Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum

About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.     

Neuroinvasion of prions: insights from mouse models

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrP(C). Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrP(C) is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.     

Circulating leucocyte subpopulations in sedentary subjects following graded maximal exercise with hypoxia

Summary Ten healthy sedentary subjects [age, 27.5 (SD 3.5) years; height, 180 (SD 5) cm; mass, 69.3 (SD 6.3) kg] performed two periods of maximal incremental graded cycle ergometer exercise in a supine position. Randomly ordered and using an open spirometric system, one exercise was carried out during normoxia [maximal oxygen consumption ( O_2max)=38.6 (SD 3.5) ml·min^–1·kg^–1; maximal blood lactate concentration, 9.86 (SD 1.85) mmol·l^–1; test duration, 22.6 (SD 2.7) min], the other during hypoxia [ O_2max=33.2 (SD 3.2) ml·min^–1· kg^–1; maximal blood lactate concentration, 10.38 (SD 2.02) mmol·l^–1; test duration, 19.7 (SD 2.8) min]. At rest, immediately (0 p) and 60 min (60 p) after exercise, counts of leucocyte subpopulations (flow cytometry), cortisol and catecholamine concentrations were determined. At 0 p in contrast to normoxia, during hypoxia there was no significant increase of granulocytes. There were no significant differences between normoxia and hypoxia in the increases from rest to 0 p in counts of monocytes, total lymphocytes and lymphocyte subpopulations [clusters of differentiation (CD), CD3⁺, CD4⁺CD45RO^–, CD4⁺CD45RO⁺, CD8⁺CD45RO^–, CD8⁺CD45RO⁺, CD3⁺HLA-DR⁺, CD3^–CD16/CD56⁺, CD3⁺CD16/CD56⁺, CD 19⁺] as well as adrenaline, noradrenaline and cortisol concentrations. The counts of CD3 ^–CD16/CD56⁺-and CD8 ⁺CD45RO⁺-cells increased most. At 60 p, CD3^–CD16/CD56⁺ and CD3⁺CD16/CD56⁺-cell counts were below pre-exercise levels and under hypoxia slightly but significantly lower than under normoxia. We concluded that the exercise-induced mobilization and redistribution of most leucocyte and lymphocyte subpopulations were unimpaired under acute hypoxia at sea level. Reduced increases of granulocyte counts during the study and reduced cell numbers of natural killer cells and cytotoxic, not major histocompatibility complex-restricted T-cells, only indicated marginal effects on the immune system.     

Comparison of lymphocyte populations bearing surface immunoglobulins in avian bone marrow, bursa, spleen and thymus.

Rabbit anti-chicken gamma-globulin was labeled with 125I and then incubated with cells from the bursa, thymus, spleen, and bone marrow of 4- and 8-week old birds. The same procedure was carried out on 11-week-old agammaglobulinemic chickens. Autoradiography revealed that the majority of large, medium, and small bursal lymphocytes bind the antibodies while labeled lymphocytes of each type in the spleen and thymus never exceeded 11 or 4 percent, respectively. Labeled medium and small lymphocytes in the bone marrow increased from 4.2 and 1.7%, respectively, at 4 weeks of age, to 9.5 and 8.8%, respectively, at 8 weeks of age. Labeled lymphocytes of all sizes were completely absent in all tissues of agammaglobulinemic chicks, including the marrow. Therefore, the increase in frequency of labeled lymphocytes in the bone marrow with age may be a result of recruitment of cells from the bursa of Fabricius. The majority of lymphocytes in the bone marrow do not label. Therefore, lymphocytes from the bone marrow may be T cells, subsets of B cells, or neither T or B cells.     

The pathogenesis of molybdenum cofactor deficiency, its delay by maternal clearance, and its expression pattern in microarray analysis

Molybdenum cofactor (Moco)-deficiency is a lethal autosomal recessive disease, for which until now no effective therapy is available. The biochemical hallmark of this disorder is the inactivity of the Moco-dependent sulfite oxidase, which results in elevated sulfite and diminished sulfate levels throughout the organism. In humans, Moco-deficiency results in neurological damage, which is apparent in untreatable seizures and various brain dysmorphisms. We have recently described a murine model for Moco-deficiency, which reflects all enzyme and metabolite changes observed in the patients, and an efficient therapy using a biosynthetic precursor of Moco has been established in this animal model. We now analyzed these mice in detail and excluded morphological brain damage, while expression analysis with microarrays indicates a massive cell death program. This neuronal damage appears to be triggered by elevated sulfite levels and is ameliorated in affected embryos by maternal clearance.     

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Plant microtubules are organized into specific cell cycle-dependent arrays that have been implicated in diverse cellular processes, including cell division and organized cell expansion. Mutations in four Arabidopsis genes collectively called the PILZ group result in lethal embryos that consist of one or a few grossly enlarged cells. The mutant embryos lack microtubules but not actin filaments. Whereas the cytokinesis-specific syntaxin KNOLLE is not localized properly, trafficking of the putative auxin efflux carrier PIN1 to the plasma membrane is normal. The four PILZ group genes were isolated by map-based cloning and are shown to encode orthologs of mammalian tubulin-folding cofactors (TFCs) C, D, and E, and associated small G-protein Arl2 that mediate the formation of alpha/beta-tubulin heterodimers in vitro. The TFC C ortholog, PORCINO, was detected in cytosolic protein complexes and did not colocalize with microtubules. Another gene with a related, although weaker, embryo-lethal phenotype, KIESEL, was shown to encode a TFC A ortholog. Our genetic ablation of microtubules shows their requirement in cell division and vesicle trafficking during cytokinesis, whereas cell growth is mediated by microtubule-independent vesicle trafficking to the plasma membrane during interphase.     

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.