Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system

A novel tumor-detection system consisting of complementmediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.     

Oxidation and Reduction Pathways in the Knowles Hydroamination via a Photoredox-Catalyzed Radical Reaction**

Systematic reaction path exploration revealed the entire mechanism of Knowles's light-promoted catalytic intramolecular hydroamination. Bond formation/cleavage competes with single electron transfer (SET) between the catalyst and substrate. These processes are described by adiabatic processes through transition states in an electronic state and non-radiative transitions through the seam of crossings (SX) between different electronic states. This study determined the energetically favorable SET path by introducing a practical computational model representing SET as non-adiabatic transitions via SXs between substrate's potential energy surfaces for different charge states adjusted based on the catalyst's redox potential. Calculations showed that the reduction and proton shuttle process proceeded concertedly. Also, the relative importance of SET paths (giving the product and leading back to the reactant) varies depending on the catalyst's redox potential, affecting the yield.     

Total Biosynthesis of Melleolides from Basidiomycota Fungi: Mechanistic Analysis of the Multifunctional GMC Oxidase Mld7

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock-in method to elucidate the biosynthetic pathway leading to 1α-hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation of the Δ⁶-protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short-chain dehydrogenase/reductase family proteins. The highlight of the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation-triggered double-bond shift accompanying dehydration and acyl-group-assisted substitution with two different nucleophiles at the C6 position to afford the Δ⁷-protoilludene derivatives, such as melleolide and armillarivin. The complex reaction mechanism was proposed by DFT calculations. Of particular importance is that product distribution is regulated by interaction with the cell membrane.     

Studies on the synthesis of compounds related to adenosine 3',5'-cyclic phosphate. IX. Synthesis and cytotoxic effect of adenosine 3',5'-cyclic alkylphosphoramidates.

A series of adenosine 3',5'-cyclic monophosphoramidates (3, cAMP amidates), including long-chain alkyl amidates, were synthesized from adenosine 3',5'-cyclic monophosphate (1, cAMP) by means of a one-pot reaction. This reaction proceeded by the treatment of cAMP tributylammonium salt (2) with phosphorus pentachloride (PCl5) and alkylamine in N,N-dimethylformamide (DMF). Compounds 3 synthesized were investigated to determine their cytotoxic activities on the growth of mouse mastocytoma P-815 cells, mouse mammary tumor FM3A cells, and human mammary tumor ZR-75 cells in culture. It was found that compounds 3h-m showed significant cytotoxic activities against these cell lines, and that cAMP decylamidate (3j) was the most cytotoxic compound (the concentration required for 50% inhibition of cell growth, ID50 = 6.0, 15.0, 2.2 microM, respectively); the antitumor effect on P-815 cells by a total packed cell volume method showed 81.8% inhibition. The cytotoxic activity of 3 increased with the increase in alkyl chain length up to 10 carbon atoms and decreased in compounds having longer alkyl chain.     

Resolution of lymphocyte subpopulations derived from rat spleen by polyamine-graft-PHEMA copolymer

The retention behavior of lymphocyte subpopulations, B cell, T cell and null cell, derived from rat spleen to polyamine-graft-poly(2-hydroxyethyl methacrylate) copolymer (HA) surface was investigated, focusing on the conformational transition of the polyamine side chain as well as the protonation of amino groups in the polyamine grafts. Furthermore, the availability of HA was discussed as a column adsorbent for separation of lymphocyte subpopulations derived from spleen. The conformational transition of polyamine grafts significantly influenced the mode of retention of lymphocyte subpopulations. When polyamine grafts existed in an aggregated conformation (protonatin degree α < 0.5), the retention of lymphocyte subpopulations was decreased in the order B cell> null cell> T cell. On the other hand, when polyamine existed in an extended conformation into the aqueous interior from the matrix interface (α > 0.5), T cell retention became greater than null cell retention, resulting in a decreased B cell> T cell> null cell order. These results indicate that the differential retention of spleen lymphocyte subpopulations is attributed to their differential responses to the change in matrix interface accompanied by the protonation of amino groups. Furthermore, spleen lymphocytes were compared with lymph node lymphocytes in terms of resolution efficacy by an HA copolymer column.     

