Intervention of α-lipoic acid ameliorates methotrexate-induced oxidative stress and genotoxicity: A study in rat intestine

Methotrexate (MTX) is an anti-metabolite, widely used in the cancer chemotherapy and rheumatoid arthritis. However, its long-term clinical use is restricted on account of its severe intestinal toxicity. The present study was aimed to investigate the intestinal toxicity of MTX and the possible protective effect of α-lipoic acid (LA) on Sprague–Dawley rats. MTX-induced intestinal toxicity was evaluated at the dose of 2.5 mg/kg for short-term (5 days treatment) and 1 mg/kg for long-term (5 days in a week for four consecutive weeks treatment) study. The possible protective effect of LA was evaluated in both short- as well as long-term study in a dose-dependent manner. MTX treatment induced diarrhoea and mortality in rats, indicating its severe toxicity in the target organ of investigation, i.e., intestine. Further, the intestinal toxicity of MTX was assessed by evaluating different parameters of oxidative stress, DNA damage, cytotoxicity as well as histological changes. Immunostaining for p53 revealed higher genotoxic assault in the intestinal cells due to MTX treatment. Pretreatment of rats with LA led to significant decrease in the oxidative stress, DNA damage, cellular damage, inflammatory changes and apoptosis as determined by malondialdehyde level, glutathione level, comet assay parameters, histological evaluation, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In the present investigation, we report that LA pretreatment ameliorates MTX-induced intestinal toxicity in rat as evident from the protection against oxidative stress, decrease in DNA damage and protection of cellular morphology as well as improvement in the stool consistency and animal survival rate.     

Development of one-step TaqMan(R) real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses

ABSTRACT: BACKGROUND: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. RESULTS: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). CONCLUSION: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.     

Physiological evidence for an interaction between helices II and XI in the melibiose carrier of Escherichia coli

The melibiose carrier from Escherichia coli is a cation-substrate cotransporter that catalyzes the accumulation of galactosides at the expense of H(+), Na(+), or Li(+) electrochemical gradients. Charged residues on transmembrane domains in the amino-terminal portion of this carrier play an important role in the recognition of cations, while the carboxyl portion of the protein seems to be important for sugar recognition. In the present study, we substituted Lys-377 on helix XI with Val. This mutant carrier, K377V, had reduced melibiose transport activity. We subsequently used this mutant for the isolation of functional second-site revertants. Revertant strains showed the additional substitutions of Val or Asn for Asp-59 (helix II), or Leu for Phe-20 (helix I). Isolation of revertant strains where both Lys-377 and Asp-59 are substituted with neutral residues suggested the possibility that a salt bridge exists between helix II and helix XI. To further test this idea, we constructed three additional site-directed mutants: Asp-59-->Lys (D59K), Lys-377-->Asp (K377D), and a double mutant, Asp-59-->Lys/Lys-377-->Asp (D59K/K377D), in which the position of these charges was exchanged. K377D accumulated melibiose only marginally while D59K could not accumulate. However, the D59K/K377D double mutant accumulated melibiose to a modest level although this activity was no longer stimulated by Na(+). We suggest that Asp-59 and Lys-377 interact via a salt bridge that brings helix II and helix XI close to one another in the three-dimensional structure of the carrier.     

Dynamics of Deinococcus radiodurans under controlled growth conditions

Deinococcus radiodurans is a potent radiation resistant bacterium with immense potential in nuclear waste treatment. In this investigation, the translational and rotational dynamics of dilute suspensions of D. radiodurans cultured under controlled growth conditions was studied by the polarized and depolarized dynamic light-scattering (DLS) techniques. Additionally, confocal laser scanning microscopy was used for characterizing the cultured samples and also for identification of D. radiodurans dimer, tetramer, and multimer morphologies. The data obtained showed translational diffusion coefficients (DT) of 1.2 x 10(-9), 1.97 x 10(-9), and 2.12 x 10(-9) cm2 /s, corresponding to an average size of 3.61, 2.22, and 2.06 microm, respectively, for live multimer, tetramer, and dimer forms of D. radiodurans. Depolarized DLS experiments showed very slow rotational diffusion coefficients (DR) of 0.182/s for dimer and 0.098/s for tetramer morphologies. No measurable rotational diffusion was observed for multimer form. Polarized DLS measurements on live D. radiodurans confirmed that the bacterium is nonmotile in nature. The dynamics of the dead dimer and tetramer D. radiodurans were also studied using polarized and depolarized DLS experiments and compared with the dynamics of live species. The dead cells were slightly smaller in size when compared to the live cells. However, no additional information could be obtained for dead cells from the polarized and depolarized dynamic light-scattering studies.     

Applying Landscape Metrics to Characterize Potential Habitat of Bonobos (Pan paniscus) in the Maringa-Lopori-Wamba Landscape, Democratic Republic of Congo

To conserve areas and species threatened by immediate landscape change requires that we make planning decisions for large areas in the absence of adequate data. Here we study the utility of broad-scale landscape metrics as predictors of species occurrence, especially for remote areas where there is a need to make the most of limited spatial and biological data. Bonobos (Pan paniscus) are endangered great apes endemic to lowland forests of the Democratic Republic of Congo. They are threatened by bushmeat hunting that is exacerbated by habitat fragmentation through slash-and-burn agriculture and timber harvest. We developed four landscape metrics —edge density (ED), COHESION, CONTAGION, and class area (CA)— that may serve as surrogates for measuring accessibility of areas to hunting in order to predict relative bonobo-habitat suitability. We calculated the metrics for the Maringa-Lopori-Wamba (MLW) landscape and evaluated them for utility in predicting bonobo-nest occupancy based on 2009 field data. Cross-validations showed that all four metrics performed similarly. However, forest ED was arguably the best predictor, with an overall classification accuracy of 72.1% in which 85% of known nest blocks (N = 124) were classified correctly. We demonstrated that for a relatively intact landscape and a mobile forest-dwelling species that is fairly tolerant of forest openings, forest fragmentation can still be an important predictor of species occurrence. We suggest that ED can be helpful when mapping bonobo habitat in MLW and can aid landscape-planning and conservation efforts. Our approach may be applied to other edge-sensitive species, especially where high-resolution data are deficient.     

Role of Microorganisms in Emission of Nitrous Oxide and Methane in Pulse Cultivated Soil Under Laboratory Incubation Condition

Soil from a pulse cultivated farmers land of Odisha, India, have been subjected to incubation studies for 40 consecutive days, to establish the impact of various nitrogenous fertilizers and water filled pore space (WFPS) on green house gas emission (N₂O & CH₄). C₂H₂ inhibition technique was followed to have a comprehensive understanding about the individual contribution of nitrifiers and denitrifiers towards the emission of N₂O. Nevertheless, low concentration of C₂H₂ (5 ml: flow rate 0.1 kg/cm²) is hypothesized to partially impede the metabolic pathways of denitrifying bacterial population, thus reducing the overall N₂O emission rate. Different soil parameters of the experimental soil such as moisture, total organic carbon, ammonium content and nitrate–nitrogen contents were measured at regular intervals. Application of external N-sources under different WFPS conditions revealed the diverse role played by the indigenous soil microorganism towards green house gas emission. Isolation of heterotrophic microorganisms (Pseudomonas) from the soil samples, further supported the fact that denitrification might be prevailing during specific conditions thus contributing to N₂O emission. Statistical analysis showed that WFPS was the most influential parameter affecting N₂O formation in soil in absence of an inhibitor like C₂H₂.     

Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.