Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H 2 O 2 production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice.Cell death is important in the development and defense of multicellular organisms (1). Cell death occurs in normal plant development and during infection by avirulent pathogens (2-5). It plays a key role in plant defense and shares several features with apoptosis in mammalian cells (3,5,6). Rapid production of reactive oxygen species (ROS) often is associated with cell death during resistance reactions to pathogens, and a plasma membrane NADPH oxidase similar to the neutrophil enzyme is suggested to be responsible for ROS production (5-7). However, the molecular mechanisms for ROS production and cell death in plants are largely unknown.In phagocytic cells, activation of NADPH oxidase leads to production of superoxide, which effectively kills invading microorganisms (8, 9). The neutrophil NADPH oxidase is a multicomplex enzyme consisting of two membrane proteins, gp91 phox and p22 phox , and three cytosolic factors, p47 phox , p67 phox , and Rac (8, 9), and the plant enzyme is thought to be similar to the neutrophil enzyme (10-13). Genes whose deduced amino acid sequences are similar to those of the Rho͞Rac family of the small GTP-binding proteins have been isolated in plants (14-18), and some were shown to play a role in the control of pollen tube growth (19). However, functions of most of these genes are not known. Genes whose amino acid sequences are similar to that of the animal gp91 phox also have been isolated from plants (20, 21), but their functions have not been investigated. In this study we analyzed functions of Rac in rice by expressing constitutively active and dominant negative Rac genes in transgenic cell cultures and plants and found that Rac plays an important role in ROS production as well as cell death in rice. MATERIALS AND METHODSBiochemical Analysis of Recombinant OsRac1. OsRac1 cDNA was cloned in an expression vector, pGEX-4T-1 (Pharmacia), and transformed into Escherichia coli. The glutathione S-transferase (GST)-fusion protein was purified by glutathioneSepharose beads (Pharmacia) and used for assays of GTPbinding and GTPase activities according to published protocol (22). For the assay of the GTP-binding activity, the binding of Rice Transformation. OsRac1-G19V was made by substitution of the glycine corresponding to G12 of human Rac1 by valine by the use of an LA PCR in vitro Mutagenesis kit (Takara Shuzo, Kyoto). OsRac1-T24N similarly ...

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Expression of aquaporin-8 mRNA has previously been shown in hepatocytes, pancreatic acinar cells, colon epithelial cells and seminiferous tubules of the testis in the rat by in situ hybridization technique. However, immunolocalization of this water channel has not yet been demonstrated. In the present study, the localization of immunoreactive aquaporin-8 and expression of the mRNA were examined in rat organs (cerebrum, cerebellum, eye, salivary gland, heart, lung, liver, pancreas, esophagus, stomach, jejunum, ileum, colon, testis, ovary, kidney, spleen and lymphnode) by immunohistochemistry using an antibody against aquaporin-8 and ribonuclease protection assay. Aquaporin-8 was distinctly immunolocalized on the apical membranes of pancreatic acinar cells and mucosal epithelium of the colon and jejunum. In the liver, the bile canalicular membrane of hepatocytes was immunostained. In the testis, immunoreactive aquaporin-8 was demonstrated on the luminal side of the seminiferous tubules. At high magnification, the peroxidase reaction products appeared on the ramified cytoplasmic membrane of Sertoli cells surrounding the residual bodies or spermatogenic cells. Specificity of the antibody was verified by Western blot analysis showing a minor approximately 28 kDa band (deduced deglycosylated form of aquaporin-8) and a major approximately 30 kDa band (glycosylated form) in these organs. The intensity of aquaporin-8 immunoreactivity was approximately comparable to that of aquaporin-8 mRNA expression in the liver, pancreas, colon, jejunum and testis. The aquaporin-8 mRNA expression in the hepatocytes was presumed to be closely associated with the structure of bile canaliculi since the message was detected in hepatocytes immediately after isolation from the liver but not in cells following cultivation for three days. The localization of immunoreactive aquaporin-8 indicated functions for this water channel in the secretion of bile and pancreatic juice, and the secretion or absorption of water in the colon and jejunum, and the maturation or liberation of spermatogenic cells in the testis.

For the developement of bio-PIXE, two problems remain to be solved; one in accelerator availability and the other in data analysis. To improve the former, a method of applying baby cyclotrons to PIXE has been developed. To solve the latter, the computer program SAPIX for x-ray spectrum analysis ,which runs on ordinary personal computers and is fairly easy to operate has been developed. Examples of spectral fitting by the SAPIX and a description of the program are given. Further, the two-detector measuring system which we have employed for the simultaneous determination of all elements is reported.