Inactivation of Cyanobacterial Nitrogenase After Exposure to Ultraviolet-B Radiation

Exposure of the N₂-fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m⁻²) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure. Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation. Received: 13 June 2002 / Accepted: 22 July 2002     

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.     

Tautomeric rearrangement of a dihydroflavin bound to monomeric sarcosine oxidase or N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous bacterial flavoenzymes that contain covalently bound flavin [8alpha-(S-cysteinyl)FAD]. Reaction of MSOX or MTOX with a small excess of sodium borohydride results in immediate flavin reduction to a species that exhibits spectral properties (lambda(max) = 405 nm with a second broad peak at 332 nm) similar to those of 3,4-dihydroflavin. The borohydride-reduced enzymes retain full catalytic activity. Substrate reduction converts the 405 nm species to an air-sensitive tetrahydroflavin that reacts with oxygen to yield unmodified oxidized enzyme. Unexpectedly, the putative 3,4-dihydroflavin bound to MSOX or MTOX is unstable in the absence of substrate. An isosbestic conversion of the 405 nm species to yield unmodified, oxidized flavin is observed when the reaction is conducted under aerobic conditions (k(obs) = 4.9 x 10(-2) min(-1)). Under anaerobic conditions, an oxygen-sensitive species resembling 1,5-dihydroflavin is formed in an isosbestic reaction that occurs at a rate similar to that of the aerobic reaction (k(obs) = 5.3 x 10(-2) min(-1)). Possible reaction of the 3,4-dihydroflavin with a second molecule of borohydride to yield an air-sensitive tetrahydroflavin is unlikely since prior scavenging of residual borohydride with excess formaldehyde had no effect on the aerobic conversion to unmodified oxidized flavin. The observed instability is attributed to a tautomeric rearrangement of the 3,4-dihydroflavin to generate 1,5-dihydroflavin, a species that is also air-sensitive. Evidence in favor of an active site facilitated tautomerization reaction is provided by the fact that the stability of the 405 nm species formed with MSOX is enhanced 200-fold upon denaturation with urea or heat. The observed tautomeric rearrangement of 3,4-dihydroflavin may provide insight regarding a related flavin tautomerization reaction that has been proposed as a key step in the biosynthesis of covalent flavin linkages.     

Immunotoxicological effects of dermal application of scum of waste crankcase oil in mice

The scum of waste crankcase oil (SWCO) forms due to weathering of waste crankcase oil, deposited on the surface of water bodies. It is known to attach to the feathers of aquatic birds and cause toxicity to the eggs of nestling birds. The water bodies contaminated with SWCO can also be a source of toxicity to the human beings and animals entering such bodies. Since SWCO used in the present study had an appreciable content of heavy metals like Zn, Pb, Cd, Mn, Cr and Ni, the present investigation was undertaken to study a probable effect on immune system of mice. Animals treated with SWCO at a dose of 0.5, 1.0, 2.0, or 4.0 g/kg body weight for 28 days, had no effect on the weight gain of vital organs. A depressing effect was observed on the cell population of spleen and thymus. The number of primary antibody (IgM) producing cells was significantly depressed in spleen. The IgM antibody titer of serum, reduction of NBT dye by peritoneal exudat cells and mounting of delayed hypersensitivity response were not affected. In view of above immunotoxic effects of SWCO, the waste crankcase oil should be carefully disposed of, away from water bodies.     

Toxicity of argemone oil: Effect on hsp70expression and tissue damage in transgenic Drosophila melanogaster(hsp70-lacZ) Bg 9

The effect of argemone oil on hsp70expression and tissue damage was investigated by studying β-galactosidase activity, Western blotting and hybridization, and trypan blue staining in the larval tissues of transgenic Drosophila melanogaster(hsp70-lacZ)Bg ⁹. Different concentrations of argemone oil were mixed with food and third-instar larvae were allowed to feed on them for different time intervals (2, 4, 24, and 48 h). Argemone oil was found to induce hsp70even in the lowest concentration of the adulterant while maximum tissue damage was observed in the higher two treatment groups. Malpighian tubules and midgut tissue reflected maximum damage as evidenced by both high β-galactosidase activity and trypan blue staining in these tissues. A prior temperature shock treatment to the larvae was enough to protect the larvae from argemone oil-induced tissue damage as evidenced by little or no trypan blue staining. The present study suggests the cytotoxic potential of argemone oil and further strengthens the evidence for the use of hsp70as a biomarker in risk assessment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of ovariectomy and estrogen supplementation on brain acetylcholinesterase activity and passive-avoidance learning in rats

The effect of ovariectomy and estrogen treatment on the brain acetylcholinesterase activity and cognition in rats was investigated in this study. Ovariectomized and nonovariectomized rats were treated subcutaneously with estradiol dipropionate for 8 d. In the single-trial, passive-avoidance test all the groups showed significant learning and retention of memory as evident by the increase in transfer latency time in trial 2 as compared with trial 1. No-transfer response was significantly increased in the estradiol-dipropionate-treated ovariectomized (80%) and nonovariectomized (60%) group as compared with the ovariectomized (30%) group. Specific activity of acetylcholinesterase was assayed spectrophotometrically in salt-soluble and detergent-soluble fractions of various brain areas: frontal cortex, cerebral cortex, striatum, hippocampus and hypothalamus, thalamus, pons, medulla, and cerebellum. The effect of ovariectomy and estradiol dipropionate was varied in both fractions of these brain areas. Estradiol dipropionate treatment could restore the acetylcholinesterase activity to the control level only in the detergent-soluble fraction of hypothalamus and salt-soluble fraction of hypothalamus, thalamus, and medulla in ovariectomized rats. The results indicate that ovariectomy alters acetylcholinesterase activity in the brain areas but not in a uniform manner and affects only qualitative aspects of cognitive function, which could be improved by estrogen supplementation.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Reversal of fluoride induced cell injury through elimination of fluoride and consumption of diet rich in essential nutrients and antioxidants

The objective of the present communication is to address the issues concerning reversal of fluoride induced cell injury and disease (i.e. fluorosis) through the elimination of fluoride and consumption of a diet containing essential nutrients and antioxidants. Humans afflicted with fluorosis, as a result of consuming fluoride contaminated water or food, have been investigated. Hospital based diagnostic procedure for early detection of fluorosis, through retrieval of history, clinical complaints, testing of blood, urine and drinking water for fluoride using ion selective electrode technology, along with X-ray of the forearm have been carried out. Confirmed cases of fluorosis were introduced to an intervention protocol consisting of (1) provision of safe drinking water with fluoride levels less than 1 mg/L and (2) counselling on nutritional supplementation with focus on adequate intake of calcium, vitamins C, E and antioxidants. The patients were monitored at frequent intervals up to one year and the results are reported. With a standardized early diagnosis, elimination of fluoride intake and supplementation of a diet rich in essential nutrients and antioxidants, we have shown that the fluorosis can be reversed.