for chromium determination in the 0.702-2.81 mg L-1 concentration range (15-65% T, 1.00 cm-width quartz cells). Analytical applications of the current method were tested with a nutritional supplement containing chromium. Results were compared with those obtained with atomic absorption spectrometry.

The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 ± 1.09%) versus controls (1.12 ± 0.18%), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 ± 0.83%) than hypertensive (8.50 ± 1.11%) and alcoholic (10.77 ± 2.09%) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 ± 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 ± 0.07), hypertensive (0.75 ± 0.07), and alcoholic (0.81 ± 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Detection of hilA gene sequences in serovars of Salmonella enterica sufigbspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.

Typhoid fever as cellular microbiological model

The knowledge about typhoid fever pathogenesis is growing in the last years, mainly about the cellular and molecular phenomena that are responsible by clinical manifestations of this disease. In this article are discussed several recent discoveries, as follows: a) Bacterial type III protein secretion system; b) The five virulence genes of Salmonella spp. that encoding Sips (Salmonella invasion protein) A, B, C, D and E, which are capable of induce apoptosis in macrophages; c) The function of Toll R2 and Toll R4 receptors present in the macrophage surface (discovered in the Drosophila). The Toll family receptors are critical in the signalizing mediated by LPS in macrophages in association with LBP and CD14; d) The lines of immune defense between intestinal lumen and internal organs; e) The fundamental role of the endothelial cells in the inflammatory deviation from bloodstream into infected tissues by bacteria. In addition to above subjects, the authors comment the correlation between the clinical features of typhoid fever and the cellular and molecular phenomena of this disease, as well as the therapeutic consequences of this knowledge.

Structure-function relationships for the interleukin 2 receptor system

Receptors for interleukin 2 (IL-2) esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta]) chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s) and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

CHARACTERIZING THE MECHANISM OF QUETIAPINE DISTRIBUTION IN LIPID-CORE NANOCAPSULES PSEUDO-PHASES USING A VALIDATED LC/UV METHOD

Quetiapine is an atypical antipsychotic used to treat schizophrenia. However, despite great interest for its chronic therapeutic use, quetiapine has some important side effects such as weight gain induction. The development of a quetiapine nanocarrier can potentially target the drug into central nervous system, resulting in a reduction of systemic side effects and improved patient treatment. In the present work, a simple liquid chromatography/ultraviolet detection (LC/UV) analytical method was developed and validated for quantification of total quetiapine content in lipid core nanocapsules as well as for determination of incorporation efficiency. An algorithm proposed by Oliveira et al. (2012) was applied to characterize the distribution of quetiapine in the pseudo-phases of the nanocarrier, leading to a better understanding of the quetiapine nanoparticles produced. The analytical methodology developed was specific, linear in the range of 0.5 to 100 µg mL−1 (r2 > 0,99), and accurate and precise (R.S.D < ±5%). The absolute recovery of quetiapine from the nanoparticles was approximately 98% with an incorporation efficiency of approximately 96%. The results indicated that quetiapine was present in a type III distribution according to the algorithm, and was mainly located in the core of the nanoparticle because of its logD in the formulation pH (6.86 ± 0.4).

Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.