Short-term culture and gammaH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.     

Expression profile of plasmid DNA obtained using spermine derivatives of pullulan with different molecular weights

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.     

Novel effect of estrogen on RANK and c-fms expression in RAW 264.7 cells

Temporomandibular disorder (TMD), a progressive disease entity, and osteoarthrosis preferentially affect females, denoting a possible role of estrogen. Using RAW 264.7 cells, the expression of estrogen receptors (ERs) alpha and beta and the consequent effect of estrogen was investigated. We present the novel detection of ER beta expression in RAW 264.7 cells. Furthermore, we innovatively demonstrated the increase in expression of both ER alpha and beta, as well as RANK and c-fms, with estrogen treatment. However, a decrease in expression of c-fms, RANK and ER beta, and nearly no change in the expression of ER alpha were experienced upon further increase in estrogen concentrations. These findings lead us to hypothesize a new mechanism of inflammation in TMD.     

Proteomic analysis of mechanisms of hypoxia-induced apoptosis in trophoblastic cells

Preeclampsia is often accompanied by hypoxia of the placenta and this condition induces apoptosis in trophoblastic cells. The aim of this study was to characterize global changes of apoptosis-related proteins induced by hypoxia in trophoblastic cells so as to clarify the mechanism of hypoxia-induced apoptosis by using the PoweBlot, an antibody-based Western array. Human choriocarcinoma cell line JAR was cultured for 24 hours under aerobic and hypoxic conditions. Hypoxia induced apoptosis accompanied by increased expression of Bcl-x, Caspase-3 and -9, Hsp70, PTEN, and Bag-1. Bad, pan-JNK/SAPK-1, Bcl-2, Bid, and Caspase-8 showed decreased expression. Hypoxia-induced apoptosis was increased with the transfection of a bag-1 antisense oligonucleotide. The bag-1 antisense oligonucleotide affected the expression of Bid, Bad, Bcl-2, JNK, and phosphorylated JNK, although expression of PTEN and Bcl-X did not change. Bag-1 may inhibit apoptosis by suppressing the expression of Bid and Bad. It may also enhance apoptosis by inhibiting the expression of Bcl-2 and by modulating phosphorylation of JNK. Both mitochondrial and stress-activated apoptosis pathways played important roles in the hypoxia induced cell death of trophoblastic cells. These findings will contribute to establish new approach to detect hypoxic stress of the placenta, which leads to preeclampsia and other hypoxia-related obstetrics complications.     

Larvivorous activity of Poecilia reticulata against Culex quinquefasciatus larvae in a polluted water drain in Hardwar, India

The efficacy of the larvivorous fish Poecilia reticulata against mosquito larvae was monitored in a drain at Bharat Heavy Electricals Limited, Hardwar, India. The water was polluted and the water flow was in some way impeded. Poecilia reticulata failed to feed on Culex quinquefasciatus larvae in this drain. Laboratory experiments also confirmed the inefficacy of P. reticulata as a predator of Cx. quinquefasciatus larvae during the first 24 h. Significant differences in the efficacy of P. reticulata against Cx. quinquefasciatus were recorded between polluted water and drinking water. Poecilia reticulata preferred to feed on other available food present in the polluted water rather than on Cx. quinquefasciatus larvae. This was verified by the identification of plankton in the gut content of the fish and by the high density of plankton present in the polluted water.     

Di(2-ethylhexyl)phthalate induces hepatic tumorigenesis through a peroxisome proliferator-activated receptor alpha-independent pathway

Di(2-ethylhexyl)phthalate (DEHP), a commonly used industrial plasticizer, causes liver tumorigenesis presumably via activation of peroxisome proliferator-activated receptor alpha (PPARalpha). The mechanism of DEHP tumorigenesis has not been fully elucidated, and to clarify whether DEHP tumorigenesis is induced via PPARalpha, we compared DEHP-induced tumorigenesis in wild-type and Pparalpha-null mice. Mice of each genotype were divided into three groups, and treated for 22 months with diets containing 0, 0.01 or 0.05% DEHP. Surprisingly, the incidence of liver tumors was higher in Pparalpha-null mice exposed to 0.05% DEHP (25.8%) than in similarly exposed wild-type mice (10.0%). These results suggest the existence of pathways for DEHP-induced hepatic tumorigenesis that are independent of PPARalpha. The levels of 8-OHdG increased dose-dependently in mice of both genotypes, but the degree of increase was higher in Pparalpha-null than in wild-type mice. NFkappaB levels also significantly increased in a dose-dependent manner in Pparalpha-null mice. The protooncogene c-jun-mRNA was induced, and c-fos-mRNA tended to be induced only in Pparalpha-null mice fed a 0.05% DEHP-containing diet. These results suggest that increases in oxidative stress induced by DEHP exposure may lead to the induction of inflammation and/or the expression of protooncogenes, resulting in a high incidence of tumorigenesis in Pparalpha-null mice.     

