Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

ER stress responses in the absence of apoptosome: A comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9^+/+ and casp9^−/− MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9^−/− cells as compared with casp9^+/+ MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9^−/− MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.     

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells

Copper oxide nanoparticles (CuO NPs) are increasingly used in various applications. Recent studies suggest that oxidative stress may be the cause of the cytotoxicity of CuO NPs in mammalian cells. However, little is known about the genotoxicity of CuO NPs following exposure to human cells. This study was undertaken to investigate CuO NPs induced genotoxic response through p53 pathway in human pulmonary epithelial cells (A549). In addition, cytotoxicity and oxidative stress markers were also assessed. Results showed that cell viability was reduced by CuO NPs and degree of reduction was dose dependent. CuO NPs were also found to induce oxidative stress in dose-dependent manner indicated by depletion of glutathione and induction of lipid peroxidation, catalase and superoxide dismutase. The expression of Hsp70, the first tier biomarker of cellular damage was induced by CuO NPs. Further, CuO NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and MSH2 expression. These results demonstrate that CuO NPs possess a genotoxic potential in A549 cells which may be mediated through oxidative stress. Our short-term exposure study of high level induction of genotoxic response of CuO NPs will need to be further investigated to determine whether long-term exposure consequences may exist for CuO NPs application.     

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA₃ gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b₅₅₉ that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ?Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b₅₅₉ into the low potential form. Structural reasons for such effects are discussed.     

TEM immunocytochemistry of a 48 kDa MW organic matrix phosphoprotein produced in the mantle epithelial cells of the Eastern oyster (Crassostrea virginica)

Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel purified 48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical microvilli of these cells. The 48 kDa phosphoprotein forms a component of the fibrous organic matrix and appears to be involved in calcium supply thus enabling crystal growth at the mineralization front.     

Structural basis for bile salt inhibition of pancreatic phospholipase A2

Bile salt interactions with phospholipid monolayers of fat emulsions are known to regulate the actions of gastrointestinal lipolytic enzymes in order to control the uptake of dietary fat. Specifically, on the lipid/aqueous interface of fat emulsions, the anionic portions of amphipathic bile salts have been thought to interact with and activate the enzyme group-IB phospholipase A2 (PLA2) derived from the pancreas. To explore this regulatory process, we have determined the crystal structures of the complexes of pancreatic PLA2 with the naturally occurring bile salts: cholate, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate. The five PLA2-bile salt complexes each result in a partly occluded active site, and the resulting ligand binding displays specific hydrogen bonding interactions and extensive hydrophobic packing. The amphipathic bile salts are bound to PLA2 with their polar hydroxyl and sulfate/carboxy groups oriented away from the enzyme's hydrophobic core. The impaired catalytic and interface binding functions implied by these structures provide a basis for the previous numerous observations of a biphasic dependence of the rate of PLA2 catalyzed hydrolysis of zwitterionic glycerophospholipids in the presence of bile salts. The rising or activation phase is consistent with enhanced binding and activation of the bound PLA2 by the bile salt induced anionic charge in a zwitterionic interface. The falling or inhibitory phase can be explained by the formation of a catalytically inert stoichiometric complex between PLA2 and any bile salts in which it forms a stable complex. The model provides new insight into the regulatory role that specific PLA2-bile salt interactions are likely to play in fat metabolism.     

Novel Aspects of Prions,Their Receptor Molecules,and Innovative Approaches for TSE Therapy

1. Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination due to the accumulation of an abnormal isoform (PrP^Sc) of the host-encoded prion protein (PrP^c). 2. This article reviews the current knowledge on PrP^c and PrP^Sc, prion replication mechanisms, interaction partners of prions, and their cell surface receptors. Several strategies, summarized in this article, have been investigated for an effective antiprion treatment including development of a vaccination therapy and screening for potent chemical compounds. Currently, no effective treatment for prion diseases is available. 3. The identification of the 37 kDa/67 kDa laminin receptor (LRP/LR) and heparan sulfate as cell surface receptors for prions, however, opens new avenues for the development of alternative TSE therapies.     

Identification and characterization of rad51 of Pneumocystis

Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis.