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NaLtural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, lami- nm can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 ± 5 and 86 ± 3 nm of height, which approximately correspond to the sizes of the~ short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment El', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, invo've only interactions between the short arms. The electrostatic surface map of laminin al LG4 -5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin inthe future. [ABSTRACT FROM AUTHOR]

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na(+)+K(+))ATPase. On the other hand, we observed a Na(+)-ATPase with the following characteristics: (1) stimulated by Na(+) or K(+), but these effects are not addictive; (2) the apparent affinity is similar for Na(+) and K(+) (K(0.5) = 13.3 +/- 3.7 and 15.4 +/- 3.1mM, respectively), as well as the V(max) (24.9 +/- 1.5 or 27.5 +/- 1.6 nmol Pi mg(-1)min(-1), respectively); (3) insensitive up to 2mM ouabain; and (4) inhibited by furosemide with an IC(50) of 0.12 +/- 0.004 mM. Furthermore, this enzyme forms a Na(+)- or K(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.

The influence of tricyclazole (5-methyl-1,2,4-triazol[3,4]benzothiazole), a specific DHN-melanin inhibitor, on the cell walls and intracellular structures of Fonsecaea pedrosoi conidia and sclerotic cells was analyzed by transmission electron microscopy (TEM), deep-etching, and field emission scanning electron microscopy. The treatment of the fungus with 16 microg mL(-1) of tricyclazole (TC) did not significantly affect fungal viability, but electron microscopy observations showed several important morphological differences between TC-treated and non-TC treated cells. Control sclerotic cells presented patched granules, with an average diameter of 47 nm, on the cell surface, which were absent in TC-treated cells. Also, TC-treated sclerotic cells showed an undulated relief. TC treatment leads to an accumulation of electron lucent vacuoles in the fungal cytoplasm of both conidia and sclerotic cells, and treated conidia observed by deep etching showed a relevant thickening of the fungal cell wall. Together, these observations support the previous data of our group that F. pedrosoi synthesizes melanin in intracellular organelles. In addition, we suggest that melanin is not only an extracellular constituent but could also be dispersing all over the cell walls and could have an effective role in cross-linking different cell wall compounds that help maintain the regular shape of the cell wall., ((c) 2006 Wiley-Liss, Inc.)