Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.     

Uterine Msx-1 and Wnt4 signaling becomes aberrant in mice with the loss of leukemia inhibitory factor or Hoxa-10: evidence for a novel cytokine-homeobox-Wnt signaling in implantation

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

Interaction among Non-toxic Phytoplankton,Toxic Phytoplankton and Zooplankton: Inferences from Field Observations

We explore the mutual dependencies and interactions among different groups of species of the plankton population, based on an analysis of the long-term field observations carried out by our group in the North–West coast of the Bay of Bengal. The plankton community is structured into three groups of species, namely, non-toxic phytoplankton (NTP), toxic phytoplankton (TPP) and zooplankton. To find the pair-wise dependencies among the three groups of plankton, Pearson and partial correlation coefficients are calculated. To explore the simultaneous interaction among all the three groups, a time series analysis is performed. Following an Expectation Maximization (E-M) algorithm, those data points which are missing due to irregularities in sampling are estimated, and with the completed data set a Vector Auto-Regressive (VAR) model is analyzed. The overall analysis demonstrates that toxin-producing phytoplankton play two distinct roles: the inhibition on consumption of toxic substances reduces the abundance of zooplankton, and the toxic materials released by TPP significantly compensate for the competitive disadvantages among phytoplankton species. Our study suggests that the presence of TPP might be a possible cause for the generation of a complex interaction among the large number of phytoplankton and zooplankton species that might be responsible for the prolonged coexistence of the plankton species in a fluctuating biomass.     

Gradient of reduced glutathione in the tail of Hemidactylus leschenaultii (Sauria: Gekkonidae)

A proximo-distal gradient of reduced glutathione (GSH), a non enzymatic antioxidant was observed in the original tails of the lizard, H. leschenaultii. In the regenerating tails, a gradual increase in the level of GSH was noted with tail elongation. In the newly regenerated tails also the level of GSH remained higher in the proximal part than the corresponding distal parts.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.     

Adenosine transport by the lung

Adenosine, a nucleoside and potent vasodilator, has been found to be taken up by the lung and converted by deamination into inosine and hypoxanthine. In a single circulation through an isolated rat lung, 69.3 +/- 3.3% of infused [14C]adenosine (10 microM) was removed from the circulation. Uptake of [14C]adenosine remained unchanged when deamination of adenosine was inhibited by 8-azaguanine or coformycin. In a single passage of adenosine through the pulmonary artery, very little of the deaminated products appeared in the pulmonary circulation, but when adenosine was recirculated through the pulmonary circulation inosine and hypoxanthine appeared in the venous effluent. These adenosine metabolites were also taken up by the lung. A major portion of the circulating adenosine was transported into the lung, where it was used to synthesize adenine nucleotides. Inhibition of adenosine kinase by iodotubercidin resulted in reduced formation of ATP and ADP. Uptake of adenosine by the lung was saturable on a concentration gradient and was a passive process because it was not affected by the absence of glucose or the presence of ouabain. Km and Vmax for adenosine transport were 0.227 mM and 4.6 mumol.min-1.g lung-1, respectively. Adenosine transport was inhibited by adenosine analogues, and the inhibitions were found to be competitive in nature. These results suggest that a specific and rate-limiting transport system exists in the lung for adenosine.