Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: the protective effect of Ellagic acid

Cyclophosphamide (CPM), an alkylating agent is used as an immunosuppressant in rheumatoid arthritis and in the treatment of several cancers as well. In this study, Ellagic acid (EA), a naturally occurring plant polyphenol, was evaluated for its antigenotoxicity and antioxidant efficacy against the CPM-induced renal oxidative stress and genotoxicity in Swiss albino mice. The mice were given a prophylactic treatment of EA orally at a dose of 50 and 100 mg/kg body weight (b wt) for seven consecutive days before the administration of a single intraperitoneal (i.p.) injection of CPM at 50 mg/kg b wt. The modulatory effects of EA on CPM-induced nephrotoxicity and genotoxicity were investigated by assaying oxidative stress biomarkers, serum kidney toxicity markers, DNA fragmentation, alkaline unwinding assay, micronuclei (MN) assay, and by histopathological examination of kidney tissue. A single intraperitoneal administration of CPM in mice increased malondialdehyde level with depletion in glutathione content, antioxidant enzymes activities, viz. glutathione peroxidase, glutathione reductase, catalase, quinone reductase, induced DNA strand breaks, and MN induction. EA oral administration at both doses caused significant reduction in their levels, restoration in the activities of antioxidant enzymes, reduction in MN formation, and DNA fragmentation. Serum toxicity marker enzymes like BUN, creatinine, and LDH were also increased after CPM treatment which was significantly decreased in EA pretreated groups. Present findings suggest a prominent role of EA against CPM-induced renal injury, DNA damage, and genotoxicity.     

Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

Nuclear transport of macromolecules is regulated by the physiological state of the cell and thus sensitive to stress. To define the molecular mechanisms that control nuclear export upon stress, cells were exposed to nonlethal concentrations of the oxidant diethyl maleate (DEM). These stress conditions inhibited chromosome region maintenance-1 (Crm1)-dependent nuclear export and increased the association between Crm1 and Ran. In addition, we identified several repeat-containing nucleoporins implicated in nuclear export as targets of oxidative stress. As such, DEM treatment reduced Nup358 levels at the nuclear envelope and redistributed Nup98. Furthermore, oxidative stress led to an increase in the apparent molecular masses of Nup98, Nup214, and Nup62. Incubation with phosphatase or β-N-acetyl-hexosaminidase showed that oxidative stress caused the phosphorylation of Nup98, Nup62, and Nup214 as well as O-linked N-acetylglucosamine modification of Nup62 and Nup214. These oxidant-induced changes in nucleoporin modification correlated first with the increased binding of Nup62 to the exporter Crm1 and second with the reduced interaction of Nup62 with other FxFG-containing nucleoporins. Together, oxidative stress up-regulated the binding of Crm1 to Ran and affected multiple repeat-containing nucleoporins by changing their localization, phosphorylation, O-glycosylation, or interaction with other transport components. We propose that the combination of these events contributes to the stress-dependent regulation of Crm1-mediated protein export.     

Neo-regeneration of urinary bladder: a desired metaplasia of autologous membrane from rectosigmoid colon containing stem cells of intestinal crypts

The current management of diseases of urinary bladder requiring resection is by augmentation cystoplasty or transplantation of ureters. Transplantation of ureters is associated with morbidity and mortality. Ideal management will be by regenerating urinary bladder in vivo. Neo-regeneration of tissues and organs like abdominal wall, aponeurosis etc., has been attempted and patented. After neo-regeneration of mesoderm tissues and organs, regeneration of urinary bladder (developed from endoderm) was. In vivo surgical techniques were developed in dogs. It is known that the embryonic morphogenesis of urinary bladder is from uro-genital sinus of hind gut. A membrane, containing endoderm stem cells in crypts of recto-sigmoid colon, was surgically isolated and colonized with remnant of urinary bladder wall after extensive resection. Experimental study was performed in dogs, for 60 days to one and a half year. Regeneration of all the layers of tissues of the wall of urinary bladder was observed. The neo-regeneration phenomenon has been recognized as "desired metaplasia". The regenerated neo tissue/organ on histological examination and cystometry studies was found compatible with normal urinary bladder. The hypothesis, neo-regeneration and desired metaplasia, is discussed.     

