High-throughput immunophenotyping by surface plasmon resonance imaging

Cell binding assays on antibody arrays permit the rapid immunophenotyping of living cells. The throughput of the analysis, however, is still limited due to our inability to perform parallel and quantitative detection of cells captured on the array. To address this limitation, we employed here an imaging technique based on surface plasmon resonance (SPR). SPR has been frequently used to monitor capture of proteins on antibody microarrays, while few cases were reported for capture of cells. Antibody arrays were prepared through the photopatterning of an alkanethiol monolayer on a gold-evaporated glass plate and the subsequent immobilization of various antibodies onto 4-9 separate spots created by photopatterning. A glass slip was mounted onto the array with a thin spacer to construct a parallel-plate chamber. Leukemia cells were injected into the chamber to conduct a binding assay, while refractive index changes at the vicinity of the array surface were monitored by SPR imaging. We observed that SPR signals were intensified on specific antibody spots but not on nonspecific spots. Confocal laser scanning microscopy revealed that the observed SPR signals were attributed to cell deformations caused by multivalent interactions with immobilized antibody, which effectively elevated the refractive index of a medium phase within an evanescent field. This effect could be suitably utilized to monitor quantitatively cell binding to multiple spots from a heterogeneous cell population.     

Potential of predominant activated sludge bacteria as recipients in conjugative plasmid transfer

We investigated the possibility of conjugative plasmid transfer to the predominant bacteria in activated sludge and the factors influencing the transfer frequency in the activated sludge process. We performed conjugative transfers of a self-transmissible, broad-host-range plasmid RP4 from Escherichia coli C600 to activated sludge bacteria by broth mating. Most of the activated sludge bacteria tested could acquire plasmid RP4, although the transfer frequencies varied from 8.8 x 10(-7) to 1.3 x 10(-2) transconjugants per recipient. The transfer frequencies in several strains were similar to, or higher than, that in intraspecific transfer to E. coli HB101. Matings under various environmental conditions showed that factors relevant to physiological activity, such as temperature and nutrient conditions, seemed to affect the transfer frequency. In addition, conjugative transfer was detected even in filtered raw and treated wastewaters. Thus, the predominant activated sludge bacteria seem to have sufficient potential as recipients in conjugative plasmid transfer under the conditions likely to occur in the activated sludge process. Transfer frequency was reduced by agitation in the presence of suspended solid. This may suggest that conjugative plasmid transfer is physically inhibited in aeration tanks.     

Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM

We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.     

Role of peritoneal lavage smears and gastric wall brushing smears in gastric cancer surgery

Examinations of peritoneal lavage smears (LC) and gastric wall brushing smear cytology (BC) appear to be important for determining accurately the stage of gastric cancer. We have been carrying out such examinations during gastric cancer surgery for 7 years. In the present study, we evaluated the results obtained from 287 patients with gastric cancer. Tumor invasion and peritoneal dissemination were correlated with a positive incidence of cancer cells in LC and/or BC. Gastric cancer showing serosal invasion was classified into positive for LC and/or BC and negative for LC and/or BC. Patients positive for LC and/or BC had a poorer prognosis. For future gastric cancer treatment, patients positive for peritoneal cytology are expected to be targeted for intensive treatment during or before surgery.     

A carboxy-terminal truncation of 99 amino acids resulting from a novel mutation (Arg555-->stop) in the CHM gene leads to choroideremia

The gene content of the MHC class I telomerically adjacent region, in linkage disequilibrium with hereditary hemochromatosis, has not been well characterized yet. In the present work, we established three bacterial clone contigs, including mainly P1-derived artificial chromosomes. These contigs cover 89% of the 1.2-Mb 6p-subtelomeric region encompassing locus D6S105. Terminal exon trapping was applied to selected clones from these contigs. Forty-six independent terminal exons were identified and mapped within the region, 2 of which matched perfectly to expressed sequence tags. These 3' exons are all expressed in human fetal brain but differentially expressed in four tissues and two cell lines. The high number of exons identified indicates that the high gene density observed in the MHC class I region extends to this telomerically adjacent region.     

Effect of hardness difference on the surface durability and surface failure of steel rollers

Experiments with pairs of rollers of different hardnesses designed to assess the effect of hardness difference on surface durability and surface failure are described. Sliding-rolling contact fatigue tests were performed with combinations of thermally refined, through-hardened and induction-hardened 0.45% C steel rollers. The mode of failure is discussed in relation to the hardness difference between the rollers and the presence of residual stresses. The effect of hardness on the modulus of elasticity was also examined. An empirical equation was devised to describe the relationship between the rolling contact fatigue limit under hertzian stress and the surface hardness.     

Synthesis of terpene alcohols from isoprene oligomers

Various terpene alcohols were prepared from isoprene oligomers via two steps: epoxidation with peracetic acid and isomerisation with aluminium triisopropoxide. For example, 2-methylene-6-methyl-7-octene-1-o1 was prepared from 2,6-dimethyl-1,7-octadiene. From 2,6-dimethyl-2,7-octadiene, 3,3,6-trimethyl-1,5-heptadiene, 2,7-dimethyl-2,6-octadiene, 2,6-dimethyl-1,3,6-octatriene, 1-methyl-4-isopropenyl-1-cyclohexene, and 1,5-dimethyl-1,5-cyclooctadiene, the corresponding alcohols were obtained, respectively.     

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.