To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

Ent-11alpha-hydroxy-16-kauren-15-one (1) induced apoptosis in a human leukemia cell line (HL-60 cells), however, the apoptosis-inducing properties of 1 and its related compounds remain to be proved. We examined the involvement of caspases, a family of cysteine aspartic proteases, which play a central role in induction of apoptosis, in apoptosis induced by the compounds in HL-60 cells. Treatment of the cells with compounds 1, 2 and 3 with the enone group at C-15/C-16 caused DNA fragmentation, a sign of induction of apoptosis, and proteolysis of poly(ADP-ribose) polymerase (PARP), a hallmark of caspase activation. Z-Asp-CH2-DCB, abroad spectrum inhibitor of caspases, abolished the appearance of DNA fragmentation and also significantly attenuated the cytotoxic effects. These data suggest that induction of apoptosis by 1 and some of its related compounds are dependent on caspases activation and might be partly involved in the cytotoxicity in HL-60 cells.

Defects in apoptosis signaling pathways contribute to tumorigenesis and drug resistance, and these defects are often a cause of failure of chemotherapy. Thus, a major goal in chemotherapy is to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We previously found that an Ent-kaurene diterpene,

The kinetics of the spontaneous and triethylamine-catalyzed reactions of 1-alkenyl isocyanates with methanol were studied and the results were compared with those of ethyl isocyanate and phenyl isocyanate. The data were treated by the equa- x ) , and k,, kl and kS of ethyl, phenyl, vinyl, propenyl, isopropenyl, P-propylvinyl, a-n-hexylvinyl isocyanate and k3 of a-n-butylvinyl, 6-phenylvinyl isocyanate were determined.The kinetics of the general reaction of isocyanates with alcohols were also discussed.The kland &-values of 1-alkenyl isocyanates were 10-300 times of those of ethyl isocyanate.

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