Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.     

FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis

Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23(-/-) or Klotho(-/-) knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.     

PYY3-36 injection in mice produces an acute anorexigenic effect followed by a delayed orexigenic effect not observed with other anorexigenic gut hormones

Peptide YY (PYY) is secreted postprandially from the endocrine L cells of the gastrointestinal tract. PYY(3-36), the major circulating form of the peptide, is thought to reduce food intake in humans and rodents via high-affinity binding to the autoinhibitory neuropeptide Y (NPY) receptor within the arcuate nucleus. We studied the effect of early light-phase injection of PYY(3-36) on food intake in mice fasted for 0, 6, 12, 18, 24, and 30 h and show that PYY(3-36) produces an acute anorexigenic effect regardless of the duration of fasting. We also show evidence of a delayed orexigenic effect in ad libitum-fed mice injected with PYY(3-36) in the early light phase. This delayed orexigenic effect also occurs in mice administered a potent analog of PYY(3-36), d-Allo Ile(3) PYY(3-36), but not following injection of other anorectic agents (glucagon-like-peptide 1, oxyntomodulin, and lithium chloride). Early light-phase injection of PYY(3-36) to ad libitum-fed mice resulted in a trend toward increased levels of hypothalamic NPY and agouti-related peptide mRNA and a decrease in proopiomelanocortin mRNA at the beginning of the dark phase. Furthermore, plasma levels of ghrelin were increased significantly, and there was a trend toward decreased plasma PYY(3-36) levels at the beginning of the dark phase. These data indicate that PYY(3-36) injection results in an acute anorexigenic effect followed by a delayed orexigenic effect.     

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.     

Differential sensitivity to cadmium in germinating seeds of three cultivars of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

The reserve mobilization was analysed in germinating seeds of faba bean (Vicia faba) exposed to treatment with a toxic cadmium concentration for 4 days. When the behaviours of three cultivars were compared with regard to the germination rate, the following order of sensitivity to cadmium was observed: Aguadulce and Luz de otoño showed 59 and 19% of inhibition from controls, respectively, while no effect was observed in the case of the local Féverole. The growth of embryo radicle was also affected in the same pattern. The differential vulnerability to Cd stress cannot be correlated to shortage in water supply of cotyledons. However, Cd-treated germinating seeds of the most sensitive cultivar (Aguadulce) showed restriction in starch mobilization and decrease in availability of soluble sugars and free amino acids. Moreover, glucose, fructose and amino acids were markedly leaked into the germination medium at the expense of the growing embryonic axis during exposure to Cd. These results provide an indication of the way in which cadmium might impair seed germination.     

Alpha-tocopheryl acetate is absorbed and hydrolyzed by Caco-2 cells comparative studies with alpha-tocopherol

Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.     

Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions.