The progression of human tuberculosis (TB) to active disease and transmission involves the development of a caseous granuloma that cavitates and releases infectious Mycobacterium tuberculosis bacilli. In the current study, we exploited genome-wide microarray analysis to determine that genes for lipid sequestration and metabolism were highly expressed in caseous TB granulomas. Immunohistological analysis of these granulomas confirmed the disproportionate abundance of the proteins involved in lipid metabolism in cells surrounding the caseum; namely, adipophilin, acyl-CoA synthetase long-chain family member 1 and saposin C. Biochemical analysis of the lipid species within the caseum identified cholesterol, cholesteryl esters, triacylglycerols and lactosylceramide, which implicated low-density lipoprotein-derived lipids as the most likely source. M. tuberculosis infection in vitro induced lipid droplet formation in murine and human macrophages. Furthermore, the M. tuberculosis cell wall lipid, trehalose dimycolate, induced a strong granulomatous response in mice, which was accompanied by foam cell formation. These results provide molecular and biochemical evidence that the development of the human TB granuloma to caseation correlates with pathogen-mediated dysregulation of host lipid metabolism.

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.

Advances in mass spectrometry and the availability of genomic databases made it possible to determine the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liquid chromatography tandem mass spectrometry of an extract of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homologue in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homology to any known peptide. Therefore, no evidence towards the physiological role of these 'orphan' peptides was available. We identified one of the eight peptides, IPNamide, in an extract of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole-mount immunocytochemistry and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the oesophagus.

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SUMMARYMalpighian tubules of the mealworm Tenebrio molitor were isolated for intracellular measurement of basolateral (Vbl) and,indirectly, apical (Vap) membrane potentials. In control Ringer (50 mmol l-1 K+, 140 mmol l-1Na+), Vbl was 24mV, cell negative, and Vap was 48 mV, cell negative with reference to the lumen. Ion substitution experiments involving K+ and Na+indicated that both Vbl and Vap were sensitive to the bathing K+ concentration, with the change in Vap being 60-77% that of Vbl. A 10-fold drop in bath [K+] irreversibly decreased fluid secretion rates from 6.38±0.95 nl min-1 (mean ± S.E.M.) to 1.48±0.52 nl min-1 (N=8). In the presence of 6 mmol l-1 Ba2+, a blocker of basal K+ channels,fluid secretion rates reversibly decreased and the hyperpolarization of both Vbl and Vap seen in 50 mmol l-1 and 140 mmol l-1 K+ indicated a favourable electrochemical gradient for basal K+ entry. In 5 mmol l-1 K+, Ba2+ induced two different responses: Vbl either hyperpolarized by approximately 10 mV or depolarised by approximately 14mV, according to the electrochemical gradient for K+, which was either inward or outward in low bath[K+]. Rubidium, a `permeant' potassium substitute, caused a hyperpolarization of Vbl, indicating the specificity of K+ channels found in Tenebrio tubule cells. Other possible K+ uptake mechanisms located in the basolateral membrane were investigated. Blocking of the putative electroneutral Na+/K+/2Cl- cotransporter by 10 μmol l-1 bumetanide reversibly decreased fluid secretion rates, with no detectable change in membrane potentials. Ouabain (1 mmol l-1), an Na+/K+-ATPase inhibitor, irreversibly decreased fluid secretion rates but had no effect on electrical potential differences either in the absence or presence of Ba2+. The results implicate K+ channels, the Na+/K+/2Cl-contransporter and the Na+/K+-ATPase in basal K+ and fluid transport of Tenebrio tubule cells.