A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Doxorubicin directly binds to the cardiac-type ryanodine receptor

The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.     

Comparative studies of genetic diversity in kenaf (Hibiscus cannabinus L.) varieties based on analysis of agronomic and RAPD data

Kenaf (Hibiscus cannabinus L.) is a fiber crop classified in the genus Hibiscus (Malvaceae), and has a great potential for its multipurpose utilization, in addition to its traditional usage. Varietal identification of kenaf is always problematic and knowledge on genetic diversity of kenaf varieties is also limited, which significantly hindered our effective utilization and conservation of the valuable kenaf germplasm. In order to find a proper method for identifying kenaf varieties and studying their variation, morpho-agronomic characters and random amplified polymorphic DNA (RAPD) markers were analyzed among 14 kenaf varieties commonly used in Japan. Data from morphological analysis showed that the included kenaf varieties could be divided into three major groups. The characters, such as middle stem diameter, whole stalk weight, and days to 50% flowering, are highly responsible for the variation of the kenaf varieties, but it is difficult to identify individual varieties merely by the morpho-agronomic characters. On the other hand, clearly separation of the kenaf varieties was achieved based on the RAPD variation patterns. Genetic relationship of the kenaf varieties can also be traced through the analysis of RAPD and morph-agronomic variation. It is concluded from the present study that RAPD analysis is an effective tool in identifying of kenaf varieties and determining their genetic relationships, particularly when combined with the analysis of morpho-agronomic characters.     

Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. I. Effect of ATP

The effect of ATP on calcium binding of the Ca2+-ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding variously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 mm) and millimolar (5 mm) ATP, respectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem. 269, 30818-30821). For example, in the sarcoplasmic reticulum membrane at pH 7.40, submillimolar ATP converted the calcium binding manner of the A form from noncooperative (Hill number (n(H)) of approximately 1) to cooperative (n(H) approximately 2), concurrent with a decrease in the apparent calcium affinity (K(0.5)) from 2-6 to 0.1-0.3 microm. The binding of the A form became almost the same as that of the B form (n(H) approximately 2, K(0.5) approximately 0.2 microm), which was not affected by ATP. Millimolar ATP further decreased the K(0.5) of the cooperative binding of the two forms to approximately 0.05 microm. Regulation of the calcium binding performance by ATP is discussed in terms of monomeric and oligomeric pathway models.     

Impaired proliferative response of V alpha 24 NKT cells from cancer patients against alpha-galactosylceramide

Human invariant V alpha 24(+) NKT cells are a relatively new subpopulation of lymphocytes. It has been reported that V alpha 24 NKT cells are significantly involved in some human diseases. We have evaluated the number and function of V alpha 24 NKT cells in both healthy volunteers and cancer patients. In this study we found that V alpha 24 NKT cells in unfractionated PBMCs obtained from cancer patients did not respond efficiently to alpha-galactosylceramide (alpha-GalCer) in vitro. Thus, their proportion after stimulation with alpha-GalCer was smaller than that found in healthy volunteers. However, the cancer patients' V alpha 24 NKT cells retained cytotoxic activity against malignant target cells, and they could efficiently proliferate to alpha-GalCer when fractionated by sorting. Furthermore, we found that addition of G-CSF to the culture could restore the low proliferative response of V alpha 24 NKT cells from cancer patients. These results suggest that some functions of NKT cells in cancer patients are impaired, and this observation carries significant implications for immunotherapy-based cancer treatments.     

Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Recovery of photosynthetic systems during rewetting is quite rapid in a terrestrial cyanobacterium, Nostoc commune

Recovery processes of photosynthetic systems during rewetting were studied in detail in a terrestrial, highly drought-tolerant cyanobacterium, Nostoc commune. With absorption of water, the weight of N. commune colony increased in three phases with half-increase times of about 1 min, 2 h and 9 h. Fluorescence intensities of phycobiliproteins and photosystem (PS) I complexes were recovered almost completely within 1 min, suggesting that their functional forms were restored very quickly. Energy transfer from allophycocyanin to the core-membrane linker peptide (L(CM)) was recovered within 1 min, but not that from L(CM) to PSII. PSI activity and cyclic electron flow around PSI recovered within 2 min, while the PSII activity recovered in two phases after a time lag of about 5 min, with half times of about 20 min and 2 h. Photosynthetic CO(2) fixation was restored almost in parallel with the first recovery phase of the PSII reaction center activity. Although the amount of absorbed water became more than 20 times the initial dry weight of the N. commune colony in the presence of sufficient water, about twice the initial dry weight was enough for recovery and maintenance of the PSII activity.