Hiroya Obama

Identification of the Domain of α-Catenin Involved in Its Association with β-Catenin and Plakoglobin (γ-Catenin)

α-Catenin is a 102-kDa protein exhibiting homology to vincuin, and it forms complexes with cadherins or the tumor-suppressor gene product adenomatous polyposis coli through binding to β-catenin or plakoglobin (γ-catenin). The incorporation of α-catenin into the cadherin-catenin complexes is a prerequisite for expression of the cell-adhesive activity of cadherins. Using an in vitro assay system involving bacterially expressed proteins, we localized a region in α-catenin required for molecular interaction with β-catenin and plakoglobin. Analysis of various truncated α-catenin molecules revealed that amino-terminal residues 48–163 are able to bind to β-catenin and plakoglobin. Consistent with the observation that β-catenin and plakoglobin bind to the same region of α-catenin, β-catenin competed with the binding of plakoglobin to α-catenin and vice versa. Under the conditions used, β-catenin bound to α-catenin with higher affinity than did plakoglobin. Scatchard analysis indicated that the affinity of the interaction between α-catenin and β-catenin or that between α-catenin and plakoglobin was moderately strong (Kd = 3.8 × 10−8 and 7.7 × 10−8, respectively). When transfected into L cells expressing E-cadherin, the amino-terminal region of α-catenin (from residue 1 to 226) formed complexes with β-catenin supporting the in vitro binding experiment results.

Blood coagulation equilibrium in rat liver microcirculation as evaluated by endothelial cell thrombomodulin and macrophage tissue factor

The regulatory mechanisms of microcirculation might differ in the liver from other organs, because macrophages are resident in the hepatic sinusoids and sinusoidal endothelial cells are unique in shape and function. Thrombomodulin expression in endothelial cells and tissue factor activity in isolated macrophages were studied in the liver and lung of rats. In normal rats, the thrombomodulin expression was minimal in hepatic sinusoids, but prominent in pulmonary capillaries, while the tissue factor activity in the presence of endotoxin was higher in pulmonary macrophages than in Kupffer cells, although the levels in the absence of endotoxin were comparable in both cells. The tissue factor activity in hepatic macrophages was increased after priming of the cells with Corynebacterium parvum or after induction of liver necrosis or cirrhosis with carbon tetrachloride. In the necrotic or cirrhotic liver, increased thrombomodulin expression was seen along capillaries extending in necrotic areas and regenerating nodules, but this increase was minimal in the Corynebacterium parvum-treated rat liver. Blood coagulation equilibrium in microcirculation regulated by endothelial cells and macrophages may differ between the liver and lung. Such equilibrium in the liver may vary depending on pathological status.

Structure-Function Analysis of Nel, a Thrombospondin-1-like Glycoprotein Involved in Neural Development and Functions

Background: Nel is a multimodular glycoprotein and plays important roles in neural development and functions. Results: The N-terminal thrombospondin-1 domain is involved in multimer formation and heparin- and retinal axon-binding. Cysteine-rich domains bind to and inhibit retinal axons. Conclusion: Different molecular interactions and functions are mediated by distinct domains of Nel. Significance: The findings provide insights into how Nel exerts diverse functions. Nel (neural epidermal growth factor (EGF)-like molecule) is a multimeric, multimodular extracellular glycoprotein with heparin-binding activity and structural similarities to thrombospondin-1. Nel is predominantly expressed in the nervous system and has been implicated in neuronal proliferation and differentiation, retinal axon guidance, synaptic functions, and spatial learning. The Nel protein contains an N-terminal thrombospondin-1 (TSP-N) domain, five cysteine-rich domains, and six EGF-like domains. However, little is known about the functions of specific domains of the Nel protein. In this study, we have performed structure-function analysis of Nel, by using a series of expression constructs for different regions of the Nel protein. Our studies demonstrate that the TSP-N domain is responsible for homo-multimer formation of Nel and its heparin-binding activity. In vivo, Nel and related Nell1 are expressed in several regions of the mouse central nervous system with partly overlapping patterns. When they are expressed in the same cells in vitro, Nel and Nell1 can form hetero-multimers through the TSP-N domain, but they do not hetero-oligomerize with thrombospondin-1. Whereas both the TSP-N domain and cysteine-rich domains can bind to retinal axons in vivo, only the latter causes growth cone collapse in cultured retinal axons, suggesting that cysteine-rich domains interact with and activate an inhibitory axon guidance receptor. These results suggest that Nel interacts with a range of molecules through its different domains and exerts distinct functions.

A transgenic mouse line with α-1,3/4-fucosyltransferase cDNA: production and characteristics

AbstractcDNA of human α-1,3/4-fucosyltransferase (Fuc-TIII) was placed under the control of the chicken β-actin promoter and cytomegalovirus enhancer, then introduced into male pronuclei of fertilized mouse eggs. A transgenic mouse line thus obtained exhibited enhanced expression of Lex (4C9) antigen in endothelial cells located in the glomerulus, sinusoidal capillaries of the liver and capillaries of the heart. Furthermore, in the transgenic mice, sialyl dimeric Lex (FH6) and sialyl Lea (2D3), antigens were strongly expressed in the glomerular endothelial cells.