Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

Infection with Trypanosoma cruzi causes acute myocarditis and chronic cardiomyopathy. Remarkable changes have been demonstrated in the structure and physiology of cardiomyocytes during infection by this parasite that may contribute to the cardiac dysfunction observed in Chagas' disease. We have investigated the expression of alpha-actinin, an actin-binding protein that plays a key role in the formation and maintenance of Z-lines, during the T. cruzi-cardiomyocyte interaction in vitro. Immunolocalization of alpha-actinin in control cardiomyocytes demonstrated a typical periodicity in the Z line of cardiac myofibrils, as well as its distribution at focal adhesion sites and along the cell-cell junctions. No significant changes were observed in the localization of alpha-actinin after 24 h of infection. In contrast, depletion of sarcomeric distribution of alpha-actinin occurred after 72 h in T. cruzi-infected cardiomyocytes, while no change occurred at focal adhesion contacts. Biochemical assays demonstrated a reduction of 46% and 32% in the expression of alpha-actinin after 24 h and 72 h of infection, respectively. Intracellular parasites were also stained with an anti-alpha-actinin antibody that recognized a protein of 78 kDa by Western blot. Taken together, our data demonstrate a degeneration of the myofibrils in cardiomyocytes induced by T. cruzi infection, rather than a disassembly of the I bands within sarcomeres.

Our data show that the pleural resident cell disappearance phenomenon noted early after inflammatory provocation depends on the activation of the fibrinogen-fibrin system, and is not required for the subsequent leukocyte recruitment.

The presence and localization of storage polysaccharides and of polysaccharides as cell structure constituents of Paracoccidioides brasiliensis yeast-like cells and protoplasts were studied using the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) technique. Yeast-like cells presented glycogen particles in the form of rosettes, which were mostly concentrated in regions of the cytoplasm. Cells in the budding process presented small amounts of glycogen in their matrix. The intracellular membranes and the attachment of the mother cell to the bud were clearly labelled by silver, demonstrating a large amount of glyco-conjugates. The protoplasts presented a small amount of glycogen in their cytoplasm, a reduction probably due to the enzymatic treatment to which the cells were submitted.

Astroglial cells are involved in directional movements of neurons such as migration of the neuronal cell body and growth of neurites. In the mammalian midbrain, medial (M) and lateral (L) radial glia and derived astrocytes differ in their ability to support neuritic growth. In previous work, we have demonstrated that the growthpermissive ability of L astrocytes and non-permissive properties of M astrocytes correlate with the respective composition of the cell surface-associated and secreted glycosaminoglycans (GAGs). Recent work also shows that the GAG-degrading enzyme heparitinase I increases the neurite growth-promoting ability of M midbrain astrocytes (Garcia-Abreu J et al. 2000 Glia 29: 260). In agreement with previous AFM studies of living glial cell lines and cells in primary culture, imaging of living L and M cells at similar load forces showed structures identified as F-actin fibers. Moreover, no systematic differences were observed between L and M pictures. By contrast, the surfaces of formaldehyde-fixed lateral (L) and medial (M) astrocytes differ by the presence of conspicuous 250 nm protrusions in the former, and of a fibrillar network in the extracellular matrix of the latter. Furthermore, we show that treatment with heparitinase I leads to disappearance of the fibrils from M cells and, consequently, to the assumption of an L-like appearance. Our results suggest that the formation of fibrils may follow from an ability to form large aggregates by association of HS carbohydrate units as has been unexpectedly detected by AFM for oligomers of polysialic acid or polysialic acid-containing carbohydrate units of N-CAM, with formation of filament bundles (Toikka J et al. 1998. J Biol Chem 273: 28557). Taken together with the functional effects of heparitinase I treatment, our present results demonstrate an important role of the extracellular matrix on the functional properties of astrocytes. They also emphasize the power and potentialities of AFM in the study of the extracellular matrix on the surface of fixed cells. - ( June 27, 2000 ) . * Supported by PRONEX/ MCT, CNPq, FAPERJ, CEPG/UFRJ. E-mail: gweissmuller@hotmail.com Aggregation of amyloid β peptide (Aβ) into fibrils and deposition as senile plaques are primarily related to neurotoxicity in Alzheimer's Disease (AD). Thus, agents interfering with aggregation may be potentially useful in preventing or decreasing Aβ toxicity. We first studied the effects of guanidine hydrochloride and temperature on the stability of fibrillar Aβ using peptides truncated at different positions. These experiments suggested that hydrophobic interactions mediated by the C-terminal portion of Aβ are important for fibril stability. Based on these results, we found two compounds capable of disaggregating amyloid fibrils at micromolar concentrations, as indicated by light scattering measurements and transmission electron microscopy. When applied to primary cultures of E18 rat hippocampal neurons, both drugs significantly reduced Aβ-induced cell death (as assayed by trypan blue ...