Functional and site-specific macromolecular micelles as high potential drug carriers

Since several years, macromolecular micelles based on amphiphilic block copolymers have attracted much interest as drug carriers. These micelles show a long term blood circulation time resulting from their small diameter and the steric repulsion created by the poly(ethylene oxide) chains which constitute micelle corona, as well as from their high thermodynamic stability. Besides this long term blood circulation time generating a passive targeting, an active targeting, chemical or physical affinity targeting, might allow the preparation of more efficient drug carriers. In order to obtain such double targeting properties, we have prepared two kinds of macromolecular micelles. The first one is based on amphiphilic poly(ethylene oxide)/poly(β-benzyl -aspartate) ---PEO/PBLA--- block copolymers having hydroxy groups at the free end of PEO chains. As a result of their structure, such micelles have hydroxy groups on their outer-shell which can be further modified in order to introduce a targeting moiety (sugar, etc.). The characteristics (diameter, critical micellar concentration (cmc), drug loading capacity) have been determined. Moreover, doxorubicin loaded -hydroxy PEO/PBLA micelles have been shown to be slightly more cytotoxic than the corresponding -methoxy PEO/PBLA micelles. The second type of micelles is based on thermosensitive amphiphilic poly(N-isopropyl acrylamide)/polystyrene ---PIPAAm/PSt--- block copolymers. Such micelles have a small diameter and a low cmc in addition to thermosensitivity properties which are similar to those of PIPAAm.     

Remarkably Selective Ag(+) Extraction and Transport by Thiolariat Ethers

Synthesis and metal binding properties of thiolariat ethers, where a sulfide side chain is introduced into a framework of a crown ether, have been performed. Remarkably high Ag(+) selectivity among heavy metal ions was observed in solvent extraction and transport across a liquid membrane using thiolariat ethers with a 15-crown-5 ring as carriers. Thiolariat ethers with a 12-crown-4 or a 18-crown-6 do not exhibit such a high Ag(+) selectivity. The former binds metal ions weakly, and the latter recognizes Pb(2+) as well as Ag(+). The corresponding oxygen analogs, i.e. lariat ethers, do not show Ag(+) selectivity. The Ag(+) binding strength of the sulfoxide and sulfone analogs is much lower than that of thiolariat ethers. Thiolariat ethers with a benzocrown framework containing a sulfide chain at the 4 position of the benzene nucleus showed very low affinity to Ag(+). Extractability and transport ability using various thiolariat ether derivatives strongly suggested that this high Ag(+) selectivity is a result of the synergistic coordination of the ring oxygen and the sulfur atom of the thiolariat ether. NMR chemical shifts of protons and carbons in the proximity of the sulfur atom of the thiolariat ether were changed significantly in accordance with the synergistic coordination described above. 1:1 Complexation between a thiolariat ether and Ag(+) were supported by a Job plot using the chemical shift of the methylene protons adjacent to the sulfur atom.     

Preparation and characterization of silica—polyimide composite membranes coated on porous tubes for CO2 separation

Silica-polyimide microcomposite membranes were prepared on γ-alumina-coated α-alumina support tubes, and their gas permeation properties were evaluated with He, N₂ and CO₂. Smoothing of the substrate surface and hybridization of silica and polyamic acid were both effective to form defect-free thin composite membranes. The CO₂ permeance of a membrane with a silica content of 68 wt% was one order of magnitude higher than that of a polyimide membrane having the same thickness. The permselectivity of CO₂ to N₂ was 30 at 30°C and 13 at 100°C. Contributions of the silica and polyimide phases to permeance of the composite membrane were analyzed with a two-phase permeation model. The effective thickness of the rate-controlling polyimide phase was less than one-tenth of the total thickness of the silica-polyimide membrane.     

Molecular structures,electronic spectra,and aromaticity of 1,6- and 1,8-dioxa analogues of pyrene and benzo[a]pyrene. Semiempirical molecular orbital calculations

The molecular structures of [1]benzopyrano[6,5,4-def][1]benzopyran, [2]benzopyrano[7,8,1,def]-1-benzopyran, 1-benzopyrano[6,5,4-mna]xanthene, and 2-benzopyrano[7,8,1-mna]xanthene have been optimized by using the PM3 semiemperical MO method. These calculated molecules have been shown to be planar. Their aromaticity has been investigated by use of the HOMA index. The molecules are found to be less aromatic than the correspolding parednt hydrocarbons, pyrene and benzo[a]pyrene. The CNDO/S MO method has been used to interpret th experimental uv-vis specrtoscopic data. The result of the PM3, CNDO/S and HOMA-index clculations are in good arteement with the experimental data.