Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.     

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.     

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

Denitrification in a seashore sandy deposit influenced by groundwater discharge

The chemical compositions of ground water and organic matter in sediments were investigated at a sandy shore of Tokyo Bay, Japan to determine the fate of ground water NO₃ ^–. On the basis of Cl^– distribution in ground water, the beach was classified into freshwater (FR)-, transition (TR)-, and seawater (SW)-zones from the land toward the shoreline. The NO₃ ^– and N₂O did not behave conservatively with respect to Cl^– during subsurface mixing of freshwater and seawater, suggesting NO₃ ^– consumption and N₂O production in the TR-zone. Absence of beach vegetation indicated that NO₃ ^– assimilation by higher plants was not as important as NO₃ ^– sink. Low NH₄ ⁺ concentrations in ground water revealed little reduction of NO₃ ^– to NH₄ ⁺. These facts implied that microbial denitrification and assimilation were the likely sinks for ground water NO₃ ^–. The potential activity and number of denitrifiers in water-saturated sediment were highest in the low-chlorinity part of the TR-zone. The location of the highest potential denitrification activity (DN-zone) overlapped with that of the highest NO₃ ^– concentration. The C/N ratio and carbon isotope ratio (¹³C) of organic matter in sediment (< 100 -m) varied from 12.0 to 22.5 and from –22.5 to –25.5, respectively. The ¹³C value was inversely related to the C/N ratio (r ² = 0.968, n = 11), which was explained by the mixing of organic matters of terrestrial and marine origins. In the DN-zone, the fine sediments were rich in organic matters with high C/N ratios and low ¹³C values, implying that dissolved organic matters of terrestrial origin might have been immobilized under slightly saline conditions. A concurrent supply of NO₃ ^– and organic matter to the TR-zone by ground water discharge probably generates favorable conditions for denitrifiers. Ground water NO₃ ^– discharged to the beach is thus partially denitrified and fixed as microbial biomass before it enters the sea. Further studies are necessary to determine the relative contribution of these processes for NO₃ ^– removal.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.     

CR2 units of CR2 complexes are possibly associated with nucleophilic agents through reactive covalent links

The human complement receptor type 2 (CR2/CD21), a transmembrane glycoprotein, associates with a variety of surface antigens and proteins in the cell membrane. We examined the possibilities that the CR2 units of CR2 complexes are associated through internal covalent links reactive with nucleophilic agents, e.g. H(2)O or methylamine, and that CR2-positive cells process anti-CR2 monoclonal antibodies (MoAbs). Data from immunoblotting and cytofluorimetry with CR2-binding site-specific MoAbs show that: (i) CR2-positive Raji cells release soluble CR2 isoforms into the medium when incubated in phosphate buffered saline; (ii) despite affecting the detection of one soluble CR2 isoform, methylamine treatment of soluble CR2 allows the detection of another of its isoforms; (iii) limited pre-treatment of cells with methylamine reveals a more heterogeneous CR2-positive cell population or enhances the detection of CR2; (iv) cell treatment with CR2-binding site-specific MoAbs enhances the detection of CR2 isoform(s). The data suggest that CR2 is shed mainly as a soluble CR2 complex, in which the CR2 units link covalently and react with nucleophilic agents. Raji cells may process bound fragments (145 kDa) that are recognised by and become bound by anti-CR2 MoAb.     

The contribution of photosynthetic pigments to the development of biochemical separation methods: 1900–1980

The role of photosynthetic pigments in the development of separation methods in biochemistry during the period 1900-1980 is described beginning with M. Tswett who introduced separation of chlorophylls and carotenoids on columns and coined the term chromatography in 1906. In Uppsala, T. Svedberg developed the ultracentrifuge in the 1920s. A. Tiselius improved electrophoresis in the 1930s and developed chromatography of proteins in the 1940s and 1950s. Others of 'The Uppsala school in separation science' include J. Porath, P. Flodin and S. Hjertén who further developed various gel chromatographic methods. Hjertén introduced free zone electrophoresis in narrow tubes, a forerunner of capillary electrophoresis. Two proteins, phycoerythrin and phycocyanin, were used as test substances in all these methodological studies. Aqueous two-phase partitioning as a separation method was introduced in 1956 by the author. In this work, chloroplast particles were used, and the method was applied for the separation and purification of intact chloroplasts, inside-out thylakoid vesicles and plasma membranes. My research was carried out in cooperation with G. Blomquist, G. Johansson, C. Larsson, B. Andersson and H.-E. Akerlund during a 20-year period, 1960-1980.