Characterization of N-terminal processing of group VIA phospholipase A<Subscript>2</Subscript> and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.     

Comparison of one-, two-, and three-dimensional iron phosphates containing ethylenediamine

A new two-dimensional (2d) iron phosphate, (C₂N₂H₁₀)Fe₂O(PO₄)₂, has been synthesized under hydrothermal conditions in the system of FeCl₃-H₃PO₄-C₂N₂H₈-H₂O. The crystal data is: space group P2₁/c, a=10.670(1) Å, b=10.897(1) Å, c=9.918(1) Å, β=105.632(1)°, Z=4. The layered structure consists of double sheet layers, of composition Fe₂O(PO₄)₂, built from FeO₅ trigonal bipyramids and PO₄ tetrahedra. The amine holds the layers together via H-bonding. The study of the magnetic properties reveals two magnetic transitions at 160 and 30 K with spin-glass-like behavior below 160 K. By varying the hydrothermal conditions, three other iron phosphates were synthesized: the one-dimensional (1d) (C₂N₂H₁₀)Fe(HPO₄)₂(OH)·H₂O, the 2d (C₂N₂H₁₀)Fe₂(PO₄)₂(OH)₂, and the three-dimensional (3d) (C₂N₂H₁₀)₂Fe₄O(PO₄)₄·H₂O. The 1d compound can be used as the starting reagent in the synthesis of both the 2d compound and the 3d lipscombite Fe₃(PO₄)₂(OH)₂ due to the similar building blocks in their structures. In the 3d phosphate (C₂N₂H₁₀)₂Fe₄O(PO₄)₄·H₂O, manganese can substitute for half of the iron atoms. Magnetic study shows ordering transitions at about 30 K, however, manganese substitution depresses the magnetic ordering temperature.     

Application of an integrated microchip system with capillary array electrophoresis to optimization of enzymatic reactions

In this work, a combination of complementary metal-oxide semiconductor (CMOS) microchip system with capillary array electrophoresis (CAE) is demonstrated as a system for optimizing conditions for enzymatic reaction. Dimethylacridinone (DDAO)-phosphate substrate and alkaline phosphatase conjugate were selected for the enzymatic reaction, which was applicable to the enzyme-linked immunosorbent assay (ELISA) technique. Laser-induced fluorometry with a miniature semiconductor laser was used to detect the enzymatic products. The speed of the enzymatic reaction between the DDAO-phosphate and the alkaline phosphatase conjugate was investigated as a function of reaction time. The microchip-CAE detection system could determine the pH condition and the concentration of enzyme that are suitable for rapid and low-cost analysis. This result shows the feasibility of using the microchip-CAE system for application to miniaturized screening systems.     

Metastasis-suppressor KAI1/CD82 induces homotypic aggregation of human prostate cancer cells through Src-dependent pathway

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.     

On the relative Wall-Witt groups

LetR* be a simplicial involutive ring. According to certain involutions onK(R*) and _ε L R _∗, there are 1/2-local splittings and . It is known [2] that _ε L _\ga ^α R _∗, the Wall-Witt group. SupposeI→R S is a split extension of discrete involutive rings withI ²=0, andI is a freeS-bimodule. Then we have and . The trace map Tr: Prim _n ∧*M(I ⊗ ℚ)→ ₀ ρ _n ;I ⊗ ℚ) is an isomorphism. We prove in Lemma 1 that the trace map Tr is ℤ/2-equivariant. In Theorem 2 we show that under a certain assumption the rational relative Wall-Witt group vanishes. Theorem 2 can be extended to a more general case (Theorem 3) by employing Goodwillie’s reduction technique [3]. This work was partially supported by KOSEF under Grant 923-0100-010-1.     

Symmetric Skorohod topology onn-variable functions and hierarchical Markov properties ofn-parameter processes

Summary We introduce a new Skorohod topology for functions of several variables. Since ann-variable function may be viewed as a one-variable function with values in the set of (n–1)-variable functions, this topology is defined by induction from the classical Skorohod topology for one-variable functions. This allows us to define the notion of completen-parameter symmetric Markov processes: Such processes are, for any 1pn, rawp-parameter Markov processes (in the sense of our previous paper [17]) with values in the space of (n–p)-variable functions. We prove, for these processes and their Bochner subordinates, a maximal inequality which implies the continuity of additive functionals associated with finite energy measures. We finally present several important examples.