Investigating serum factors promoting erythrocytic growth of Plasmodium falciparum

The elucidation of factors inducing the growth of Plasmodium falciparum can provide critical information about the developmental mechanisms of this parasite and open the way to search for novel targets for malaria chemotherapy. The ability of components of a growth-promoting factor derived from bovine serum and various related substances to sustain growth of P. falciparum was characterized. A simple total lipid fraction (GFS-C) containing non-esterified fatty acids (NEFAs) as essential factors was noted to promote the parasite's growth. Various proteins from a variety of animals were tested, indicating the importance not only of GFS-C, but also of specific proteins, such as bovine and human albumin, in the parasite growth. Several combinations of the NEFAs tested sustained low parasite growth. Among various phospholipids and lysophospholipids tested, lysophosphatidylcholine containing C-18 unsaturated fatty acids was found to sustain the complete development of the parasite in the presence of bovine albumin. Several other lysophospholipids can partially support growth of P. falciparum.     

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

Lysophosphatidic acid inhibits anti-Fas-mediated apoptosis enhanced by actin depolymerization in epithelial ovarian cancer

Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.     

Uncovering RNAi mechanisms in plants: biochemistry enters the foray

In plants, the RNA interference (RNAi) machinery responds to a variety of triggers including viral infection, transgenes, repeated elements and transposons. All of these triggers lead to silencing outcomes ranging from mRNA degradation to translational repression to chromatin remodeling. Thus, plants offer us a potentially unique opportunity to understand the full range of RNAi effector mechanisms. In this review, we discuss the recent developments in our understanding of plant RNAi mechanisms from a biochemical perspective.     

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.     

Identification of dynein light chain 2 as an interaction partner of p21-activated kinase 1

p21-Activated kinase 1 (PAK1), a member of the evolutionarily conserved PAK family of serine/threonine kinases, is essential for a variety of cellular functions. Our previous studies showed that PAK1 participated in the apoptotic pathway mediated by p110C. To further investigate its functions, we used the yeast two-hybrid system to screen a human fetal brain cDNA library and identified dynein light chain 2 (DLC2)/myosin light chain (MLC) as an interacting partner of PAK1. The association of PAK1 with DLC2 was further confirmed by in vitro binding assay. With the stimulation of EGF, PAK1 interacted with HA-DLC2 in vivo and relocalized in cytoplasm near the perinuclear location in confocal microscope analysis. The deletion analysis showed that the interaction of DLC2 with PAK1 occurred within the residues 210-332 of PAK1. For that studies showed that DLC2 was a subunit of myosin complex, so it is possible that PAK1 binds to DLC2 and transports by myosin complex.     

Complementation analyses suggest species-specific functions of the SNF5 homology domain

Inactivation on both alleles of the hSNF5/INI1 tumor suppressor gene which encodes a subunit of the human SWI/SNF chromatin remodelling complex occurs in most malignant rhabdoid tumors. No paralog of hSNF5/INI1 is identified in the human genome. In contrast, it has two homologs in the yeast Saccharomyces cerevisiae, SNF5 and SFH1 which encode core components of the ySWI/SNF and RSC complexes, respectively. The homology mainly concerns an approximately 200 amino acid region termed the SNF5 homology domain. We have tested the ability of the hSNF5/INI1-wild type gene product and of chimerical constructs in which the yeast SNF5 domains were replaced by that of the human protein, to complement yeast snf5 and sfh1 phenotypes. Neither growth deficiencies on different carbon sources of snf5 yeasts nor the lethality of the sfh1 phenotype could be rescued. This strongly suggests that the SNF5 homology domain presents species-specific functions.     

Light-induced changes of photosystem II activity in dark-grown Scots pine seedlings

Both chlorophyll a and b and polypeptides of the photosynthetic apparatus are found in gymnosperm seedlings. germinated and grown in absolute darkness. The photosystem II (PSII) activity is, however, limited, probably due to an inactive oxygen evolving system. In the present study dark-grown seedlings of Scots pine ( Pinus sylvestris L.) were transferred to light and changes in antenna size and the activation process of PSII were investigated using fluorescence measurements and quantitative western blotting. It was found that the activation process is rapid, requires very little light and that strong light inhibits the process. It takes place without any changes in the primary reactions of PSII. Furthermore, all polypeptides except the major light-harvesting chlorophyll a/b -binding protein complex of PSII (LHCII) were present in dark-grown seedlings in amounts comparable to the light treated control. The dark-grown seedlings had the same LHCII polypeptide composition as light treated seedlings, and the LHCII present seemed to be fully connected to the reaction centre. The results indicate that activation of PSII in dark-grown conifer seedlings resembles the photoactivation process of angiosperms. This implies that the fundamental processes in the assembly of the photosystem II complex is the same in all plants, but that the regulation differs between different taxa.     

Interaction between beta-cyclodextrin and 1,10-phenanthroline: uncommon 2:3 inclusion complex in the solid state

The crystallographic structure of the complex formed by beta-cyclodextrin with 1,10-phenanthroline has been studied by X-ray diffraction. The result shows that the complex adopts an uncommon 2:3 stoichiometry in solid state, that is, every complex unit contains three 1,10-phenanthroline molecules and two beta-cyclodextrin molecules, where two 1,10-phenanthroline molecules individually occupy two cyclodextrin cavities, and the third guest molecule is located in the interstitial space between two head-to-head cyclodextrin molecules. The intermolecular hydrogen bonds between the adjacent complex units further link these individual monomers to a channel-type assembly. Furthermore, 1H and 2D NMR spectroscopy has been employed to investigate the inclusion behavior between the host beta-cyclodextrin and guest 1,10-phenanthroline in aqueous solution.     

Inclusion complexes of V-amylose with undecanoic acid and dodecanol at atomic resolution: X-ray structures with cycloamylose containing 26 D-glucoses (cyclohexaicosaose) as host

Crystal structures are reported of cycloamylose containing 26 D-glucose residues (CA26, cyclohexaicosaose, C156H260O130) in complexes with undecanoic acid (CA26 x 2C10H21COOH x 34.95 H2O, orthorhombic P2(1)2(1)2(1), one CA26 and two bound undecanoic acids F1 and F2 in the asymmetric unit, resolution 0.95 angstroms) and with dodecanol ((CA26)(0.5) x C12H25OH x 32.0H2O, monoclinic C2, half a CA26 binding one dodecanol, A, in the asymmetric unit, resolution 1.0 angstroms). The macrocycle of CA26 is folded like the figure '8' into two 10 D-glucoses long left-handed V-amylose helices forming approximately 5A wide V-channels that are occupied by undecanoic acid (F1, F2) or dodecanol (A) as guest molecules. The functional head groups of the guests near the O(6) ends of the V-channels are hydrogen bonded with d-glucose O(6)n-H; the aliphatic termini beyond C(9) protrude from the O(2), O(3) ends. Parts of the aliphatic chains enclosed in the V-channels are all-trans except for one torsion angle each (approximately 130 degrees ) in undecanoic acid molecules F1 and F2. There are several (guest)C-H...O hydrogen bonds to O(4) and O(6) of CA26 in both complexes, and H...H van der Waals interactions with d-glucose C(3)-H and C(5)-H dominate. C(5)-H determine the position of the aliphatic chains of undecanoic acid F1 and of dodecanol A in contrast to F2 where both C(3)-H and C(5)-H contribute equally, probably because the V-channel is narrower than in F1 and in dodecanol. Complexes of polymeric V-amylose with fatty acids and alcohols studied by X-ray fiber diffraction could not provide the here described high resolution.