Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation.

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H(2)O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4 -- used as an indicator strain, B. licheniformis T6-5, and B. firmus H(2)O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H(2)O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H(2)O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H(2)O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.

Production of antimicrobial substances by Bacillus subtilis LFE-1, B. firmus HO-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil.

AIMS: Forty Bacillus strains isolated from a Brazilian oil reservoir were tested against each other to select strains producing antimicrobial substances (AMS). Three strains, Bacillus subtilis (LFE-1), Bacillus firmus (H2O-1) and Bacillus licheniformis (T6-5), were selected due to their ability to inhibit more than 65% of the Bacillus strains tested. These three strains were also investigated for their capability to inhibit sulphate-reducing bacteria (SRB). Furthermore, physiological and biochemical characteristics of the antimicrobial compounds produced by the selected strains were determined. METHODS AND RESULTS: Among the forty strains tested, 36 (90%) strains were able to inhibit at least one Bacillus strain used as indicator in plate assays and three of them (LFE-1, T6-5 and H2O-1) were able to inhibit 65, 70 and 97.5% of the 40 strains studied here respectively. Clear zones of inhibition were observed when H2O-1 was tested against SRB-containing consortium T6-lab and Desulfovibrio alaskensis strain NCIMB 13491, while strain T6-5 was able to inhibit only the D. alaskensis strain. The three substances showed to be insensitive to different enzymes and chemicals, were heat stable and the substances produced by strains T6-5 and H2O-1 were active over a wide pH range. CONCLUSIONS: Three different AMS produced by Bacillus strains from an oil reservoir, two of them with activity against SRB, are presented here. SIGNIFICANCE AND IMPACT OF THE STUDY: The preliminary characterization of these AMS points to their potential use as biocides in the petroleum industry for controlling problems associated with SRB.

Differential expression of mRNA coding for the alpha-2-macroglobulin family and the LRP receptor system in C57BL/6J and C3H/HeJ male mice.

Expression of mouse A2M (MAM), murinoglobulin (MUG), the A2M receptor or LDL-Receptor related protein (A2MR/LRP) and the Receptor Associated Protein (RAP) were measured by northern blotting of mRNA isolated from liver, heart and peritoneal macrophages from C3H/HeJ and C57BL/6J (B6) mice. Marked differences between males of the two mouse strains were observed for MAM and MUG mRNA levels in liver, which were reflected in plasma levels of both proteinase inhibitors, as confirmed by immune-electrophoresis. C3H/HeJ mice had higher levels of the MAM and MUG mRNA and their corresponding plasma proteins than B6 mice. B6 mice expressed higher levels of LRP mRNA relative to C3H/HeJ mice but had lower levels of RAP mRNA. LRP receptor activity, assayed by fluoresceinated-A2M binding, was higher in B6 cells. The present data contribute to the knowledge of genetic background characteristics among male mouse of these two strains, which can take part in many biological events such as lipid metabolism, inflammation and immune response to different infectious agents.

Trypanosoma cruzi: acute infection affects expression of alpha-2-macroglobulin and A2MR/LRP receptor differently in C3H and C57BL/6 mice.

Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.

A cardiomyocyte mannose receptor system is involved in Trypanosoma cruzi invasion and is down-modulated after infection.

Mannosyl binding sites were detected "in vitro" on cardiomyocytes (CM) surface using horseradish peroxidase (HRP) as the ligand. Binding assays revealed a specific recognition system, which was time- and concentration-dependent. The binding required physiological pH and was inhibited by EDTA and trypsin treatments. HRP binding was reduced by pre-incubations with low concentrations of D-mannose. Ultrastructural analysis of the endocytic process was followed using HRP coupled to colloidal gold particles (HRP-Au). The tracer was found within caveolae characterizing early steps of the receptor-mediated endocytosis. The addition of 10 mM D-mannose to the interaction medium blocked Trypanosoma cruzi uptake by CM. The labeling of CM with a subsaturating concentration of HRP-Au before their infection showed, by ultrastructural studies, that its association with trypomastigote forms occurred frequently near to HRP-gold particles that could also be seen to comprise the parasitophorous vacuole. After infection of CM with T. cruzi, a considerable reduction on HRP binding was noticed. Binding was almost completely restored by treating the infected cultures with the trypanocidal drug Nifurtimox. Our "in vitro" findings suggest that cardiomyocyte's mannose receptors localized at the sarcolemma mediates T. cruzi recognition and can be down-modulated by parasite infection.