T-cell receptor tetramer binding or the lack there of does not necessitate antigen reactivity in T-cell receptor transduced T cells

Genetic transfer of T-cell receptor (TCR) chains provides a means of transferring tumor antigen specificity onto an alternate T-cell population. To determine which tumor reactive TCRs are best suitable for such adoptive transfer, careful evaluation of the resulting TCR modified populations need to be performed. We have previously cloned, and expressed TCRs from melanoma, EBV, HCV, and HPV reactive T-cell clones and found that several routine indicators of T-cell function do not always predict the relative strength of a TCR. Using a combination of tetramer binding assays and antigen recognition assays, we identified TCRs that fall into three classes. One class of TCR did not bind tetramers yet resulted in cells with high avidity for antigen. A second TCR class bound tetramer but did not secrete cytokines in response to antigen. Finally, the third class of TCRs bound tetramer and reacted to antigen as would be expected. We conclude that tetramer binding is not always a good indicator of the function of a cloned TCR or the avidity of a TCR gene modified T cell. Furthermore, our data indicate that the use of tetramer binding alone to identify antigen reactive TCRs may result in the exclusion of TCRs that may be highly reactive or cross reactive to the relevant tumor antigen.     

Oxidative stress provokes atherogenic changes in adipokine gene expression in 3T3-L1 adipocytes

Increased oxidative stress has been associated with obesity-related disorders. In this study, we investigated how oxidative stress, in different ways of exposure, regulates gene expression of various adipokines in 3T3-L1 adipocytes. Exposure to 100-500microM H(2)O(2) for 10min, as well as exposure to 5-25mU/ml glucose oxidase for 18h, similarly decreased adiponectin, leptin, and resistin mRNAs, and increased plasminogen activator inhibitor-1 mRNA. Secretion levels of adipokines were also changed by oxidative stress in parallel with mRNA expression levels. Although a peak increase in plasminogen activator inhibitor-1 mRNA was achieved between 4 and 8h after exposure to H(2)O(2) for 10min, significant decreases in adiponectin and resistin mRNA were observed after 16h, while leptin mRNA was decreased earlier. Our results suggest that oxidative stress, even of short duration, has a significant impact on the regulation of various adipokine gene expressions favoring atherosclerosis.     

Immunization with heat shock protein 105-pulsed dendritic cells leads to tumor rejection in mice

Recently, we reported that heat shock protein 105 (HSP105) DNA vaccination induced anti-tumor immunity. In this study, we set up a preclinical study to investigate the usefulness of dendritic cells (DCs) pulsed with mouse HSP105 as a whole protein for cancer immunotherapy in vivo. The recombinant HSP105 did not induce DC maturation, and the mice vaccinated with HSP105-pulsed BM-DCs were markedly prevented from the growth of subcutaneous tumors, accompanied with a massive infiltration of both CD4+ T cells and CD8+ T cells into the tumors. In depletion experiments, we proved that both CD4+ T cells and CD8+ T cells play a crucial role in anti-tumor immunity. Both CD4+ T cells and CD8+ T cells specific to HSP105 were induced by stimulation with HSP105-pulsed DCs. As a result, vaccination of mice with BM-DCs pulsed with HSP105 itself could elicit a stronger tumor rejection in comparison to DNA vaccination.     

Reactive oxygen species modify oligosaccharides of glycoproteins in vivo: a study of a spontaneous acute hepatitis model rat (LEC rat)

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, spontaneously develops hepatitis as the result of abnormal copper accumulation in liver. The findings of this study show that copper, hydrogen peroxide, and lipid peroxides accumulate to drastically high levels in LEC rat serum in acute hepatitis but not chronic hepatitis. The effect of these reactive oxygen species (ROS) on oligosaccharides of glycoproteins in the LEC rat serum was examined. Lectin blot and lectin ELISA analyses showed that sialic acid and galactose residues of serum glycoproteins including transferrin were decreased in acute hepatitis. Further analyses of oligosaccharide structures of transferrin demonstrated that di-sialylated and asialo-agalacto biantennary sugar chains, but not tri-sialylated sugar chain, exist on transferrin in the acute hepatitis rats. In addition, treatment of non-hepatitis rat serum with copper ions and hydrogen peroxide decreased tri-sialylated sugar chain of the normal transferrin and increased di-sialylated and asialo-agalacto biantennary sugar chains. This is the first evidence to show that ROS result in the cleavage of oligosaccharides of glycoproteins in vivo, and indicate this cleavage of oligosaccharides may contribute the development of acute hepatitis.     

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.     

Biosynthesis of archaeal membrane lipids: digeranylgeranylglycerophospholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.     

Role of AKT in cyclic strain-induced endothelial cell proliferation and survival

Endothelial cells (ECs) are exposed to repetitive cyclic strain (CS) in vivo by the beating heart. The aim of this study was to assess the influence of CS amplitude and/or frequency on EC proliferation and survival and to determine the role of AKT in CS-induced EC proliferation and survival. Cultured bovine aortic ECs were exposed to 10% strain at a frequency of 60 (60 cpm-10%) or 100 (100 cpm-10%) cycles/min or 15.6% strain at a frequency of 60 cycles/min (60 cpm-15.6%). AKT, glycogen synthase kinase (GSK)-3, BAD, and cleaved caspase-3 were activated by CS in ECs. Increasing the magnitude or frequency of strain resulted in an earlier phosphorylation of GSK-3, although the magnitude of phosphorylation was similar. After CS at 60 cpm-10% for 24 h, the number of nontransfected ECs was significantly increased by 8.5% (P < 0.05). We found that the number of apoptotic ECs was slightly decreased with exposure to CS. ECs transfected with kinase-dead AKT (KA179) as well as plasmids containing a point mutation in the pleckstrin homology domain of AKT (RC25) not only prevented AKT, GSK-3, and BAD phosphorylation but also inhibited the CS-induced increase in cell number as well as the CS-induced protection against apoptosis (both P < 0.05). The ratio of 5'-bromo-2'-deoxyuridine-positive cells was increased when ECs transfected with RC25 and KA179 as well as nontransfected ECs and ECs transfected with Lipofectamine 2000 were exposed to CS. We conclude that AKT is important in enhancing the survival of ECs exposed to CS but is not involved in EC proliferation. apoptosis; glycogen synthase kinase     

Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagic Escherichia coli strains

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.     

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.