In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br.

This work evaluated the in vitro cytotoxic activity of laticifer proteins (LP) recovered from the latex of the medicinal plant Calotropis procera. The LP displayed considerable cytotoxicity with IC(50) values ranging from 0.42 to 1.36 microg/ml to SF295 and MDA-MB-435 cell lines, respectively. In healthy peripheral blood mononuclear cells exposed to LP (10 microg/ml) for 72 h, no noticeable effects on viability or cell morphology were seen. The fractionating of LP on an ion exchange chromatography gave rise to a new fraction (PI) that retained almost all cytotoxicity. The cytotoxic effects of both LP and PI were diminished when previously treated with pronase, or 2-mercaptoethanol, suggesting a protein nature of active molecules, however, pre-incubation with dithiothreitol (DTT) only reduced PI activity. PI did not exhibit cysteine proteinase activity, indicating that cysteine proteinases, abundantly found in LP, are not implicated in LP cytotoxicity. In this study, using HL-60 cell as a model, LP was shown to inhibit DNA synthesis. This is probably due to alterations in the topology of DNA, since it was observed that LP is able to interfere in topoisomerase I activity by somehow acting upon DNA. LP provoked reduction in cell number but it did not cause any significant increase in the number of non-viable cells. These findings corroborated with the morphologic analysis, where cells treated with LP showed morphology of apoptotic process with abundant vacuoles, chromatin condensation and fragmentation of the nuclei. The results of this study suggests that LP is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines.

Biochemical characterisation of the trehalase of thermophilic fungi: an enzyme with mixed properties of neutral and acid trehalase.

The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.

Human platelets produce granulocyte-macrophage colony-stimulating factor and delay eosinophil apoptosis.

An association between eosinophils and platelets has been described in several diseases, most notably asthma. Although the mechanisms through which platelets influence eosinophil behavior are not well defined, platelets seem to contribute to the selective accumulation of eosinophils at sites of allergic inflammation by virtue of their ability to produce eosinophil chemotactic factors. We report here for the first time that platelets delay apoptosis, thus enhancing eosinophil survival. A marked inhibition of spontaneous apoptosis was observed using eosinophil:platelet ratios of 1:50, 1:25, 1:10, and 1:5. Moreover, promotion of eosinophil apoptosis by either pronase or dexamethasone was also inhibited greatly in the presence of platelets. The antiapoptotic effect mediated by platelets was dependent on the release of soluble products and was significantly inhibited by neutralizing antibodies directed to GM-CSF. Studies performed by flow cytometry, directed to analyze the cellular source of this cytokine, demonstrated that intracytoplasmic GM-CSF is present in resting platelets. Moreover, GM-CSF was found in platelet supernatants, at concentrations able to prevent eosinophil apoptosis. Our findings support a novel mechanism through which platelets may contribute to eosinophil accumulation at allergic inflammatory sites.

Neutrophil apoptosis induced by proteolytic enzymes.

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.

Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro.

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood-dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N-HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo.

Ultrastructural studies on the dimorphism of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum.

Whole cells or cell walls of the yeastlike and mycelial forms of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum were treated successively with sodium hydroxide, beta-1,3-glucanase and pronase. The microfibrils in the insoluble residues, probably composed of chitin, were examined in the electron microscope. In the yeastlike form, tightly interwoven, randomly oriented microfibrils were seen. On the other hand, in the mycelial form, a large portion of microfibrils tended to lie in a more or less longitudinal orientation. A role of chitin in the conversion from the yeastlike form to the mycelial form is discussed.

[Immunology of histoplasmosis: isolation of a polysaccharide-protein complex with immuno-specific activity from Histoplasma capsulatum]. / Inmunologia de la histoplasmosis: aislamiento de un complejo polisacárido-proteína con actividad inmunoespecífica a partir de Histoplasma capsulatum.

A polysaccharide protein complex (PPC) with a high specificity in immunodiffusion in gel (IDG) was isolated from a Histoplasma capsulatum strain. Although unaffected by proteolytic enzymes (pronase) and heat, the PPC antigen activity was destroyed by beta-glucosidase. These results suggest that the specificity of the PPC antigen might be located in the polysaccharide fraction. Polyacrylamide gel electrophoresis of the PPC antigen gave only one brand detected by Schiff's reagent (PAS).