Capturing cold-stress-related sequence diversity from a wild relative of common bean (Phaseolus angustissimus).

One restriction to the cultivation of common bean, Phaseolus vulgaris L., is its limited tolerance to low temperatures. In the present study, subtraction suppression hybridization was employed to enrich for stress responsive genes in both a chilling-susceptible common bean and a relatively more chilling-tolerant wild bean species, Phaseolus angustissimus. For each species, approximately 11 000 expressed sequence tags were generated. Comparative sequence analysis of the EST collection with the available annotated genome sequences of the model Fabaceae species Medicago truncatula and Glycine max identified protein homologues for approximately 65% and 80% of the Phaseolus sequences, respectively. This difference reflects the closer phylogenetic relationship between the genera Phaseolus and Glycine compared with Medicago. Annotation of the Phaseolus sequences was facilitated through this comparative analysis and indicated that several heat shock proteins, cytochrome P450s, and DNA binding factors were uniquely found among the sequences from the wild species P. angustissimus. The Phaseolus sequences have been made available on a GBrowse implementation using M. truncatula as the reference genome, providing rapid access to the sequence data and associated comparative genome data.

Cloning and characterization of the vacuolar ATPase B subunit from Plasmodium falciparum.

The transvacuolar pH gradient determines, to a significant extent, the distribution of the antimalarial drug chloroquine in Plasmodium falciparum. A proton pump, similar to the vacuolar ATPase found in many cell types, appears to regulate a pH gradient across the membranes of acidic compartments of the parasite. In order to understand and define the components involved in the maintenance of the vacuolar pH gradient, we have cloned and characterized a gene, designated VAP B, encoding a P. falciparum homologue of the B subunit of the vacuolar ATPase. The VAP B gene encodes a protein of 494 amino acids which has between 69% and 74% amino acid identity with the sequences of vacuolar ATPase B subunits of other organisms. The VAP B gene exists as a single copy gene on chromosome 4 that gives rise to a RNA transcript of 2.4 kb. Antibodies raised to the VAP B protein react specifically with a protein of 56-kDa, consistent with the size predicted from the gene sequence and with the homologous protein from other organisms. The 56-kDa protein is expressed throughout the asexual life cycle and subcellular localization by indirect immunofluorescence shows that the protein has a heterogeneous distribution over most of the parasite. This suggests that the function of the vacuolar proton ATPase is not confined to the regulation of the pH of the digestive vacuole.

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

Nucleotide binding properties of a P-glycoprotein homologue from Plasmodium falciparum.

The Plasmodium falciparum P-glycoprotein homologue 1 (PGH1) is structurally similar to several members of the ATP-binding cassette (ABC) superfamily of membrane transporters. We have examined whether the nucleotide binding domains predicted from the deduced amino sequence are functional by photoaffinity labeling of purified parasite digestive vacuoles with the analogue 8-azido-alpha-[32P]ATP (8-N3-ATP). This reagent labels a 160-kDa protein in vacuoles from both a chloroquine sensitive and a chloroquine-resistant parasite isolate. The 160-kDa protein could be immunoprecipitated with affinity-purified antibodies against the P. falciparum P-glycoprotein homologue (PGH1). Inhibition of photoaffinity labeling of PGH1 could be achieved with ATP, ADP, GTP and GDP but not with AMP or GMP. In order to map the 8-N3-ATP binding sites on PGH1, photoaffinity-labeled PGH1 was digested with trypsin and immunoprecipitated with site-specific antibodies. Taken together, these results indicate that 8-N3-ATP specifically labels PGH1 and that one binding site resides within the amino terminal half of the molecule. This supports the contention that PGH1 is involved in a nucleotide-regulated transport function across the membrane of the digestive vacuole.

Cloning and characterization of a vacuolar ATPase A subunit homologue from Plasmodium falciparum.

The distribution of the antimalarial drug chloroquine is determined to a significant extent by a transvacuolar pH gradient in Plasmodium falciparum. A proton pump similar to the vacuolar ATPase found in many cell types has been suggested to maintain a pH gradient across the membranes of acidic compartments in the parasite. In order to understand and define the components involved in the mechanism of acidification of parasite vesicles, we have cloned and characterized a gene, designated VAP-A, encoding a P. falciparum homologue of the catalytic A subunit of the vacuolar ATPase. The VAP-A gene encodes a polypeptide of 611 amino acids which shows between 56 to 61% amino acid identity over its entire length with the sequences of vacuolar ATPase A subunits from several species. The VAP-A gene exists as a single copy gene on P. falciparum chromosome 13 and gives rise to a transcript of 3.7 kb. Antibodies raised against a VAP-A gene segment expressed in Escherichia coli react specifically with a 67-kDa polypeptide, consistent with the size predicted from the sequence and with the size of the corresponding polypeptide in other organisms. The 67-kDa protein is present throughout the asexual erythrocytic cycle and is expressed at similar levels in 5 P. falciparum isolates of differing chloroquine sensitivity. Sequence analysis of the coding region of the VAP-A gene from 2 chloroquine-sensitive and 3 chloroquine-resistant isolates has shown no changes that are linked to chloroquine resistance. Therefore, a proposed chloroquine resistance-linked vacuolar acidification defect does not involve mutations in the VAP-A gene in the isolates we have studied.

