Modulation of radiation-induced intestinal injury by radioprotective agents: a cellular and molecular perspectives.

The gastrointestinal (GI) system has rapidly proliferating and differentiating cells, which make it one of the most radiosensitive organs in the body. Exposure to high dose of ionising radiation (IR) during radiotherapy may generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) including radicals, cause some side effects such as nausea, vomiting, diarrhoea, pain, ulceration, mal-absorption etc. Irradiation disrupts GI system by damaging proliferating stem cells of the crypts that alters the histology and physiology of intestine. Radiation damage reflects the qualitative and quantitative changes in intestinal epithelial stem cells like enterocytes, enteroendocrine cells, goblet cells and Paneth cells. The damaging effects of radiation to bio-molecules and cellular structures can alter gene signalling cascades and grounds genomic instability, protein modifications, cell senescence and cell death. The signalling pathways of GI tract includes Wnt, BMP, Hedgehog, PTEN/PI3K and Notch plays an important role in self-renewal of intestinal stem cells (ISCs) and maintaining the balance between self-renewal and differentiation of ISCs. Various radiation countermeasures including radioprotectors and mitigators are under development phase globally but still not approved for clinical applications during any radiation emergencies. In view of above, present review highlights cellular and molecular interruptions of GI system due to acute and chronic GI radiation injury, role of radioprotectors in signalling cascade modulations in GI epithelium and involvement of ISC markers in radioprotection.

An In Silico Approach for Identification of the Pathogenic Species, Helicobacter pylori and Its Relatives.

Helicobacter is an economically important genus within the phylum Proteobacteria and include many species which cause many diseases in humans. With the conventional methods, it is difficult to identify them easily due to the high genetic similarity among its species. In the present study, 361 16S rRNA (rrs) gene sequences belonging to 45 species of genus Helicobacter were analyzed. Out of these, 264 sequences of 10 clinically relevant species (including Helicobacter pylori) were used. rrs gene sequences were analyzed to obtain a phylogenetic framework tree, in silico restriction enzyme analysis and species-specific conserved motifs. Protein sequences of another housekeeping gene, hsp60 were also subjected to phylogenetic analysis to supplement the data obtained using rrs sequences. Using these approaches, six out of ten species (including H. pylori) were easily segregated, whereas four species namely H. bilis, H. cinaedi, H. felis and Candidatus H. heilmannii were found to be heterogeneous. The above approaches have also helped in segregating unclassified sequences, thus proving them as an easy diagnostic method for identifying members of genus Helicobacter up to species level.

A semiquinone glucoside derivative provides protection to male reproductive system of the mice against gamma radiation toxicity.

Present investigation was carried out to evaluate the radioprotective efficacy of a novel Semiquinone glucoside derivative (SQGD), isolated from Bacillus sp. INM-1, in the male reproductive system of BALB/c mice. Animals were administered 50 mg/kg b.wt. (i.p.) SQGD 2 h before whole body γ-irradiation (10 Gy). Radiation-induced cellular toxicity and its modulation by SQGD pretreatment was evaluated in the mice testes by quantitative histological and protein expression analysis. SQGD pretreatment protects irradiated mice from radiation-induced testicular atrophy and germ cells degeneration, which may lead to emptiness of seminiferous tubules. Significant decrease in P53 and P21((Cip/WAF-1)) expression was observed in the irradiated mice pretreated (2 h) by SQGD at 6 h compared with only irradiated mice. However, contrary to P53, expressions of P21 at latter time, that is, 24-72 h was found to be increased significantly in the irradiated mice pretreated by SQGD. Significant increase in the intact PARP-1 protein expression were observed in the testes of the mice pretreated by SQGD 2 h before irradiation at 24-72 h compared with the only irradiated mice, whereas significant increase in PARP-1 cleaved fragment was noticed at 24 h. Similarly, significant increase in NF-kB and BCL-2/BAX expressions ratio was noticed in SQGD-treated mice (± irradiation) compared with irradiated mice, suggested a role of SQGD in the activation of prosurvival signaling in the testicular germinal cells population of the irradiated mice and thus contributed to protection against lethal γ-irradiation.

Semiquinone fraction isolated from Bacillus sp. INM-1 protects hepatic tissues against γ-radiation induced toxicity.

Present study was focused on evaluation of a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 for its ability against γ radiation induced oxidative stress in irradiated mice. Animals were divided into four group, i.e., (i) untreated control mice; (ii) SQGD treated (50 mg/kg b. wt. i.p.) mice; (iii) irradiated (10 Gy) mice; and (iv) irradiated mice which were pre-treated (-2 h) with SQGD (50 mg/kg b. wt. i.p.). Following treatment, liver homogenates of the treated mice were subjected to endogenous antioxidant enzymes estimation. Result indicated that SQGD pre-treatment, significantly (P < 0.05) induced superoxide dismutase (SOD) (19.84 ± 2.18% at 72 h), catalase (CAT) (26.47 ± 3.11% at 12 h), glutathione (33.81 ± 1.99% at 24 h), and glutathione-S-transferase (24.40 ± 2.65% at 6 h) activities in the liver of mice as compared with untreated control. Significant (P < 0.05) induction in SOD (50.04 ± 5.59% at 12 h), CAT (62.22 ± 7.50 at 72 h), glutathione (42.92 ± 2.28% at 24 h), and glutathione-S-transferase (46.65 ± 3.25 at 12 h) was observed in irradiated mice which were pre-treated with SQGD compared with only irradiated mice. Further, significant induction in ABTS(+) radicals (directly proportional to decrease mM Trolox equivalent) was observed in liver homogenate of H2 O2 treated mice which were found to be significantly inhibited in H2 O2 treated mice pre-treated with SQGD. Thus, it can be concluded that SQGD treatment neutralizes oxidative stress caused by irradiation not only by enhancing endogenous antioxidant enzymes but also by improving total antioxidant status of cellular system and thus cumulative effect of the phenomenon may contributes to radioprotection.

Radioprotection to small intestine of the mice against ionizing radiation by semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1.

Ionizing irradiation induces severe damage to the intestinal crypt cells which are responsible for renovation and maintenance of the intestinal cellular architecture. Therefore, protection of intestinal cells and tissue against lethal irradiation using a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 is the prime focus of the present investigation. BALB/c mice were administered by SQGD (50 mg/kg.b.wt. i.p.) 2 h before whole body irradiation (10 Gy), and histological analysis of the jejunum section was carried out and compared to the irradiated mice. Significant (p < 0.0001) increase in villus length, number of cells per villus, crypts numbers per villus section, total cells counts and mitotic cell counts per crypt and low goblet cells per villus section, and low apoptotic index per crypt section were observed in the irradiated mice pre-treated by SQGD at 48-168 h. Significant induction in NF-kß at 24 h and Bcl-2/Bax ratio was observed in irradiated mice pre-treated by SQGD compared to only irradiated animals. SQGD pre-treatment before irradiation was found instrumental to reverse the radiation-induced degenerative changes by replenishment of the damaged cells by enhancing mitotic, proliferating, pro-survival, and apoptosis inhibitory activities probably through modulation of cell cycle arrest in G(1)/S phase in the intestinal cellular milieu.

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1.

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30-90 microg/ml) of SQGD for different time (3-12h) intervals at 37°C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30µg/ml) stimulation for 3h at 37°C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3-6h, which was found decrease at 12h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30-90 microg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25mg/kgbwt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH. Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.

Antioxidative and radioprotective activities of semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1.

A semiquinone glucoside derivative (SQGD) was isolated from a radioresistant bacterium Bacillus sp. INM-1 and its antioxidant and radioprotective activities were evaluated using in vitro assays. Natural stable free radical properties of SQGD in solid as well as in solution form were estimated using Electron Paramagnetic Resonance (EPR) spectrometry. Results of the study were demonstrated high reducing power (1.267 ± 0.03356 U(abs)) and nitric oxide radicals scavenging activity (34.684 ± 2.132%) of SQGD. Maximum lipid peroxidation inhibitory activity of SQGD was found to be 74.09 ± 0.08% at 500 µg/ml concentration. Similarly, significant (39.54%; P < 0.05) protection to the liposomal artificial membrane against gamma radiation was observed by SQGD in terms of neutralization of gamma radiation-induced TBARS radicals in vitro. OH(-) radicals scavenging efficacy of SQGD was estimated in terms of % inhibition in deoxy D: -ribose degradation by non-site-specific and site-specific assay. The maximum (54.01 ± 1.01%) inhibition of deoxy D: -ribose degradation was observed in non-site-specific manner, whereas, site-specific inhibition was observed to be 46.36 ± 0.5% at the same concentration (250 µg/ml) of SQGD. EPR spectroscopic analysis of the SQGD indicated ~80% reduction of DPPH radicals at 6.4% concentration. EPR spectral analysis of SQGD was revealed an appearance of very strong EPR signal of 2.00485 (crystalline form) and 2.00520 (solution form) g(y) tensor value, which were an established characteristic of o-semiquinone radicals. Therefore, it can be concluded that SQGD is a natural stable o-semiquinone-type radical, possessing strong antioxidant activities and can effectively neutralize radiation induced free radicals in biological system.