Characterization of water distribution and activities of enzymes during germination in magnetically-exposed maize (Zea mays L) seeds.

Magnetic seed treatment is one of the physical pre-sowing seed treatments to enhance the performance of crop plants. In our earlier experiment, we found significant increase in germination and vigour characteristics of maize (Zea mays L.) seeds subjected to magnetic fields. Among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 100 mT for 2 h and 200 mT for 1 h exposure. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions. Thus, in the present study, changes in water uptake during imbibition and its distribution and activities of germinating enzymes during germination were investigated in maize seeds exposed to static magnetic fields of 100 and 200 mT for 2 and 1 h respectively by nuclear magnetic resonance (NMR) spectroscopy. The magnetically-exposed seed showed higher water uptake in phase II and III than unexposed seed. The longitudinal relaxation time T1 of seed water showed significantly higher values and hence greater molecular mobility of cellular water in magnetically-exposed seeds as compared to unexposed. Component analysis of T2 relaxation times revealed the early appearance of hydration water with least mobility and higher values of relaxation times of cytoplasmic bulk water and hydration water in magnetically-exposed over unexposed seeds. Activities of -amylase, dehydorgenase and protease during germination were higher in magnetically-exposed seeds as compared to unexposed. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions.

Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.

Ionizing radiation induces blockade of c-Jun N-terminal kinasedependent cell death pathway in amanner correlated with p21Cip/WAF1 induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H2O2. This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H2O2 failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H2O2 treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H2O2, but elevated cellular levels of p21Cip/WAF1, a p53 target that can suppress H2O2-induced cell death by blocking JNK activation. Consistently, H2O2-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.

Involvement of protein kinase C pathway in UVC-stimulated phospholipase D2 activity in Vero 76 cells

Phospholipase D (PLD) activity is known to be related to oxidant-induced cellular signaling and membrane disturbance. Previously, an induction of PLD activity in various cell lines by X-ray irradiation was observed. In this study, we examined the effect of UVC radiation on the PLD activity in Vero 76 cells. At a dose of 10 kJ/m2 of UVC irradiation, the PLD activity was stimulated approximately 10-fold over the basal activity. This UVC-induced PLD activity was found to be dependent on the presence of extracellular calcium and was inhibited by catalase as well as amifostine-an intracellular thiol antioxidant. Pretreatments with Ro32-0432-a selective inhibitor of protein kinase C (PKC)-and downregulation of PKC by preincubation of phorbol 12-myristate 13-acetate significantly inhibited the UVC-induced PLD activity. UVC-stimulated PLD activity was observed only in murine PLD2 (mPLD2)-transfected Vero 76 cells and not in human PLD1 (hPLD1)-transfected cells. Transient incorporation of PKC with mPLD2 and the phosphorylation of mPLD2 by a and b forms of PKC by UVC irradiation were observed. These results suggest that the UVC-stimulated PLD activity in Vero 76 cells is mediated through transient phosphorylation of PLD2 by the translocation of PKC to PLD2.

Change of telomerase activity in rectal cancer with chemoradiation therapy

Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the exception of proliferative stem cells, germ cells and activated lymphocytes. Measuring telomerase activity clinically may provide useful diagnostic and prognostic information of cancer. The purpose of this study was to investigate the change in telomerase activity following chemoradiation in rectal cancer, which almost always produces positive enzymatic activity. A total of 24 tumor tissue samples were used in this study, consisting of 12 paired specimens before and 4 week after chemoradiation. Telomerase activity was determined by PCR-based telomeric repeat amplification protocol (TRAP) assay. The telomerase activity was positive in 10 out of 12 patients (83%) in pre-irradiated and post-irradiated states. The levels of telomerase activity was decreased in 8 out of 10 patients after chemoradiation (80%) and two cases showed no change in enzymatic activity. One case showed no activity in either sample. The other case showed no enzymatic activity in the pre-irradiated sample, but showed weak activity in the post-irradiated sample. These data indicate that telomerase activity in rectal cancer is reduced after neoadjuvant chemoradiation therapy, possibly suggesting a mechanism of downstaging following chemoradiation therapy in cancer.