NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP ؉ -dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP ؉ -dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor ␥ were markedly elevated. The hepatic and epididymal fat pad contents of acetylCoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.Abnormal lipid metabolism is frequently associated with obesity and hyperlipidemia. In fat and cholesterol biosynthesis, NADPH is an essential cofactor for numerous enzymes. For instance, 3-L-hydroxylacyl-coenzyme A dehydrogenase and enoyl-coenzyme A reductase in fatty acid synthesis and hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, require NADPH for their enzyme activities. It has been demonstrated that glucose-6-phosphate dehydrogenase (G6PDH), 1 6-phosphogluconate dehydrogenase, and malic enzyme are considered as the major enzymes producing cytosolic NADPH (1). Nevertheless, the activities of these enzymes were markedly lower than that of cytosolic NADP ϩ -dependent isocitrate dehydrogenase (IDPc) in the rat liver (1, 2). Consistent with this observation, McLean and co-workers (3) reported that certain adaptive changes in the pentose phosphate pathway dehydrogenases did not take place in parallel with fat synthesis in adipose tissue and suggested that a major source of NADPH for fat synthesis could be IDPc. It is worthy of note that IDPc is expressed mainly in lipogenic tissues such as liver and adipocytes, whereas G6PDH and 6-phosphogluconate dehydrogenase are expressed ubiquitously (4, 5). These data indicate that NADPH-producing IDPc may ...

Key Words: atherosclerosis Ⅲ immunity Ⅲ tumor necrosis factor receptor superfamily 14 Ⅲ matrix metalloproteinases Ⅲ foam cells T umor necrosis factor (TNF)-␣ and CD40L play pivotal roles in the atherogenesis. TNF-␣ was found to be expressed in atherosclerotic plaques, 1,2 and TNF-␣ was also found to be colocalized with foam cells, smooth muscle cells (SMCs), 3,4 and mast cells. 5 CD40, a member of the TNF receptor superfamily (TNFRSF), is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. CD40 binds to a ligand (CD40L) which is a member of the TNF superfamily (TNFSF). 6 In atherosclerotic plaques, the expression of CD40L in T cells and the coexpression of CD40 and CD40L in vascular endothelial cells, SMCs, and macrophages were detected. 7 The interaction between CD40 and CD40L, similar to the interaction between TNF-␣ and its receptor, elicits diverse biological responses involved in atherosclerosis, such as the secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs), and the expression of adhesion molecules and tissue factor. 8,9 These responses are known to make the plaque unstable. See page 1873Recently, the list of molecules belonging to TNFRSF has expanded significantly. TNFRSF14 (HVEM/HveA/ LIGHTR/TR2/ATAR) was initially identified as a cellular coreceptor for herpes simplex virus entry, hence, the name HVEM (herpes virus entry mediator, later named HveA [herpes virus entry protein A]). 10 TNFRSF14 has a wide tissue distribution and is prominently expressed by cells in lymphoid tissue, such as the spleen, and on peripheral blood leukocytes. TNFRSF14 mRNA was detected on resting and activated CD4ϩ and CD8ϩ T cells, on CD19ϩ B cells, and on monocytes. 14 We hypothesized that TNFRSF14, like the CD40/CD40L system, has a role in atherosclerosis. We analyzed the expression of TNFRSF14 in atherosclerotic plaques and the expression of proatherogenic cytokines and MMPs after stimulation of TNFRSF14 in THP-1 cells. Methods Histological AnalysisFor immunohistochemical analysis, carotid endarterectomy specimens were obtained from 13 patients, aged 63 to 81 years, who underwent the surgery at Samsung Seoul Hospital. The study was approved by an institutional review committee, and the subjects gave informed consent. Atherosclerotic plaque specimens were washed with saline and embedded in OCT (Miles Laboratories) to make frozen sections. Standard 5-m sections were stained by use of the LSAB kit (DAKO) according to the manual provided by the manufacturer. Double staining of CD68 and TNFRSF14 was performed by using an Animal Research Kit (DAKO) according to the manual provided by the manufacturer. Cell CultureHuman monocytic leukemia THP-1 cells 15 were obtained from the American Type Culture Collection. For the analysis of peripheral blood monocytes, whole blood was collected either in heparin Vacutainer or CTAD Diatubes (Becton Dickinson/Diagnostica Stago) containing dipyridamole and theophylline to pr...

The kinesin family of motor proteins, which contain a conserved motor domain of approximately 350 amino acids, generate movement against microtubules. Over 90 members of this family have been identified, including motors that move toward the minus or plus end of microtubules. The Kar3 protein from Saccharomyces cerevisiae is a minus end-directed kinesin family member that is involved in both nuclear fusion, or karyogamy, and mitosis. The Kar3 protein is 729 residues in length with the motor domain located in the C-terminal 347 residues. Recently, the three-dimensional structures of two kinesin family members have been reported. These structures include the motor domains of the plus end-directed kinesin heavy chain [Kull, F. J., et al. (1996) Nature 380, 550-555] and the minus end-directed Ncd [Sablin, E. P., et al. (1996) Nature 380, 555-559]. We now report the structure of the Kar3 protein complexed with Mg.ADP obtained from crystallographic data to 2.3 A. The structure is similar to those of the earlier kinesin family members, but shows differences as well, most notably in the length of helix alpha 4, a helix which is believed to be involved in conformational changes during the hydrolysis cycle.