Adrenomedullin in the eye

Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. We showed the presence of high concentrations of adrenomedullin-immunoreactivity in the vitreous fluid, and the levels were elevated in patients with proliferative vitreoretinopathy. Furthermore, adrenomedullin mRNA expression levels were elevated in the tissues of intraocular tumors and orbital tumors. Adrenomedullin is produced and secreted by cultured human retinal pigment epithelial (RPE) cells. Inflammatory cytokines and hypoxia are strong stimulators for the adrenomedullin expression in retinal pigment epithelial cells. Adrenomedullin stimulated the proliferation of retinal pigment epithelial cells both under normoxia and hypoxia. Dexamethasone (DEX) increased the adrenomedullin expression in two cultured cell lines of human retinal pigment epithelial cells; ARPE-19 cells and D407 cells, while it had no noticeable effects on the cytokine-induced adrenomedullin expression. These findings suggest that adrenomedullin is involved in the pathophysiology of inflammatory and neoplastic eye diseases as an autocrine or paracrine growth stimulator. The findings on glucocorticoid-induced AM expression raise the possibility that it may be related to the pathogenesis of some eye diseases, such as central serous chorioretinopathy and multifocal posterior pigment epitheliopathy, which are frequently seen in patients treated with high doses of glucocorticoids.     

Adoptive immunotherapy of cancer using activated Autologous lymphocytes-current status and new strategies-

After the discovery of interleukin-2 (IL-2), lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), and cytotoxic T lymphocytes (CTLs) sensitized with the mixed lymphocyte-tumor culture (MLTC) system have been conducted in adoptive immunotherapy (AIT) trials during past 15 years. Although the overall response rate of tumor shrinkage was marginal (9%), locoregional administration of TILs for malignant effusions was effective (77%) for a decrease or disappearance of the effusions even in terminally-ill patients, resulting in an improvement of QOL. Recent advances for molecular understanding of antigen presentation and recognition have promoted us to enhance the efficacy of AIT by using cultured dendritic cells (DCs) for generating antigen-specific CTLs in vitro. The peptide-pulsed DC-activated killer (PDAK) cells showed tumor recognition against antigen-expressing cells, and were efficiently propagated with the IL2 plus immobilized anti-CD3 antibody (IL-2/CD3) culture system. Clinical trials using PDAK cells against patients with lung metastases are now progressed, in which peptides suitable for generating CTLs were chosen in individual patients using the method designated as host-oriented peptide evaluation (HPOE) approach. Moreover, DCs were introduced with tumor-derived RNA, which was amplified with the T7 promoter system, and then were used for stimulating lymphocytes. The tumor RNA-introduced DC-activated killer (TRiDAK) cells showed tumor-specific interferon-gamma spots even in a patient in whom we failed to generate PDAK cells using DCs and peptides, suggesting that the clinical trial of AIT using TRiDAK cells is warranted for the treatment of patients with metastatic cancer. Thus, more understanding of antigen-presentation and -recognition mechanisms and immune regulation systems may promote clinical applications of AIT to establish a novel modality of cancer treatment.     

Detection of 1270 nm emission from singlet oxygen and photocytotoxic property of sugar-pendant 60 fullerenes

Sugar-pendant [60] fullerene derivatives have been prepared from carbohydrate-linked azides 1a-e. Both monosugar (4a-e) and bissugar derivatives (5a-e) produce singlet oxygen ((1)O(2)) under laser irradiation (355 nm) proved by the direct observation of (1)O(2) emission at 1270 nm. Monosugar derivatives exhibit photocytotoxicity varying by the attached sugar molecule.     

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (ΔSO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ΔSO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ΔSO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.Dissimilatory metal-reducing bacteria (DMRB) conserve energy for growth by coupling the oxidation of organic compounds to the reduction of metal compounds (29). DMRB are of great interest not only for their importance in the biogeochemical cycling of metals (25) but also for their utility in biotechnological processes, such as microbial fuel cells (MFCs) (24, 40). In recent years, the ability of many DMRB, including members of the genera Shewanella (5, 12, 20, 31), Geobacter (2), Aeromonas (34), Desulfobulbus (19), and Phodoferax (9), to generate current in MFCs has been described.Among DMRB, Shewanella oneidensis MR-1 is one of the most extensively studied due to its metabolic versatility (28), annotated genome sequence (17), and genetic accessibility. In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to an anode without an exogenously added mediator (20), it has become a model organism for the study of microbial current generation in MFCs. Extensive studies have been performed to understand the mechanisms of extracellular electron transfer (EET) to solid materials, such as MFC anodes and metal oxides, in strain MR-1. Multiple mechanisms, including direct EET through the physical contact of bacterial cells via outer membrane (OM) cytochromes (42) and conductive nanowires (16) and mediated EET via self-produced electron shuttles such as quinones and flavins (27, 30, 39, 41), have been identified.Although OM cytochromes and electron shuttles have been identified to play central roles in EET, it is reasonable to speculate that this complex catabolic process is also influenced by other (extra)cellular components. To date, only limited studies have been done to investigate other cellular components involved in EET (7). A useful approach for identifying unknown cellular components (and genes) associated with a particular phenotype involves the construction and screening of a random mutant library for obtaining mutants with altered phenotypes. In the present study, we constructed a random transposon (Tn) insertion mutant library of S. oneidensis MR-1 and obtained mutants with altered colony morphologies (rough morphotypes) after the selection of mutants in an MFC. Analyses of one of such mutants suggest that cell surface capsular polysaccharides affect not only the adhesion of cells to graphite anodes but also the current generation in MFCs.     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.     

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.