Feasibility and Efficacy of Inspiratory Muscle Training in Patients with Head and Neck Cancer receiving Concurrent Chemoradiotherapy

Objectives: Patients with head and neck cancer (HNC) undergoing concurrent chemoradiotherapy (CCRT) often experience pulmonary symptoms. This study evaluated if a 7-week inspiratory muscle training (IMT) program during CCRT is feasible, adherent, and safe in patients with HNC. This study also evaluated the effect of IMT on diaphragm thickness, mobility, and cardiorespiratory parameters in patients with HNC receiving CCRT. Methods: Ten participants with advanced stage HNC receiving CCRT were recruited for the study. Feasibility, adherence, and safety of the intervention were the primary outcomes. Changes in diaphragm thickness and mobility, maximal inspiratory pressure, maximal expiratory pressure, forced vital capacity, forced expiratory volume in first second and functional capacity using 6-MWT were measured at baseline and post 7 weeks of CCRT. IMT was performed at one session per day for 5 days a week for 7 weeks. Eight sets of two minutes of inspiratory manoeuvres with one minute rest period between them with intensity of 40% MIP were given. Results: Ten participants  were included in this study out of the 13 patients screened, indicating the feasibility to be 76.9%. Participants completed a total of 260 training sessions out of the 350 planned sessions denoting the adherence level as 74%. Diaphragm thickness and MEP remained significantly unchanged while significant decline was seen in diaphragm mobility, MIP,FVC, FEV1 and 6-MWD at the end of 7 weeks. No adverse events were reported following the intervention. Conclusion: Inspiratory muscle training did not show significant effect on the diaphragm thickness, mobility, and cardiorespiratory parameters; however, it was feasible, adherent, and safe in patients with HNC receiving CCRT.     

Fertilizer Industry Effluent Induced Biochemical Changes in Fresh water Teleost, <Emphasis Type="Italic">Channa striatus</Emphasis> (<Emphasis Type="Italic">Bloch</Emphasis>)

The industrial activities pose threat to the life of aquatic organisms in many ways. This research communication presents an account of the impact of fertilizer industry effluent upon the levels of protein and the activity of lactate dehydrogenase (EC 1.1.1.28, LDH), a terminal key enzyme in glycolytic pathway, in different organs of a fresh water teleost fish, Channa striatus (Bloch). The fish exposed to different sublethal concentrations of fertilizer industry effluent (3.5, 4.7 and 7.0% v/v) equivalent to 1/20th, 1/15th and 1/10th of LC₅₀ value (70% v/v) for varying treatment periods (96 h and 15 days) exhibited decrease in the level of protein (8–76%) in different organs of the effluent treated fish. At highest effluent concentration (7% v/v) treatment for short (96 h) or long (15 days) duration, the liver of the fish registered significant (p < 0.001) decrease (62–76%) in protein content as compared to control, whereas other organs of the fish showed only 38–52% decrease in the level of protein. The industrial effluent also caused marked reduction in the activity of LDH in different fish tissues when compared to the control. The treatment of fish with 7% effluent concentration for 96 h caused 78% decrease (p < 0.001) in the LDH activity in fish muscle whereas after 15 days the effect was maximum in fish brain as it exhibited 86% decrease (p < 0.001) in LDH activity as compared to control. The effect of effluent on the activity of LDH and protein content in different body tissues of the fish was dependent on concentration and duration of exposure. The significant reductions in the activity of LDH and level of protein in fish tissues due to treatment with the fertilizer industry effluent indicated the possibility of impairments in energy metabolism and protein turnover, respectively, in C. striatus.     

Quality assurance of annual HIV sentinel surveillance 2006: experience of regional institute, central zone

National AIDS Control Organisation (NACO) identified five regional institutes (RIs) to monitor and supervise the 2006 round of annual HIV sentinel surveillance. The task mandated was quality control of both epidemiological data collection and HIV testing. The team at RI consisted of epidemiologist and microbiologist. We describe here the process of quality control and the quality of surveillance in the states of Uttar Pradesh, Uttarakhand, Bihar, Jharkand, and Delhi. The supervisors visited almost 90% of the sentinel sites. Performance of vast majority of the sentinel sites (92%) was satisfactory. The testing laboratories were found to be adhering to standard operating procedures. Concordance rate of test results between testing laboratory and the designated reference laboratory was high. Overall, the quality of sentinel surveillance was good. The lacunae found during the visit have been enumerated along with the recommendations for future surveillance round.     

The effect of ultrapure dialysate on improving renal anemia

BACKGROUND: Renal anemia is a very serious problem in hemodialysis patients. In this regard, the investigation was focused on whether ultrapure dialysate could improve renal anemia and the mechanism of renal anemia. METHODS: Ultrapure dialysate was used over a 2 years period for 61 patients on maintenance hemodialysis. During this period, the changes in renal anemia and red blood cell life span were investigated. The changes in the latter were evaluated using the creatine concentration in red blood cell. RESULTS: The hemoglobin concentration, RBC count, and hematocrit concentration before the use of the ultrapure dialysate were 9.1 +/- 0.2 g/dL, 309.9 +/- L7.2 x 10(4)/microL, and 28.8 +/- 0.6%, respectively. These values significantly increased to 10.2 +/- 0.1 g/dL, 349.7 +/- 5.6 x 10(4)/microL, and 32.6 +/- 0.3%, respectively, after 2 years of ultrapure dialysate use. The increase in reticulocyte count indicated enhanced erythropoiesis by ultrapure dialysate. The red blood cell life span evaluated by creatine concentration in red blood cell was also prolonged after the use of ultrapure dialysate. CONCLUSIONS: Ultrapure dialysate is considered to improve the renal anemia of dialysis patients by promoting erythropoiesis and prolonging red blood cell life span.