A spectroscopic and photophysical study on molecular recognition via hydrogen-bonding and pi-pi stacking interactions.

Spectroscopic and photophysical properties of a Kemp's tricarboxylic acid derivative having an anthracene chromophore (I) upon recognition of 9-butyladenine (BA) in chloroform were studied in detail. Molecular recognition of BA by I via hydrogen-bonding and pi-pi stacking interactions were sensed successfully on the basis of absorption and fluorescence spectroscopies, by which the binding constant of the I:BA complex was determined to be 240 M(-1). The fluorescence quantum yield and lifetime of I in the absence of BA were 0.24 and 5.6 ns, respectively, while those in the presence of an enough amount of BA increased to 0.35 and 13 ns, respectively. These values demonstrated that the nonradiative decay rate constant of I decreased from 13.6 x 10(7) to 5.0 x 10(7) s(-1) upon binding with BA. Such changes in the photophysical properties of I before and after complexation with BA were discussed in terms of hydrogen-bonding and pi-pi stacking interactions between I and BA. In particular, intramolecular hydrogen-bonding between the amide and imide groups in was shown to play important roles in determining the photophysical characteristics of I before complexation, while intermolecular hydrogen-bonding between I and BA governed the excited-state properties of the I:BA complex. The change in the hydrodynamic diameter of I before and after complexation with BA was also discussed on the basis of the results by fluorescence dynamic anisotropy measurements.     

Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica.

S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5'- and 3'-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity.     

Enzymatic production of γ-L-glutamyltaurine through the transpeptidation reaction of γ-glutamyltranspeptidase from Escherichia coli K-12

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C₁₈ columns, and identified by means of NMR and a polarimeter.     

Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone

Summary Regenerants derived from hairy roots of Ajuga reptans var. atropurpurea induced by infection with Agrobacterium rhizogenes MAFF03-01724 harboring pRi revealed a dwarfing response, i.e. decrease in leaf size, reduction in internode distance, and increase in leaf number. These morphogenic alterations were accompanied by an increase in root mass and lack of floral differentiation. In the pRi-transformed regenerants, the proportion of root mass to whole plant mass was higher than that of the untransformed ones, although both kinds of regenerants were comparable on a fresh weight basis. High capacity of rooting and 20-hydroxyecdysone production associated with the original hairy root line were stably maintained in clonal regenerants.     

The Effect of Lipid Peroxide on the Lipid and Carbohydrate Metabolism in Rat Liver

Feeding tests were carried out on rats to clarify the mechanisms of fatty liver formation induced by autoxidized methyl linoleate. Lipid peroxides prepared by autoxidation of highly purified methyl linoleate were given orally to rats. Triglyceride and glycogen contents in liver were determined and enzyme activities including triglyceride synthetase and α-glycerophosphate dehydrogenase were also examined. The following results were obtained. 1. Triglyceride accumulation in rat liver fed autoxidized methyl linoleate was observed. 2. Increase in triglyceride content in rat liver was soon followed by the decrease of hepatic glycogen. 3. When rats were starved prior to introduction of autoxidized methyl linoleate, hepatic triglyceride accumulation did not occur. 4. The activities of α-glycerophosphate dehydrogenase and triglyceride synthetase in liver, and those of glutamic oxalacetic transaminase and leucine aminopeptidase in plasma were practically similar among the rats of test groups fed fresh or autoxidized methyl linoleate and the control fed diet without methyl linoleate. 5. The addition of l-carnitine which is a stimulator of fatty acid oxidation retarded the accumulation of the hepatic triglyceride mentioned above.     

STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca²⁺ sensor that activates Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry (SOCE). We investigated the role of STIM1 in [Ca²⁺]_i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca²⁺-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca²⁺]_i followed by sustained [Ca²⁺]_i elevation. Sustained increases in [Ca²⁺]_i due to PDGF-BB were significantly inhibited by a Ca²⁺ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca²⁺]_i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca²⁺ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.