The tonoplast-associated citrate binding protein (CBP) of Hevea brasiliensis. Photoaffinity labeling, purification, and cloning of the corresponding gene.

A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree (Hevea brasiliensis Muell. Arg.) and of malate and citrate transport into barley (Hordeum vulgare L.) vacuoles revealed very similar transport specificities. In order to identify proteins mediating the transport, two photoreactive analogues (N'-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate/citrate were synthesized and found to efficiently inhibit citrate uptake into barley vacuoles (Ki = 18 microM) and Hevea lutoid vesicles (Ki = 27 microM). In vacuoles from both plant species, these photoaffinity probes specifically labeled a single protein with a molecular mass of 23.6 kDa. This citrate binding protein (CBP) was purified to homogeneity from Hevea lutoids, and amino acid sequences were determined for NH2-terminal and tryptic peptides. Using degenerate oligonucleotides of the NH2-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of 238 amino acids, containing an NH2-terminal 31-amino acid signal sequence for endoplasmic reticulum targeting, a prerequisite for vacuolar localization. The mature CBP does not show significant sequence similarities to any known primary protein structure and thus represents a member of a novel class of proteins.

Uptake and killing of Leishmania mexicana amazonensis amastigotes by human skin fibroblasts.

This paper describes the in vitro infection of human established fibroblast lines by Leishmania mexicana amazonensis amastigotes. Intracellular parasites were located within vacuoles. The proportion of infected cells reached a peak of about 50% on Days 2 or 3, and decreased to almost 0 on Days 6 or 8. Transmission electron microscopy was used to document different stages of the degeneration of L. m. amazonensis amastigotes on Days 4 and 5. The proportion of infected fibroblasts decreased in both the control and the irradiated cells, indicating that dilution of the parasites by cell multiplication was not the main factor in the observed decrease of the infection with time. This conclusion was also supported by studies with fibroblasts prelabelled with 3H thymidine. In these studies, total cell associated radioactivity as well as the total and TCA precipitable radioactivity of the culture medium were similar for infected and non-infected fibroblasts during the course of infection. The abortive infection of fibroblasts by L. m. amazonensis provides a potentially useful model for the study of the relationship between host cells and intracellular parasites.