Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.     

Mammalian Granulocyte–Macrophage Colony-stimulating Factor Receptor Expressed in Primary Avian Hematopoietic Progenitors: Lineage-specific Regulation of Proliferation and Differentiation

The cytokine Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure–function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R α chain specifically interfered with EpoR signaling, an activity neither seen for the β_c subunit of the receptor complex alone, nor for the α chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R α chain in defining cell type–specific functions of the GM-R.     

The Interleukin-17 Gene of Herpesvirus Saimiri

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.     

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.     

Informed Choice in the German Mammography Screening Program by Education and Migrant Status: Survey among First-Time Invitees

Breast cancer is the most prevalent cancer among women and mammography screening programs are seen as a key strategy to reduce breast cancer mortality. In Germany, women are invited to the population-based mammography screening program between ages 50 to 69. It is still discussed whether the benefits of mammography screening outweigh its harms. Therefore, the concept of informed choice comprising knowledge, attitude and intention has gained importance. The objective of this observational study was to assess the proportion of informed choices among women invited to the German mammography screening program for the first time. A representative sample of 17,349 women aged 50 years from a sub-region of North Rhine Westphalia was invited to participate in a postal survey. Turkish immigrant women were oversampled. The effects of education level and migration status on informed choice and its components were assessed. 5,847 (33.7%) women responded to the postal questionnaire of which 4,113 were used for analyses. 31.5% of the women had sufficient knowledge. The proportion of sufficient knowledge was lower among immigrants and among women with low education levels. The proportion of women making informed choices was low (27.1%), with similar associations with education level and migration status. Women of low (OR 2.75; 95% CI 2.18–3.46) and medium education level (OR 1.49; 95% CI 1.27–1.75) were more likely to make an uninformed choice than women of high education level. Turkish immigrant women had the greatest odds for making an uninformed choice (OR 5.30, 95% CI 1.92–14.66) compared to non-immigrant women. Other immigrant women only had slightly greater odds for making an uninformed choice than non-immigrant women. As immigrant populations and women with low education level have been shown to have poor knowledge, they need special attention in measures to increase knowledge and thus informed choices.     

Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease

Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N(pro) and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N(pro) and E(rns) RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.     

Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Phenylthiazolyl-hydrazide and its derivatives are potent inhibitors of tau aggregation and toxicity in vitro and in cells

One of the key pathological features of Alzheimer's disease is the aggregation of tau protein. We are therefore searching for compounds capable of inhibiting this reaction. On the basis of an initial screen of 200000 compounds [Pickhardt, M., Gazova, Z., von Bergen, M., Khlistunova, I., Wang, Y., Hascher, A., Mandelkow, E. M., Biernat, J., and Mandelkow, E. (2005) Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells, J. Biol. Chem. 280, 3628-3635], we performed an in silico screen and predicted a new phenylthiazolyl-hydrazide (PTH) compound as a possible hit [Larbig, G., Pickhardt, M., Lloyd, D. G., Schmidt, B., and Mandelkow, E. (2007) Screening for inhibitors of tau protein aggregation into Alzheimer paired helical filaments: A ligand based approach results in successful scaffold hopping. Curr. Alzheimer Res. 4 (3), 315-323.]. Synthesis of this compound showed that it was indeed active in terms of inhibiting de novo tau aggregation and disassembling preformed aggregates (IC50 = 7.7 microM and DC50 = 10.8 microM). We have now synthesized 49 similar structures and identified the core of the PTHs to be crucial for activity, thus representing a lead structure. Analysis of the binding epitope by saturation transfer difference NMR shows strong interactions between the tau protein and the ligand in the aromatic regions of the inhibitor. By chemical variation of the core, we improved the inhibitory potency five-fold. The compounds showed a low toxicity as judged by an N2A cell model of tau aggregation and lend themselves for further development.