Ceramide mediates growth inhibition of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> parasite

In mammalian cells, ceramide mediates death by chemotherapeutic drugs. We analysed, for the first time, the role of ceramide in inhibiting growth of the malaria-causing parasite Plasmodium falciparum. Added exogenously, ceramide significantly decreased the number of parasites, and this effect was abolished by sphingosine-1-phosphate, a biological antagonist of ceramide action. Ceramide can induce death of cancer cells by decreasing glutathione levels, and in our work it induced dose- and time-dependent depletion of glutathione in P. falciparum parasites. N-acetylcysteine, a precursor of glutathione, abrogated the cytotoxic effect of ceramide. Thus, ceramide can mediate growth inhibition of P. falciparum parasites by decreasing glutathione levels. The antimalarial drugs artemisinin and mefloquine induced the death of P. falciparum parasites by sphingomyelinase-generated ceramide and by decreasing parasite glutathione levels. Altogether, ceramide was identified as a signalling molecule capable of inducing growth inhibition of P. falciparum malarial parasites.     

Hepatic effects of <Emphasis Type="Italic">Cimicifuga racemosa</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Extracts of Cimicifuga racemosa are used frequently for menopausal complaints. Cimicifuga is well tolerated but can occasionally cause liver injury. To assess hepatotoxicity of cimicifuga in more detail, ethanolic C. racemosa extract was administered orally to rats, and liver sections were analyzed by electron microscopy. Tests for cytotoxicity, mitochondrial toxicity and apoptosis/necrosis were performed using HepG2 cells. Mitochondrial toxicity was studied using isolated rat liver mitochondria. Microvesicular steatosis was found in rats treated with > 1,000 mg/kg [DOSAGE ERROR CORRECTED] body weight cimicifuga extract. In vitro, cytotoxicity was apparent at concentrations > or =75 microg/mL, and mitochondrial beta-oxidation was impaired at concentrations > or =10 microg/mL. The mitochondrial membrane potential was decreased at concentrations > or =100 microg/mL, and oxidative phosphorylation was impaired at concenq trations > or =300 microg/mL. The mechanism of cell death was predominantly apoptosis. C. racemosa exerts toxicity in vivo and in vitro, eventually resulting in apoptotic cell death. The results are compatible with idiosyncratic hepatotoxicity as observed in patients treated with cimicifuga extracts.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> possesses a unique dual-specificity serine/threonine and tyrosine kinase,Pfnek3

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg²⁺ and Mn²⁺ cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3’s dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

<Emphasis Type="Italic">C</Emphasis>-<Emphasis Type="Italic">Raf</Emphasis> deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K⁺ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf ^+/? mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.     

<Emphasis Type="Italic">LAPTM4A</Emphasis> interacts with <Emphasis Type="Italic">hOCT2</Emphasis> and regulates its endocytotic recruitment

Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein–protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP⁺ uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.     

Defining motility in the <Emphasis Type="Italic">Staphylococci</Emphasis>

The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions.     

<Emphasis Type="Italic">O</Emphasis>-Mannosylation and human disease

Glycosylation of proteins is arguably the most prevalent co- and post-translational modification. It is responsible for increased heterogeneity and functional diversity of proteins. Here we discuss the importance of one type of glycosylation, specifically O-mannosylation and its relationship to a number of human diseases. The most widely studied O-mannose modified protein is alpha-dystroglycan (α-DG). Recent studies have focused intensely on α-DG due to the severity of diseases associated with its improper glycosylation. O-mannosylation of α-DG is involved in cancer metastasis, arenavirus entry, and multiple forms of congenital muscular dystrophy [1, 2]. In this review, we discuss the structural and functional characteristics of O-mannose-initiated glycan structures on α-DG, enzymes involved in the O-mannosylation pathway, and the diseases that are a direct result of disruptions within this pathway.     

Cellulase genes from the parabasalian symbiont <Emphasis Type="Italic">Pseudotrichonympha grassii</Emphasis> in the hindgut of the wood-feeding termite <Emphasis Type="Italic">Coptotermes formosanus</Emphasis>

Cellulase genes of Pseudotrichonympha grassii (Hypermastigida: Eucomonymphidae), the symbiotic flagellate in the hindgut of the wood-feeding termite Coptotermes formosanus, were isolated and characterized. The nucleotide sequences of the major cellulase component in the hindgut of C. formosanus were determined based on its N-terminal amino acid sequence. The five isolated nucleotide sequences (PgCBH-homos) had an open reading frame of 1350 bp showing similarity to catalytic domains of glycoside hydrolase family (GHF) 7 members, and primary structure comparison with GHF7 members whose tertiary structures are well-characterized revealed the overall similarity between PgCBH-homo and the catalytic domain of a processive cellulase Cel7A (formerly CBHI) from the aerobic fungus Trichoderma reesei. Functional expression of PgCBH-homos in Escherichia coli, using the carboxymethylcellulose-Congo red assay, demonstrated the actual cellulolytic activity of PgCBH-homo. RT-PCR showed that PgCBH-homos were expressed, from the three flagellates in the hindgut, specifically in P. grassii. Received 10 July 2002; accepted 26 July 2002 RID="*" ID="*"Corresponding author.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Flagellin glycosylation with pseudaminic acid in <Emphasis Type="Italic">Campylobacter</Emphasis> and <Emphasis Type="Italic">Helicobacter</Emphasis>: prospects for development of novel therapeutics

Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme–substrate and –inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.     

A new lysozyme from the eastern oyster (<Emphasis Type="Italic">Crassostrea virginica</Emphasis>) indicates adaptive evolution of <Emphasis Type="Italic">i</Emphasis>-type lysozymes

A new lysozyme (cv-lysozyme 2) with a MALDI molecular mass of 12 984.6 Da was purified from crystalline styles and digestive glands of eastern oysters (Crassostrea virginica) and its cDNA sequenced. Quantitative real time RT-PCR detected cv-lysozyme 2 gene expression primarily in digestive gland tissues, and in situ hybridization located cv-lysozyme 2 gene expression in basophil cells of digestive tubules. Cv-lysozyme 2 showed high amino acid sequence similarity to other bivalve mollusk lysozymes, including cv-lysozyme 1, a lysozyme recently purified from C. virginica plasma. Differences between cv-lysozyme 2 and cv-lysozyme 1 molecular characteristics, enzymatic properties, antibacterial activities, distribution in the oyster body and site of gene expression indicate that the main role of cv-lysozyme 2 is in digestion. While showing that a bivalve mollusk employs different lysozymes for different functions, findings in this study suggest adaptive evolution of i type lysozymes for nutrition. Received 30 August 2006; received after revision 14 October 2006; accepted 6 November 2006     

Neuroprotective effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this review, we summarize the experimental results on the mechanism of neuroprotection induced by standardized extract of Ginkgo biloba leaves (EGb 761) and its constituents. The effects described mostly in animals include those on cerebral blood flow, neurotransmitter systems, cellular redox state and nitric oxide level. Furthermore, we discuss the current status of clinical trials as well as undesired side effects of EGb 761.Received 21 November 2002; received after revision 8 March 2003; accepted 17 March 2003     

Cellular microbiology of intracellular <Emphasis Type="Italic">Salmonella enterica</Emphasis>: functions of the type III secretion system encoded by <Emphasis Type="Italic">Salmonella</Emphasis> pathogenicity island 2

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.Received 11 June 2004; received after revision 8 July 2004; accepted 12 July 2004     

Molecular neurophysiology of taste in <Emphasis Type="Italic">Drosophila</Emphasis>

The recent identification of candidate receptor genes for sweet, umami and bitter taste in mammals has opened a door to elucidate the molecular and neuronal mechanisms of taste. Drosophila provides a suitable system to study the molecular, physiological and behavioral aspects of taste, as sophisticated molecular genetic techniques can be applied. A gene family for putative gustatory receptors has been found in the Drosophila genome. We discuss here current knowledge of the gustatory physiology of Drosophila. Taste cells in insects are primary sensory neurons whereupon each receptor neuron responds to either sugar, salt or water. We found that particular tarsal gustatory sensilla respond to bitter compounds. Electrophysiological studies indicate that gustatory sensilla on the labellum and tarsi are heterogeneous in terms of their taste sensitivity. Determination of the molecular bases for this heterogeneity could lead to an understanding of how the sensory information is processed in the brain and how this in turn is linked to behavior.Received 12 May 2003; received after revision 9 June 2003; accepted 13 June 2003     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Hemoglobin catabolism and the killing of intraerythrocyticPlasmodium falciparum by chloroquine

To evaluate how chloroquine kills malaria parasites, hemoglobin catabolism was studied at the various stages of intraerythrocytic parasite development. We found that hemoglobin catabolism is switched off whenPlasmodium falciparum parasites mature to the late trophozoite or early schizont stages and is switched on again during the ring stage. When hemoglobin catabolism is switched off, the parasites are resistant to the morphologic effects of chloroquine. Although the ring stage parasites failed to mature in the presence of chloroquine, some of them switched on hemoglobin ingestion and became stuffed with hemoglobin-filled vesicles, indicating a distal block in catabolism. In fact, we demonstrated a high-grade block in hemozoin production during a 22 h incubation of synchronized ring forms; ferriprotoporphyrin IX (FP) incorporation into the -hematin of hemozoin decreased from 900 to 50 pmol/10⁶ parasitized erythrocytes. We propose that the primary effect of chloroquine on hemoglobin catabolism is to block FP polymerization to -hematin. Secondarily, toxic FP and FP-chloroquine complexes accumulate and are available to exert their several toxicities, which include inhibition of hemoglobindegrading proteases and membrane damage. As a consequence, maturation is arrested and eventually the parasites die and lyse.