Effects of isoflavone compounds on the activation of phospholipase C.

The effect of isoflavone compounds, genistein and daidzein, on the breakdown of inositol phospholipids in 3T3 cells was studied. Genistein (100 micrograms/ml) inhibited the stimulation of the production of inositol phosphates by bombesin. The stimulated production of inositol phosphates by AlF-4 was also inhibited by genistein (IC50 = 0.6 micrograms/ml) and daidzein (IC50 = 2 micrograms/ml). However, the catalytic activity of phospholipase-C (PLC) in 3T3 cell extracts was not inhibited by these isoflavones. These results suggest that the isoflavones inhibited the activation of PLC at the G-protein or downstream of the sequences in signal transduction. In permeabilized 3T3 cells, the inhibition of AlF-4 plus adenosine triphosphate (ATP)-dependent PLC was recovered by increasing ATP but not AlF-4. Genistein also inhibited the activity of adenosine 5'-[3-O-thiotriphosphate] (ATP[S])-dependent PLC. The effect of genistein and other inhibitors of protein tyrosine kinases and phosphatases suggests that protein tyrosine phosphorylation is not involved in the activation of PLC in 3T3 cells and that AlF-4- and ATP[S]-mediated activation of PLC involves a different mechanism from the tyrosine kinase-mediated activation of PLC. Daidzein and genistein seem to interrupt the ATP-dependent step of PLC activation by a putative G-protein.     

Multiple binding of inhibitors in the complex formed by bovine trypsin and fragments of a synthetic inhibitor, 4-[4-(N,N- dimethylcarbamoxylmethoxycarbonylmethyl)phenoxycarbonylphenyl+ ++] guanidinium methanesulfonate (FOY-305)

The crystal of bovine trypsin complexed with a potent inhibitor, 4-[4-(N,N- dimethylcarbamoylmethoxycarbonylmethyl)phenoxycarbonylphenyl ]guanidinium (FOY-305) in the novel orthorhombic from with a low molecular packing density was studied by the X-ray diffraction method. Using synchrotron radiation, the intensity data were collected to 1.8 A resolution. The structure was solved by molecular replacement methods, and refined to an R-factor = 18.0% for 14364 reflections by the restrained least-squares method. The final difference Fourier maps revealed that hydrolyzed inhibitor fragments bind with the protein at multiple sites around the active center of trypsin. The structural feature in the crystalline state probably corresponds to a statistical average of several complexes which would be formed between the inhibitor and trypsin during the binding and releasing process in solution.     

Studies on reactions of the N-phosphonium salts of pyridines—VII : Preparation of peptides and active esters of amino acids by means of diphenyl and triphenyl phosphites in the presence of tertiary amines

Peptides and active esters of amino acids were produced in high yields from carboxyl and amino or hydroxyl components in pyridine with an equivalent amount of diphenyl phosphite or half an equivalent amount of triphenyl phosphite and tertiary amines. Condensation reactions competed with the reaction with a phenoxy group of the phosphite to produce the phenyl ester and were governed by the tertiary amine employed in the reaction. The reactions are assumed to proceed via the N- phosphonium salts of pyridines, similar to those obtained by the oxidation of phosphorus compounds with mercuric salts in pyridine.     

Evolution of fluorescein as a platform for finely tunable fluorescence probes

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.     

High‐viscosity polyesters from secondary alicyclic 1,4‐cyclohexanedicarboxylic acids and hydroquinones with thionyl chloride/pyridine condensing agent

High‐viscosity polyesters can be prepared by the polycondensation of secondary aliphatic trans‐cyclohexanedicarboxylic acid (t‐CHDC) and hydroquinones using SOCl₂/pyridine condensing agent at 80°C. Incorporation of its cis‐isomer significantly affected η_inh and the thermal property of the resulting copolymers. The copolymers of t‐CHDC, hydroquinones, and p‐hydroxybenzoic acid having different monomer sequences were prepared by changing the order of introduction of monomers.     

Random copolyamides of terephthalic acid,p‐phenylenediamine and p‐aminobenzoic acid soluble in N‐methylpyrrolidone/CaCl2

Moderate‐molecular‐weight copolyamides soluble in N‐methylpyrrolidone (NMP) containing dissolved CaCl₂ can be obtained by polycondensation of terephthalic acid (TPA), p‐phenylenediamine (PPD), and p‐aminobenzoic acid (PABA) with triphenyl phosphite/pyridine in NMP. The randomly copolymerized polymers contain more than 40 mol‐% of PABA and are easily soluble in NMP.     

Synthesis of Controlled Block and Graft Copopolymers. II. Block and Graft Polymerization Initiated by Monohalo-Containing Polymer/Manganese Carbonyl Systems

Abstract

Block polymerization of methyl methacrylate initiated with monohalo-terminated polystyrene/manganese carbonyl systems were investigated. The rate of polymerization was lower than that of the trihalo-terminated polystyrenelmanganese carbonyl system. The molecular weight of the block copolymers obtained was independent of the conversion. To define the polymerization mechanism, 1-bromobutane was employed as a model compound for monohalo-terminated polystyrene. Polymerization kinetics followed a modified rate equation based on the Michaelis-Menten mechanism. The molecular weight of the block co-polymer could be regulated by varying the ratio of monohalo-terminated polymer to methyl methacrylate. Graft polymerization of methyl methacrylate initiated with chloromethylstyrene-styrene copolymer/ manganese carbonyl was also carried out. The polymerization behavior was strongly affected by the concentration of manganese carbonyl. Characterization of graft copolymers by GPC and halogen analysis showed that the number of grafting points per backbone polymer molecule increased when the concentration of manganese carbonyl was raised, but that the branches became shorter.     

Metabolism of methylated arsenic compounds by arsenic-resistant bacteria (Klebsiella oxytoca and Xanthomonas sp.)

Two bacteria exhibiting resistance to toxic arsenic were isolated. These had been contaminated with arsenic in a Chlorella sp. culture medium containing arsenic. The two bacteria were identified as Klebsiella oxytoca and Xanthomonas sp., and grew well in a peptone medium at neutral pH at 30°C, reaching the stationary phase in ca 100h and 70h, respectively. The growth of the bacteria was not affected by arsenic(V) concentrations in the medium as high as 1000mg dm^?3. The bacteria bioaccumulated arsenic, a part of the arsenic being methylated. The bioaccumulation exhibited its peak around the turing point from the log phase to the stationary phase. The relative content of methylated arsenic in the excrement was greater than that in the bacterial cells. Adaptation treatment of inorganic arsenic caused an increase in the bioaccumulation of inorganic arsenic by K. oxytoca. Such a situation was not observed in the case of Xanthomonas sp. The bacteria also bioaccumulated methylated arsenic compounds, and demethylation of these species was observed. When the bacteria were killed by ethanol, arsenic was not taken up by the cells.