Plasmodium falciparum is the causative agent of the most severe form of human malaria. The rapid multiplication of the parasite within human erythrocytes requires an active production of new membranes. Phosphatidylcholine is the most abundant phospholipid in Plasmodium membranes, and the pathways leading to its synthesis are attractive targets for chemotherapy. In addition to its synthesis from choline, phosphatidylcholine is synthesized from serine via an unknown pathway. Serine, which is actively transported by Plasmodium from human serum and readily available in the parasite, is subsequently converted into phosphoethanolamine. Here, we describe in P. falciparum a plant-like S-adenosyl-L-methionine-dependent three-step methylation reaction that converts phosphoethanolamine into phosphocholine, a precursor for the synthesis of phosphatidylcholine. We have identified the gene, PfPMT, encoding this activity and shown that its product is an unusual phosphoethanolamine methyltransferase with no human homologs. P. falciparum phosphoethanolamine methyltransferase (Pfpmt) is a monopartite enzyme with a single catalytic domain that is responsible for the three-step methylation reaction. Interestingly, Pfpmt activity is inhibited by its product phosphocholine and by the phosphocholine analog, miltefosine. We show that miltefosine can also inhibit parasite proliferation within human erythrocytes. The importance of this enzyme in P. falciparum membrane biogenesis makes it a potential target for malaria chemotherapy.M alaria, the world's most important parasitic disease, is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. Plasmodium falciparum is responsible for the most severe clinical cases of human malaria and kills Ͼ1 million children annually (1). The worldwide emergence of drugresistant P. falciparum strains has made treatment and prophylaxis of malaria increasingly difficult, thus emphasizing the need for new chemotherapeutic strategies to combat this disease. Previous studies in P. falciparum have indicated that the enzymes for synthesis of the major phospholipids are critical for the rapid multiplication of the parasite within human erythrocytes and display properties that are different enough from their human counterparts to be considered good targets for chemotherapy (2-5). Accordingly, compounds that interfere with membrane biogenesis inhibit parasite multiplication in vitro and clear malaria infection in mice and monkeys (3). In most eukaryotic organisms, phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) are the major phospholipids of cellular membranes. Whereas PtdCho and PtdEtn represent 44% and 18% of total phospholipids in yeast, respectively (6), these two phospholipids represent 40-50% and 35-40% of the total phospholipids in P. falciparum (5). How the parasite maintains such unusually high levels of PtdEtn and the implications of such a lipid composition on parasite development and survival are not known.Genetic and biochemical studies in various organisms reveal...

Breast-milk contains significant numbers of bifidobacteria and the maternal allergic status further deranges the counts of bifidobacteria in breast-milk. Maternal fecal and breast-milk bifidobacterial counts impacted on the infants' fecal Bifidobacterium levels. Breast-milk bacteria should thus be considered an important source of bacteria in the establishment of infantile intestinal microbiota.

Our findings suggest that activation of the IL-23/IL-17 axis is fundamentally connected to the etiology of CD and may represent the basis for the relapsing nature of the disease by increasing the sensitivity of epithelium to microbial LPS.

Long-term exposure of bacteria to bile may cause metabolic changes affecting their adhesive properties irreversibly. This may be taken as a criterion to define the probiotic properties of different strains.

Twenty-five Bifidobacterium strains isolated from infant feces were identified by sugar fermentation patterns and whole-cell protein analysis. Using gradient SDS-PAGE, six characteristic protein bands of the genus were detected in 40 strains of bifidobacteria but not in lactobacilli. Computerized numerical analysis enabled strains to be grouped in two main clusters. Strains of Bifidobacterium bifidum belong to a well-differentiated cluster that joins the cluster of the remaining species at 0.582 similarity. The predominant species among isolated strains from infant feces were B. bifidum, B. longum, and B. breve. Probiotic and technological indicators such as surface properties, inhibitory capacity, resistance to bile and low pH, and ability to grow under aerobic conditions were studied. Not all desirable characteristics were present in a single strain. In general, adherent and inhibitory strains were not resistant to bile, low pH, and aerobic conditions. Only 10 of 40 strains were resistant to 0.5% bile.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.

The action of bile on bifidobacteria was studied by measuring changes in zeta potential, hydrophobicity and adherence to enterocyte-like cells in vitro. Highly hydrophobic strains shocked with bile displayed more negative zeta potential values and a decrease in adherence. When a non-hydrophobic non-adherent strain (CIDCA 5324) was shocked with bile, an increase in hydrophobicity was observed. However, no changes of zeta potential or adherence properties were apparent. The action of the bile components was different from the action of whole bile. Cholate and deoxycholate produced a decrease in the negativity of zeta potential values of all strains studied whereas taurocholate displayed a shift in zeta potential of hydrophobic strains to more negative values, thus explaining the decrease in the autoaggregation by charge repulsion. However, the decrease in zeta potential caused by cholate and deoxycholate did not increase autoaggregation in a hydrophobic non-adherent strain (CIDCA 531). This suggests that other forces are contributing to autoaggregation.

A rapid method of screening for lactic acid bacteria with high inhibitory power was developed. The methodology employed was the agar-diffusion assay, which was standardized for the indicator strain, medium composition, and incubation conditions. The assay was performed in nutrient agar at 30°C with 108 spores of Bacillus subtilis per plate as indicator strain. The inhibition produced by supernatants of lactic acid bacteria cultures harvested at stationary phase was determined. The inhibitory powers of different strains were compared with a standard curve obtained with racemic lactic acid. Results obtained with lactic acid and supernatants of bacterial cultures demonstrate that the diameter of the inhibition zone (d) was related to the pH by the exponential relation d = a exp (− b pH). Results obtained with strains that produced inhibitory substances other than lactic acid could not be fitted into the standard linear curves obtained in the plot of ln d versus pH of lactic acid.