The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice

Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-gamma, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.     

Kinetic analysis of multisite phosphorylation using analytic solutions to Michaelis-Menten equations

Phosphorylation-induced expression or modulation of a functional protein is a common signal in living cells. Many functional proteins are phosphorylated at multiple sites and it is frequently observed that phosphorylation at one site enhances or suppresses phosphorylation at another site. Therefore, characterizing such cooperative phosphorylation is important. In this study, we determine a temporal progress curve of multisite phosphorylation by analytically integrating the Michaelis-Menten equations in time. Using this theoretical progress curve, we derive the useful criterion that an intersection of two progress curves implies the presence of cooperativity. Experiments generally yield noisy progress curves. We fit the theoretical progress curves to noisy progress curves containing 4% Gaussian noise in order to determine the kinetics of the phosphorylation. This fitting correctly identifies the sites involved in cooperative phosphorylation.     

Tk-PTP,protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.     

Terpenoids from Tripterygium doianum (Celastraceae)

The extract of Tripterygium doianum (Celastraceae) afforded three triterpenoids [3beta-acetoxy-11-ursen-13alpha,30-olide, 25-chloro-24-hydroxytirucall-7-en-3-one and tirucall-7-en-3,24-dione], two sesquiterpenoids [5alpha-acetoxy-1beta,8alpha-bis-cinnamoyl-4alpha-hydroxydihydroagarofuran and 5alpha-acetoxy-1beta-benzoyl-8alpha-cinnamoyl-4alpha-hydroxydihydroagarofuran] and nine known triterpenoids. Their structures were established based on spectroscopic studies.     

Stilbenoids from the stem of Gnetum latifolium (Gnetaceae)

An acetone extract of the stem of Gnetum latifolium Blume afforded the stilbene trimer (latifolol) together with five known stilbenoids (gnetin E, gnetin D, gnetin C, (-)epsilon -viniferin and resveratrol). Their structures were elucidated on the basis of spectral evidence, in particular by using 2D NMR methods.     

Isolation and identification of an allelopathic substance from peel of Citrus junos

The inhibitory effect of Citrus junos peel on plant growth using lettuce (Lactuca sativa L.) as a bioassay material was investigated, since the powder of the peel had been found to inhibit growth of weeds. Basic, neutral and acidic fractions were separated from the aqueous fraction obtained from the methanol extract of C. junos peel. All fractions inhibited the growth of lettuce seedlings, but by far the greatest inhibition was observed with the neutral fraction. Thus, the latter was further purified and an allelopathically active substance was isolated. The structure of the substance was determined from high-resolution MS and 1H and 13C NMR spectral data as abscisic acid-beta-D-glucopyranosyl ester (ABA-GE). ABA-GE inhibited hypocotyl and root growth of lettuce seedlings at concentrations greater than 0.3 microM, and the concentrations for 50% inhibition of hypocotyl and root growth were 2.3 and 1.4 microM, respectively. The effectiveness of ABA-GE on inhibition of growth and the occurrence of ABA-GE in the peel itself suggested that ABA-GE may play an important role in the allelopathic potential of C. junos peel. The peel may be potentially useful for weed management in a field setting.     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Med,a cell-surface localized protein regulating a competence transcription factor gene,comK, in Bacillus subtilis

Med was found as a positive regulator for comK, a master regulator for late competence genes. It was found by Western analysis that the ComK level was decreased in a med mutant. Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med. The results obtained are consistent with the localization of Med at the cell surface. An implication of the cell-surface localization of Med is discussed in terms of comK regulation.