Polyphosphorylated phosphoinositides (PIPs) are potent second messengers, which trigger a wide variety of signaling and trafficking events in most eukaryotic cells. However, the role and metabolism of PIPs in malaria parasite Plasmodium have remained largely unexplored. Our present studies suggest that PfPI3K, a novel phosphatidylinositol-3-kinase (PI3K) in Plasmodium falciparum, is exported to the host erythrocyte by the parasite in an active form. PfPI3K is a versatile enzyme as it can generate various 3′-phosphorylated PIPs. In the parasite, PfPI3K was localized in vesicular compartments near the membrane and in its food vacuole. PI3K inhibitors wortmannin and LY294002 were effective against PfPI3K and were used to study PfPI3K function. We found that PfPI3K is involved in endocytosis from the host and trafficking of hemoglobin in the parasite. The inhibition of PfPI3K resulted in entrapment of hemoglobin in vesicles in the parasite cytoplasm, which prevented its transport to the food vacuole, the site of hemoglobin catabolism. As a result, hemoglobin digestion, which is a source of amino acids necessary for parasite growth, was attenuated and caused the inhibition of parasite growth.

In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium , the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasite's erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.

Second messengers such as phosphoinositides and calcium are known to control diverse processes involved in the development of malaria parasites. However, the underlying molecular mechanisms and pathways need to be unraveled, which may be achieved by understanding the regulation of effectors of these second messengers. Calcium-dependent protein kinase (CDPK) family members regulate diverse parasitic processes. Because CDPKs are absent from the host, these kinases are considered as potential drug targets. We have dissected the function of an atypical CDPK from Plasmodium falciparum, PfCDPK7. The domain architecture of PfCDPK7 is very different from that of other CDPKs; it has a pleckstrin homology domain adjacent to the kinase domain and two calcium-binding EF-hands at its N terminus. We demonstrate that PfCDPK7 interacts with PI(4,5)P₂ via its pleckstrin homology domain, which may guide its subcellular localization. Disruption of PfCDPK7 caused a marked reduction in the growth of the blood stage parasites, as maturation of rings to trophozoites was markedly stalled. In addition, parasite proliferation was significantly attenuated. These findings shed light on an important role for PfCDPK7 in the erythrocytic asexual cycle of malaria parasites.

(1) Background: Withania somnifera Dunal (Ashwagandha) is a widely used medicinal herb in traditional medicinal systems with extensive research on various plant parts. Surprisingly, seeds of W. somnifera have never been investigated for their therapeutic potential. (2) Methods: W. somnifera seeds were extracted for fatty acids (WSSO) using super critical fluid extraction, and was analyzed by gas chromatography. Its therapeutic potential in psoriasis-like skin etiologies was investigated using a 12-O tetradecanoyl phorbol 13-acetate (TPA)-induced psoriatic mouse model. Psoriatic inflammation along with psoriatic lesions and histopathological scores were recorded. WSSO was also tested on murine macrophage (RAW264.7), human epidermoid (A431), and monocytic (THP-1) cells, stimulated with TPA or lipo poly-saccharide (LPS) to induce pro-inflammatory cytokine (IL-6 and TNF-α) release. NFκB promoter activity was also measured by luciferase reporter assay. (3) Results: Topical application of WSSO with concurrent oral doses significantly reduced inflammation-induced edema, and repaired psoriatic lesions and associated histopathological scores. Inhibition of pro-inflammatory cytokines release was observed in WSSO-treated A431 and THP-1 cells, along with reduced NFκB expression. WSSO also inhibited reactive nitrogen species (RNS) in LPS-stimulated RAW264.7 cells. (4) Conclusion: Here we show that the fatty acids from W. somnifera seeds have strong anti-inflammatory properties, along with remarkable therapeutic potential on psoriasis-like skin etiologies.

Recent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite ( Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117, 503-514 ). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level. We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4. PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family.

Psoriasis is a chronic inflammatory skin disease characterized by circumscribed, red, thickened plaques with overlying silvery white scales. It is associated with the release of pro-inflammatory mediators that lead to the development of edema and distress. Here we show the anti-inflammatory and anti-psoriatic efficacies of a neutraceutical sea buckthorn oil (SBKT) derived from the fruit pulp of Hippophae rhamnoides. Chemical analysis of the SBKT showed the presence of 16 major saturated, mono-, and polyunsaturated fatty acids components, imparting significant nutritional values. Efficacy of the SBKT in modulating psoriasis and associated inflammation was first tested in vitro using human monocytic (THP-1) cells. SBKT induced cytotoxicity at a dose of ≥25 µl/ml. Treatment of the lipopolysaccharide-stimulated THP-1 cells with SBKT subdued the enhanced release of intracellular reactive nitrogen species and expression of NF-κB protein, in a concentration-dependent manner. This was accompanied by a reduction in the release of downstream pro-inflammatory cytokines: Interleukin-1ß and interleukin-6. Tumor necrosis factor-α released in the stimulated THP-1 cells were also inhibited by SBKT dose of 5 µl/ml. In vivo oral and topical treatment with SBKT in the Carrageenan-stimulated paw edema model, showed a significant decrease in paw volume and edema. In the 12-O tetradecanoyl phorbol 13-acetate (TPA) stimulated CD-1 mice psoriasis-like model, concurrent oral and tropical SBKT treatments substantially reduced ear edema and ear biopsy weights. Histopathologically, significant reduction in ear epidermal thickness and skin lesion scores was observed in the SBKT-treated animals. In conclusion, SBKT showed anti-inflammatory and anti-psoriasis-like efficacies in healing chemical-induced inflammation and psoriasis. The possible mode of action of SBKT was found through inhibition of reactive nitrogen species, and downregulation of NF-κB protein and pro-inflammatory cytokines. Thus, the present data suggest that Sea buckthorn oil can be used as an anti-inflammatory and anti-psoriatic nutraceutical.