Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.     

Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid

The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.     

Toll-like receptor 2 activation implicated in oral squamous cell carcinoma development

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity; however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2 was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived from bacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signal transduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly, ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of caspase recruitment domain–containing protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. These findings suggest that the activation of TLR2 by bacterial components can enhance the progression of OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of the miR-146a pathway.     

n-acylation of β-amino alcohol by acyl migration following enzyme-catalyzed esterification

Lipase-catalyzed n-acylations of β-amino alcohols such as ethanolamine and l-serine were investigated. To prepare n-acyl derivatives by taking advantage of the acyl migration, we first carried out a screening of suitable enzymes for the desired reaction. As a result, we found a higher activity for n-acylation with Lipase L. This lipase had higher hydrolytic activity for the o-acyl compound but not the n-acyl compound. The observation shows that n-acylation results from the esterification and successive acyl migration into the amino group. Using Lipase L, we then investigated the n-acylation of ethanolamine or l-serine with fatty acids as acyl donors. The reaction parameters for the n-acylation were clarified.     

Nitrogenous nutrient uptake by phytoplankton and ammonium regeneration by microbial assemblage in Lake Biwa

In situ rates of nitrate, ammoniwn and urea uptake by the phytoplanktonassemblage, and the regeneration rate of ammonium by the microbialassemblage, in Lake Biwa were measured using the nitrogen 15tracer method from 1985 to 1987. The rate of total nitrogen(sum of ammonium, nitrate and urea) uptake was in the rangeof 62–594 ng N^–1 r^–1 h^–1. The percentagecontribution of ammonium uptake was 41–92%, that of urea4–58% and that of nitrate <1–28% of total uptake.The annual mean new production which was supported by nitrateuptake was 18% of the total production in 1986. The phytoplanktonassemblage in Lake Biwa preferentially utilized regeneratednitrogen, such as ammonium and urea, whose concentration wasmuch lower than that of nitrate throughout the observation penodwithout in summer. The in situ nitrogen uptake rate was almostsufficient to meet the nitrogen requirement of the phytoplanktonassemblage, except in midsummer when the nitrate concentrationwas below the detection limit of 0.3 µg N r^–1. Inthe trophogemc layer, the rate of ammonium regeneration was66–272 ng N 1^–1 h^–1 Although the ambient ammoniumconcentration in the trophogenic layer was maintained at aroundthe half-saturation constant for ammonium uptake kinetics, theammomum uptake rates were always highly correlated with ammoniumregeneration rates. From the size fractionation experimentsand estimates from the literature, it was suggested that themicrobial assemblage <1 µm may have been the most importantagent responsible for the ammonium regeneration processes inthe trophogenic layer.     

Differential Regulation of HLA-DR Expression and Antigen Presentation in Toxoplasma gondii-Infected Melanoma Cells by Interleukin 6 and Interferon γ

CD4⁺ cytotoxic T lymphocytes (CTL) clones, YT-4 and YT-9, specific for Toxoplasma gondii (T. gondii)-infected melanoma SK-MEL 28 (P36), were generated from the peripheral blood lymphocytes (PBL) of a patient with chronic toxoplasmosis. These CTL clones were shown to secrete significant amounts of interleukin 6 (IL-6) and interferon γ (IFN-γ) upon antigen (Ag)-specific stimulation. Downregulation of human leukocyte antigen (HLA)-DR surface expression and HLA-DR mRNA levels in P36 cells were observed when P36 cells were infected with T. gondii. Such downregulated HLA-DR expressions of 71 gondii-infected P36 cells were upregulated by treatment with both recombinant IL-6 (rIL-6) and recombinant IFN-γ (rIFN-γ). The antigen-presenting ability of T. gondii-infected P36 cells to T. gondii-infected cell-specific CTL was enhanced by rIFN-γ but not by rIL-6. The present study reveals the existence of differential regulation of HLA-DR expression and Ag presentation in T. gondii-infected melanoma cells by IL-6 and IFN-γ.     

Characterization of the Isoenzymes of cyclic nucleotide phosphodiesterase in human platelets and the effects of E4021

In extracts of human platelets, three isoenzymes of cyclic nucleotide phosphodiesterase (PDE), namely, PDE2, PDE3, and PDE5, were identified; activities of PDE1 and PDE4 were not detected. In human platelets, the cGMP-hydrolytic activity was about six times higher than the cAMP-hydrolytic activity, and PDE5 and PDE3 are the major phosphodiesterase isoenzymes that hydrolyze cGMP and CAMP, respectively. PDE5 exhibited organ-specific expression in humans, and platelets were among the tissues richest in PDE5. A novel inhibitor of PDE5, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl] piperidine-4-carboxylate sesquihydrate (E4021), was a potent and highly selective inhibitor of human platelet PDE5. However, E4021 (up to 10 μM) did not inhibit 9,11-epithio-11,12-methano-thromboxane A₂-induced platelet aggregation, in vitro. E4021 plus SIN-1 (3-morpholino-sydnonimine), at concentrations that had little effect individually, inhibited aggregation. These results suggest the unique distribution of phosphodiesterase isoenzymes in human platelets and the PDE5 inhibitors might be useful as a new class of antiplatelet drugs.