Cerebral and intestinal perfusion and metabolism in normocythemic hyperviscous hypoxic newborn pigs.

We studied the effects of hypoxia on cerebral cortical and intestinal perfusion and metabolism in normocythemic hyperviscous newborn pigs. Seven pigs were made hyperviscous by an injection of cryoprecipitate, increasing viscosity from 5.8 +/- 0.9 to 9.0 +/- 1. 2 (SD) cycles/s. Six normoviscous pigs received 0.9% NaCl. Reducing the inspired O(2) decreased the arterial O(2) content (Ca(O(2))) from 9.5 +/- 1.6 to 3.6 +/- 1.3 ml O(2)/100 ml. Increases in brain and decreases in gastrointestinal blood flow at the lower Ca(O(2)) values were similar between the groups. During hypoxia, blood flow to stomach, distal intestinal mucosa, and large intestines was lower (-50, -23, and -28%, respectively) in the hyperviscous than normoviscous group. At the lower Ca(O(2)) values, cerebral cortical vascular resistance decreased in both groups and intestinal vascular resistance increased (+257%) in the hyperviscous but not in the normoviscous group. During hypoxia, systemic oxygen delivery decreased, extraction increased, and uptake did not change; cerebral cortical O(2) delivery, extraction, and uptake did not change; and intestinal O(2) delivery decreased, extraction increased, and uptake did not change in both groups. Our study demonstrated that 1) during hypoxia, increases in systemic O(2) extraction compensated for decreases in delivery and systemic uptake did not change; vasodilation sustained cerebral cortical O(2) delivery and preserved metabolism; increases in intestinal oxygen extraction offset decreases in delivery and uptake was preserved; and 2) nonpolycythemic hyperviscosity did not have a major influence on cardiovascular or metabolic responses to hypoxia, except for modest effects on intestinal resistance and perfusion to certain gastrointestinal regions. We conclude that, under normocythemic conditions, a moderate increase in viscosity does not have a major impact on hemodynamic or metabolic adjustments to hypoxia in newborn pigs.     

Effect of the postfeeding interval on olfactory responses of thrips to herbivore‐induced cotton plants

We investigated the responses of 3 thrips species, Frankliniella schultzei Trybom, F. occidentalis Pergrande, and Thrips tabaci Lindeman (Thysanoptera: Thripidae) to herbivore‐damaged and undamaged cotton seedlings (Gossypium hirsutum L. [Malvales: Malvaceae]) at a range of time intervals following damage by adult Tetranychus urticae (Koch), adult T. ludeni (Zacher) (Acari: Tetranychidae) or Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae in olfactometer assays. The intensity/frequency of the response of thrips to herbivore‐induced plants decreased with time and ultimately disappeared in all cases; however, the rate at which the response declined was related to the herbivore species that inflicted the damage. All 3 species of thrips were attracted to plants damaged by T. urticae for longer than they were to plants damaged by T. ludeni. The duration for which damaged plants remained attractive was also affected by the degree of damage inflicted on cotton seedlings. For example, F. schultzei was attracted to plants damaged by a higher density of two‐spotted spider mites (100/plant) for much longer than to plants damaged by a lower density of these mites (50/plant). The results reinforce previous studies that demonstrate that arrangement of variables influences the responses of thrips to their herbivore‐induced cotton host plants. Results also show that these responses are variable in time following herbivore damage to cotton plants, which further demonstrates how difficult it is to generalize about the functional significance of these interactions.     

Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.     

Efficient <Emphasis Type=Italic>in vitro</Emphasis> plant regeneration from shoot apices and gene transfer by particle bombardment in <Emphasis Type=Italic>Jatropha curcas</Emphasis>

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5–6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm⁻³ kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.     

Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.     

Stereo-selectivity of human serum albumin to enantiomeric and isoelectronic pollutants dissected by spectroscopy, calorimetry and bioinformatics

1-naphthol (1N), 2-naphthol (2N) and 8-quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (K(b)) of these pollutants to HSA were moderate (10(4)-10(5) M(-1)). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39-5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy-entropy compensation (EEC). The difference observed between ΔC(p) (exp) and ΔC(p) (calc) are suggested to be caused by binding-induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants.