The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) Leishmania is a trypanosomatid (a flagellated, parasitic protozoon) that causes a variety of diseases in humans and other mammals in the tropics and subtropics. The parasite alternates between sandfly and mammalian hosts and has several developmental forms. The promastigote is a motile, flagellated form that multiplies in the gut of the fly, the metacyclic is a nondividing, infective form that resides in the mouthparts of the fly, and the amastigote is a nonmotile form that lives and replicates in the phagolysosomal compartment of mammalian macrophages.A number of molecules have been shown to be important factors in the virulence of Leishmania. Two major surface molecules, lipophosphoglycan and the metalloproteinase gp63, are the most studied (for reviews, see refs.

The cpb genes of Leishmania mexicana encode stageregulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOHterminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn 18 , Asp 60 , Asn 61 , Ser 64 , and Tyr 84 ) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin Llike cysteine proteinases to diverse functions in parasitic protozoa.

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (deltaCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. deltaCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, deltaCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of deltaCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyl-diazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with deltaCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

The ryanodine (RY) receptors in beta-cells amplify signals by Ca2+-induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP-phosphodiesterases (cAMP-PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose-response relationship of caffeine-induced inhibition of cAMP-PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context-dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of beta-cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9-Methyl-7-bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP-PDEs, but it stimulated secretion in a glucose-dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of beta-cells mediate a distinct glucose-dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose-dependent manner.