KNOWLEDGE PROCESS ANALYSIS： FRAMEWORK AND EXPERIENCE

We present a step-by-step approach for constructing a framework for knowledge process analysis （KPA）. We intend to apply this framework to the analysis of own research projects in an exploratory way and elaborate it through the accumulation of case studies. This study is based on a methodology consisting of knowledge process modeling, primitives synthesis, and reflective verification. We describe details of the methodology and present the results of case studies： a novel methodology, a practical work guide, and a tool for KPA; insights for improving future research projects and education; and the integration of existing knowledge creation theories.     

Degranulating activity in hybrid cells derived from rat peritoneal mast cells and ehrlich ascites tumor cells

Summary Fusion of rat mast cells and Ehrlich ascites tumor cells was mediated by HVJ. Compound 48/80-induced degranulation occurred in the fused cells formed from two mast cells and one tumor cell, but not in the fused cells from one mast cell and two or more tumor cells.I am grateful to DrK. Utsumi for valuable discussions and for the donation of HVJ.     

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.     

Eph- and ephrin-dependent mechanisms in tumor and stem cell dynamics

The erythropoietin-producing hepatocellular (Eph) receptors comprise the largest family of receptor tyrosine kinases (RTKs). Initially regarded as axon-guidance and tissue-patterning molecules, Eph receptors have now been attributed with various functions during development, tissue homeostasis, and disease pathogenesis. Their ligands, ephrins, are synthesized as membrane-associated molecules. At least two properties make this signaling system unique: (1) the signal can be simultaneously transduced in the receptor- and the ligand-expressing cell, (2) the signaling outcome through the same molecules can be opposite depending on cellular context. Moreover, shedding of Eph and ephrin ectodomains as well as ligand-dependent and -independent receptor crosstalk with other RTKs, proteases, and adhesion molecules broadens the repertoire of Eph/ephrin functions. These integrated pathways provide plasticity to cell–microenvironment communication in varying tissue contexts. The complex molecular networks and dynamic cellular outcomes connected to the Eph/ephrin signaling in tumor–host communication and stem cell niche are the main focus of this review.     

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-alpha. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-alpha ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.     

Schwann cell mitochondria as key regulators in the development and maintenance of peripheral nerve axons

Formation of myelin sheaths by Schwann cells (SCs) enables rapid and efficient transmission of action potentials in peripheral axons, and disruption of myelination results in disorders that involve decreased sensory and motor functions. Given that construction of SC myelin requires high levels of lipid and protein synthesis, mitochondria, which are pivotal in cellular metabolism, may be potential regulators of the formation and maintenance of SC myelin. Supporting this notion, abnormal mitochondria are found in SCs of neuropathic peripheral nerves in both human patients and the relevant animal models. However, evidence for the importance of SC mitochondria in myelination has been limited, until recently. Several studies have recently used genetic approaches that allow SC-specific ablation of mitochondrial metabolic activity in living animals to show the critical roles of SC mitochondria in the development and maintenance of peripheral nerve axons. Here, we review current knowledge about the involvement of SC mitochondria in the formation and dysfunction of myelinated axons in the peripheral nervous system.     

Endogenous inhibitor of ecdysone synthesis in crabs

Summary Attempts to isolate the molt-inhibiting hormone (MIH) of crustaceans from crab eyestalks (ES) resulted in the characterization of xanthurenic acid as an inhibitor of ecdysone biosynthesis in the cultured Y-organ-complex (YOC) homogenate. It was also found that 3-hydroxyl-l-kynurenine present in the ES is transformed into xanthurenic acid in the YOC and body fluid. Its mode of inhibitory action in ecdysone biosynthesis is probably inactivation of cytochrome P-450.Acknowledgments. We thank Prof. M. Oka (Nagasaki University) and Dr J. Cappuzo (Oceanographic Inst.) for information on Y-organ and molting stages, Dr J. Termini and Mr J. Cesarelli (Columbia University) for organ excision of blue crabs and experiments in the early stage of this study, the Japan Sea Farming Association for aqua culture of crabs, Prof. Y. Umebachi (Kanazawa University) for a gift of 3-OH-l-Kyn, and Mr J. Rudloe (Gulf Specimen Co.) for collection and shipping of some selected crabs. This study was partially supported by NIH grant AI 10 187 (to K. N., at Columbia University).     

Immuno-electron microscopic localization of fibronectin on rat mast cells

Summary Rabbit anti-rat plasma fibronectin (pFN) causes histamine release from rat mast cells in the presence of complement. Fibronectin (FN) on rat mast cells, as shown by immuno-electron microscopy, is principally localized on cell folds, so they may play a role of attachment in the matrix of connective tissue.Acknowledgment. This work is supported by grants (No. 56770014 and No. 544018) from the Ministry of Education, Science and Culture of Japan.