Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.

The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DNA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence.

Malaria: targeting parasite and host cell kinomes.

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays.

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance.

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A.

Pbcrk-1, the Plasmodium berghei orthologue of P. falciparum cdc-2 related kinase-1 (Pfcrk-1), is essential for completion of the intraerythrocytic asexual cycle.

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P.

Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems.

A study of selected Plasmodium yoelii messenger RNAs during hepatocyte infection.

We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap-1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation.

Quantitative assessment of murine retrovirus LP-BM5 infection in MAIDS by PCR and anion exchange HPLC.

In vitro amplification of DNA by PCR is a powerful tool to detect small amounts of DNA. It is now widely used for detection of pathogenic agents from extracellular fluids and organs. The use of anion exchange HPLC to quantify the PCR product resulting from the specific amplification of the DNA from the replicative-defective viral DNA responsible for MAIDS is described.

A method for the quantitative assessment of malaria parasite development in organs of the mammalian host.

A non-radioactive PCR method was developed to quantify the development of malaria parasites in the infected host. This was achieved by using Plasmodium genus-specific primers corresponding to the parasite's small subunit ribosomal RNA genes. The quantification of the PCR product was performed by high performance liquid chromatography, and calibration curves were obtained by amplification from defined quantities of purified Plasmodium genomic DNA.

High and low molecular weight tau proteins are differentially expressed from a single gene.

Both high and low molecular weight (HMW and LMW) tau proteins are expressed in the immature and adult mouse spinal cord. Northern blot analysis, performed with probes complementary to domains common and uncommon to the LMW and HMW entities, suggested that HMW tau proteins found in the immature mouse spinal cord are not translated from the single transcript of 6 kb expressed at these stages, but are transported within this nervous structure by axons arising in the periphery. In contrast, another minor transcript of 8 kb was detected in the adult mouse spinal cord by a HMW tau specific probe, suggesting that a small fraction of the HMW tau forms present in adulthood are translated within mouse spinal cord neurons.

[High molecular weight tau proteins and acquisition of neuronal polarity in peripheral nervous system].

Several variants of the microtubule-associated tau proteins, are expressed during brain development and in adulthood. These entities are required to define the polarity of the neuron and the architecture of the axon but differ in sequence and in their microtubule polymerizing activity. Here, we describe a new group of high molecular weight tau proteins that contain one or two additional exons of 711 and 198 bp in their middle region and a variable N-terminal domain.

Effect of prostaglandin E2 on proline uptake and protein synthesis by cultured human mesangial cells.

PGE2 production by glomeruli is increased in a variety of glomerular diseases. Potentially, this process may affect mesangial cell protein synthesis and mesangial cell growth. Thus studies have been undertaken, using cultured human mesangial cells, to assess the effects of PGE2 on proline uptake, protein synthesis and cell proliferation.

Characterization of atrial natriuretic factor receptors in human glomerular epithelial and mesangial cells.

To evaluate the distribution and functions of receptors of atrial natriuretic factor (ANF) in human glomeruli, we studied the binding sites of ANF-(1-28) in homogeneous populations of human glomerular epithelial cells or mesangial cells. 125I-labeled ANF bound specifically to both cell types. Equilibrium saturation binding curves suggested one group of receptor sites in mesangial cells (Kd = 99 +/- 32 pmol/l, Bmax = 15.

Quantification of collagen synthesis by cultured human glomerular cells.

This study examines the amount of total collagen and its different fractions synthesized by cultured human glomerular epithelial and mesangial cells. Two quantitative techniques were used, namely estimation of proline (Pro) plus hydroxyproline (Hyp) present in the collagenase-sensitive proteins and ELISA or RIA of the different types of collagen. In addition, the pattern of collagen synthesis for both cell types was further examined using immunofluorescence methods and polyacrylamide gel electrophoresis.

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide.

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM.

Stimulation of guanylate cyclase by atrial natriuretic factor in isolated human glomeruli.

A 23 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) stimulated guanylate cyclase activity in isolated human glomeruli in a concentration- and time-dependent manner. ANF activated particulate guanylate cyclase whereas it had no effect on soluble guanylate cyclase. These results demonstrate that the glomerulus is a target structure for ANF in humans.

Leukotriene C4 binds to human glomerular epithelial cells and promotes their proliferation in vitro.

In human and experimental glomerulonephritis, glomerular hypercellularity results both from accumulation of macrophages and proliferation of resident glomerular cells. The recent identification of macrophage-derived factors that stimulate mesangial and epithelial cell proliferation suggests that these factors might contribute to the hypercellularity. To determine the identity of such macrophage-derived growth factors, we studied the effect of leukotrienes (LTs), products that are released from macrophages and leukocytes, on proliferation of human glomerular epithelial cells in culture.

Vasoconstrictor-evoked prostaglandin synthesis in cultured human mesangial cells.

We examined the influence of angiotensin II (ANG II), arginine vasopressin (AVP), and platelet activating factor (PAF) on prostaglandin (PG) synthesis and cell contractility in human glomerular mesangial cells in culture. Addition of sodium butyrate to the culture medium for 40 h significantly increased synthesis of both 6-keto-PGF1 alpha and PGE2 in the presence of exogenous arachidonic acid and of PGE2 under basal conditions. To optimize conditions in all further experiments, cells cultured with butyrate were studied.

Increased prostaglandin production by glomeruli isolated from rats with streptozotocin-induced diabetes mellitus.

Abnormalities in glomerular function have been observed frequently in the early stages of both clinical and experimental diabetes mellitus. Because prostaglandins (PGs) are present in the glomerulus and have profound effects on glomerular hemodynamics, and because abnormalities of PG metabolism have been noted in other tissues from diabetics, we studied PG biosynthesis in glomeruli obtained from rats in the early stages of experimental diabetes mellitus. Streptozotocin, 60 mg/kg, was administered intravenously to male Sprague-Dawley rats.

Response of adenylate cyclase to parathyroid hormone and prostaglandins by human isolated glomeruli.

A role for hormonal substances and other biochemical messengers in the regulation of the glomerular filtration rate has been inferred from the results of micropuncture studies in the rat and from the demonstration of hormone-responsive adenylate cyclase activity in glomeruli isolated from the renal cortex of rats and rabbits. To investigate whether such hormonal factors may contribute to the regulation of glomerular function in humans, we studied the response of adenylate cyclase activity to the administration of human PTH-(1-34) and prostaglandins (PG) by glomeruli isolated from the renal cortex of four human kidneys. PTH and PGs (PGE2, PGI2, and, to a lesser extent, PGF2 alpha) stimulated human glomerular adenylate cyclase activity.

Prostaglandin synthesis by human glomerular cells in culture.

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable.

Angiotensin II receptors in human isolated renal glomeruli.

125I-Labeled angiotensin II ([125I]A II) binds specifically to glomeruli isolated from human kidneys that were obtained at nephrectomy or early autopsy. Equilibrium was reached after 30 min, and specific binding represented more than 90% of the total binding. Dissociation after dilution with the addition of an excess of unlabeled hormone was more rapid than after dilution alone.

Histamine H2 receptors in rat renal glomeruli.

The aim of this study was to demonstrate histamine-H2 receptors in glomeruli isolated from rat renal cortex and to correlate binding to stimulation by histamine of glomerular cyclic AMP concentration. Binding studies were performed at 10-12 degrees C using [3H]cimetidine as a tracer. Specificity of binding relies on the following: inhibition of [3H]cimetidine binding by the unlabelled drug, other H2-antagonists and agonists in contrast with the very weak inhibitory effects of H1 agonists and antagonists; reversibility of steady-state binding after addition of unlabelled drug; half inhibition of the glomerular cyclic AMP response to histamine at concentrations of cimetidine close to the KD value derived from the binding studies (3 microM); calculated KD value in agreement with the therapeutical concentration of cimetidine and the physiological concentration of histamine.

Stimulation by oxygen radicals of prostaglandin production by rat renal glomeruli.

Polymorphonuclear leukocytes secreting oxygen radicals are found in the glomerular capillaries at an early stage of experimental acute glomerulonephritis. The aim of this work was to study the effects of these radicals on prostaglandin (PG) production by the glomeruli. Glomeruli were isolated from rat renal cortex and incubated in the presence of a biochemical system capable of generating oxygen radicals (addition to 100 microM xanthine of increasing concentrations of xanthine oxidase).

Altered PGE2 production by glomeruli and papilla of rats with hereditary diabetes insipidus.

PGE2 synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of Brattleboro heterozygous control rats. Incubations were carried out in isotonic buffer at 37 degrees C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE2 production was measured in the supernatant by specific radioimmunoassay.

Hepatic binding sites for angiotensen II in the rat.

1. 125I-labelled (Asn1,Val5)-angiotensin II (125I-labelled AII) incubated with purified rat liver membranes was degraded with time, as estimated by three techniques: binding to an excess of specific antibody, polyacrylamide-gel electrophoresis and rebinding to fresh membranes. Degradation was inhibited in the presence of an excess of beta 1-24-corticotrophin but still very marked.