Characterization of Ca(2+)-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni.

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

Drug resistance and the P-glycoprotein homologues of Plasmodium falciparum.

The chloroquine resistance phenotype of Plasmodium falciparum shares many similarities to multi-drug resistance in tumour cells and this has led to the identification of two mdr-like genes (pfmdr1, pfmdr2) from this human pathogen. The pfmdr1 gene has been linked to the chloroquine resistance phenotype, although a genetic cross appears to contradict these results. Analysis of drug resistant mutants selected in vitro has shown that the level of expression of the pfmdr1 gene can affect resistance to chloroquine, mefloquine, halofantrine and quinine. Mefloquine isolates from the field appear to always contain amplified levels of the pfmdr1 gene and also overexpress the transcript. They are also resistant to halofantrine suggesting a true multi-drug resistant phenotype.

A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole.

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.

The pfmdr gene homologues of Plasmodium falciparum.

Chloroquine resistance in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumour cells which is mediated by P-glycoprotein. P. falciparum has two mdr-like genes (pfmdr 1 and pfmdr 2) and pfmdr 1 has been linked to the chloroquine resistance phenotype. We show that pfmdr 1 encodes a protein of 160,000 Daltons that is expressed at higher levels in a chloroquine resistant cloned isolate. The pfmdr 2 gene is located on chromosome 14 and it is in equal copy number in chloroquine resistant and sensitive isolates. Therefore amplification of pfmdr 2 is not linked to chloroquine resistance. This is in contrast to the pfmdr 1 gene which has been shown to be amplified in some chloroquine resistant isolates.

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy.

Studies on schistosome protective immune responses have focused mainly on antigens of the parasite's syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.

Biochemical properties of a 24 kilodalton membrane glycoprotein antigen complex from Schistosoma mansoni.

Antibodies were affinity purified from crude antiserum by elution from the 24 kDa region of preparative one-dimensional Western blots containing immobilized adult Schistosoma mansoni inner bilayer membrane proteins. They were shown to be specific for a single acidic polypeptide complex, Smgp24, following immunoblotting from two-dimensional polyacrylamide gels. These antibodies were then used to detect the presence of the Smgp24 complex in fractions prepared from lectin affinity chromatography, phase separation in Triton X-114 and chemical and enzymatic carbohydrate modification treatments. The 24 kDa antigen was bound and specifically eluted from both concanavalin A and lentil lectin affinity matrices. In addition, the electrophoretic mobility of the antigen was shifted to approximately 20 kDa after treatment with endoglycosidase F and N-glycanase, but was not appreciably altered following treatment with endoglycosidase H, neuraminidase, or sodium meta-periodate. The 20 kDa species produced by endoglycosidase F or N-glycanase treatment no longer bound to the lectin affinity resins. The Smgp24 complex also partitioned almost quantitatively into the detergent-enriched phase after phase separation in Triton X-114 solutions. These results indicate that the Smgp24 complex is an antigenic integral membrane glycoprotein and may consist of a single polypeptide backbone which is extensively post- or co-translationally modified.

Developmental and immune regulation of gene expression in Hymenolepis diminuta.

Gene expression and its regulation was studied in the rat tapeworm, Hymenolepis diminuta, during strobilization. RNAs extracted from the different developmental stages of the parasite were translated in vitro in a rabbit reticulocyte lysate system. Two-dimensional patterns of the translational products were compared with the 2-dimensional patterns obtained by metabolic labelling with [35S]methionine. The results indicated a post-transcriptional regulation of gene expression during strobilization in this parasite. Gene expression and its regulation was also studied in H. diminuta obtained from 'non-permissive' hosts (mice) and immune suppressed mice and compared with the parasites of the same age obtained from 'permissive' rats hosts. The 2-dimensional patterns of the in vitro translation products, obtained by translating the RNA of different groups of parasites, were compared with the patterns of gene products obtained from parasites using [35S]methionine metabolic labelling. The results indicated a massive post-transcriptional regulation of gene expression, the latter inhibited in parasites obtained from normal mice, but not in immune suppressed mice.