Detection of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photoinhibition or UV radiation

In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement in vivo is of special importance. Here we report experiments with two dansyl-based ROS sensors, the singlet oxygen specific DanePy and HO-1889NH, which is reactive to both singlet oxygen and superoxide radicals. Here we report in vivo detection of (1)O(2) and O(2)(-*) by fluorescence quenching of two dansyl-based ROS sensors, the (1)O(2) specific DanePy and HO-1889NH, which was reactive with both (1)O(2) and O(2)(-*). The ROS sensors were administered to spinach leaves through a pinhole, and then the leaves were exposed to either excess photosynthetically active radiation or UV (280-360 nm) radiation. Microlocalization of the sensors' fluorescence and its ROS-induced quenching was followed with confocal laser scanning microscopy and with fluorescence imaging. These sensors were specifically localized in chloroplasts. Quenching analysis indicated that the leaves exposed to strong light produced (1)O(2), but hardly any O(2)(-*). On the other hand, the dominant ROS in UV-irradiated leaves was O(2)(-*), while (1)O(2) was minor.     

Effects of 20 days horizontal bed rest on kinesthesia during knee flexion and two-point discrimination in skin of young subjects

Investigating the effect of 20 days bed rest (BR) on kinesthesia and two-point discrimination, 10 young volunteers participated in this study as subjects. Angle position sensation of right knee was measured in the prone position during flexion and extension monitored by a goniometer after two-point discrimination in skin was determined on the same lower leg. Flexed constant error was unchanged but directional constant error and absolute error were increased after BR (p<0.05). Two-point discrimination was unchanged after BR, which brought about a decline of the orientation of moving the joint indicated as over shooting of the angle during knee flexion, while it did not affect superficial sensation observed by two-point discrimination. Probably, an adjustment of deep sensation to the knee joint is lowered by the reduction in kinesthesia as well as the sensory disturbance during BR, which is independent on information from superficial sensation.     

Two novel steroidal alkaloid glycosides from the seeds of Lycium barbarum

Two novel steroidal alkaloid glycosides, lycioside A (1) and lycioside B (2) were isolated from the seeds of Lycium barbarum. Their structures were determined by various spectroscopic analyses. Compounds 1 and 2 showed inhibitory activities with the IC(50) values of 75.3 and 72.8 μM against rat intestinal sucrase, and 63.4 and 59.1 μM against rat intestinal maltase.     

In vitro maturation and ultrastructural observation of cryopreserved minke whale (Balaenoptera acutorostrata) follicular oocytes

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.     

Landscape structure affects food quality of sika deer (<Emphasis Type="Italic">Cervus nippon</Emphasis>) evidenced by fecal nitrogen levels

Evaluating the quality of wildlife habitat is essential for understanding and predicting population dynamics in heterogeneous environments. We used fecal nitrogen levels as an indicator of habitat quality of sika deer (Cervus nippon) and explored important landscape elements influencing nitrogen levels, taking deer density into account. We established 92 plots differing in deer density and landscape structure on the Boso Peninsula, central Japan, and collected fecal samples along a 1-km transect at each plot. The regression models involving two independent variables, i.e., deer density and the length of forest edge within an area of 100 or 200 m from the transect, were selected based on the Akaike’s Information Criterion (AIC). Levels of fecal nitrogen were positively correlated with the length of the forest edge and negatively correlated with population density of deer. The area of 100 or 200 m from the transect most likely reflected the behavioral scale of the deer. Coverage of palatable understory vegetation increased with proximity to forest edge and decreased with deer density. Variability in the level of fecal nitrogen could thus be explained by food availability in the landscape. These results suggest that landscape alterations increase the carrying capacity of sika deer and thereby increase impacts upon the ecosystem.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Urinary proteomics revealed prostaglandin H(2)D-isomerase, not Zn-α2-glycoprotein, as a biomarker for active lupus nephritis

Although renal histopathology is the gold standard for the diagnosis and prognosis of lupus nephritis (LN), the invasiveness of renal biopsy warrants the discovery of novel non-invasive diagnostic and prognostic biomarkers. In the present study, urine samples from 10 LN patients (5 active and 5 inactive) were analyzed by two-dimensional gel electrophoresis (2-DE) to screen for potential biomarkers of active LN. Quantitative analysis and statistics revealed 16 protein spots whose levels significantly differed between groups. These proteins were successfully identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Among these potential candidates, differential levels of urinary Zn-α2-glycoprotein (ZA2G) and prostaglandin H(2)D-isomerase (PGDS) were further validated by enzyme-linked immunosorbent assay (ELISA) in another independent group of 78 subjects, including 30 active LN, 26 inactive LN, 14 non-LN glomerular diseases, and 8 healthy normal individuals. Whereas ZA2G levels were elevated in urine of patients with active LN and non-LN glomerular diseases, PGDS was elevated only in the urine of the active LN group. Urinary PGDS, not ZA2G, may serve as a biomarker for active LN and upon validation in larger studies, may become the non-invasive test to evaluate the disease activity in future management of LN.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.     

Interleukin-12 genetic administration suppressed metastatic liver tumor unsusceptible to CTL

A cytokine gene therapy approach was conducted against metastatic lesions of cytotoxic T lymphocyte (CTL)-unsusceptible tumor in mice. The EBV-based and conventional plasmid vectors that encode murine interleukin-12 (IL-12) gene (pGEG.mIL-12 and pG.mIL-12, respectively) were intravenously transfected into the mice that had received a subcutaneous inoculation of M5076 sarcoma cells. The pGEG.mIL-12 transfection drastically suppressed the subcutaneous as well as hepatic metastatic tumors, resulting in significant prolongation of survival period of the animals. Although single administration with pG.mIL-12 was not effective, repetitive transfection with the plasmid significantly prolonged the longevity of the mice-bearing the metastatic liver tumors. Multiple transfection with either pGEG.mIL-12 or pG.mIL-12 also suppressed peritoneal carcinomatosis in mice that had been injected with M5076 cells into the peritoneal cavity. It was suggested that a high level IL-12 production elicited by the intravenous delivery of the cytokine gene may be quite effective in inhibiting metastatic and CTL-unsusceptible neoplasms.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.