Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Dhanwantaram kashayam,an Ayurvedic polyherbal formulation,reduces oxidative radicals and reverts lipids profile towards normal in diabetic rats

BackgroundHyperglycemia and hyper oxidative stress are indicators of diabetes mellitus which is also accompanied with decreased levels of antioxidant enzymes. While oxidative stress is important in increasing insulin secretion and controlling blood sugar level at the same time excess oxidative stress leads to the destruction of beta cells of pancreas resulting in to low insulin production and hyperglycemia. A balance between the levels of oxidative radicals and insulin production is needed, but is not defined yet. Hyperglycemia also leads to hyperlipidemia which can contribute to various health conditions like cardiovascular diseases.ObjectivesThis study was designed to study the oxidative stress and lipid levels in diabetic rats. This also was designed to elucidate the effect of Dhanwantaram Kashayam, an Ayurvedic polyphenolic derived from plants on lipid metabolism and oxidative radical scavenging in diabetic rats.MethodsRats were made diabetic by injecting streptozotocin. Different enzymes involved in oxidative radical scavenging and lipid profiles including triglycerides, total cholesterol, free fatty acids and phospholipids were estimated using standard methods reported elsewhere.ResultsLevel of antioxidant enzymes were lower in diabetic rats compared to normal controls. Administration of Dhanwantaram Kashayam restored the enzyme activity as well as reduced levels of different lipids in diabetic rats.ConclusionsAdministration of Dhanwantaram Kashayam increased the activity levels of antioxidant enzymes and reduced the levels of total cholesterol, phospholipids and triglycerides. The results of this study point to the possibility of developing Dhanwantaram Kashayam as a dietary supplement which can alleviate the complications associated with diabetes or prevent them altogether.     

The field-dependence of the solid-state photo-CIDNP effect in two states of heliobacterial reaction centers

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect is studied in photosynthetic reaction centers of Heliobacillus mobilis at different magnetic fields by ¹³C MAS (magic-angle spinning) NMR spectroscopy. Two active states of heliobacterial reaction centers are probed: an anaerobic preparation of heliochromatophores (“Braunstoff”, German for “brown substance”) as well as a preparation of cells after exposure to oxygen (“Grünstoff”, “green substance”). Braunstoff shows significant increase of enhanced absorptive (positive) signals toward lower magnetic fields, which is interpreted in terms of an enhanced differential relaxation (DR) mechanism. In Grünstoff, the signals remain emissive (negative) at two fields, confirming that the influence of the DR mechanism is comparably low.     

Genistein: A Potent Anti-Breast Cancer Agent

Genistein is an isoflavonoid present in high quantities in soybeans. Possessing a wide range of bioactives, it is being studied extensively for its tumoricidal effects. Investigations into mechanisms of the anti-cancer activity have revealed many pathways including induction of cell proliferation, suppression of tyrosine kinases, regulation of Hedgehog-Gli1 signaling, modulation of epigenetic activities, seizing of cell cycle and Akt and MEK signaling pathways, among others via which the cancer cell proliferation can be controlled. Notwithstanding, the observed activities have been time- and dose-dependent. In addition, genistein has also shown varying results in women depending on the physiological parameters, such as the early or post-menopausal states.     

Enhancement of camptothecin-induced topoisomerase I cleavage complexes by the acetaldehyde adduct N2-ethyl-2'-deoxyguanosine

The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N²-ethyl-dG adducted oligonucleotides. However, the N²-ethyl-dG adduct enhanced CPT-induced Top1–DNA cleavage complexes depending on the relative position of the N²-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N²-ethyl-dG adduct was present immediately 3′ from the Top1 site (position +1). In addition, when the N²-ethyl-dG adduct was located at the −5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N²-ethyl-dG adduct located at the 5′ end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1–DNA complex, thereby inhibiting further the religation (closing) reaction.     

Monomeric 14-3-3 protein is sufficient to modulate the activity of the Drosophila slowpoke calcium-dependent potassium channel

Drosophila 14-3-3zeta (D14-3-3zeta) modulates the activity of the Slowpoke calcium-dependent potassium channel (dSlo) by interacting with the dSlo binding protein, Slob. We show here that D14-3-3zeta forms dimers in vitro. Site-directed mutations in its putative dimerization interface result in a dimerization-deficient form of D14-3-3zeta. Both the wild-type and dimerization-deficient forms of D14-3-3zeta bind to Slob with similar affinity and form complexes with dSlo. When dSlo and Slob are expressed in mammalian cells, the dSlo channel activity is similarly modulated by co-expression of either the wild-type or the dimerization-deficient form of D14-3-3zeta. In addition, dSlo is still modulated by wild-type D14-3-3zeta in the presence of a 14-3-3 mutant, which does not itself bind to Slob but forms heterodimers with the wild-type 14-3-3. These data, taken together, suggest that monomeric D14-3-3zeta is capable of modulating dSlo channel activity in this regulatory complex.     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

Total and semisynthesis and in vitro studies of both enantiomers of 20-fluorocamptothecin

Both enantiomers of 20-fluorocamptothecin and the racemate have been prepared by total synthesis. The (R)-enantiomer is essentially inactive in a topoisomerase-I/DNA assay, while the (S)-enantiomer is much less active than (20S)-camptothecin. The lactone ring of 20-fluorocamptothecin hydrolyzes more rapidly than that of camptothecin in PBS. The results provide insight into the role of the 20-hydroxy group in the binding of camptothecin to topoisomerase-I and DNA.     

Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization

Taxol is the best anticancer agent that has ever been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we report with mechanism-based evidence that curcumin, a nontoxic food additive commonly used by the Indian population, sensitizes tumor cells more efficiently to the therapeutic effect of Taxol. A combination of 5 nm Taxol with 5 microm curcumin augments anticancer effects more efficiently than Taxol alone as evidenced by increased cytotoxicity and reduced DNA synthesis in HeLa cells. Furthermore, our results reveal that this combination at the cellular level augments activation of caspases and cytochrome c release. This synergistic effect was not observed in normal cervical cells, 293 cells (in which Taxol down-regulates nuclear factor-kappaB (NF-kappaB)), or HeLa cells transfected with inhibitor kappaBalpha double mutant (IkappaBalpha DM), although the transfection itself sensitized the cells to Taxol-induced cytotoxicity. Evaluation of signaling pathways common to Taxol and curcumin reveals that this synergism was in part related to down-regulation of NF-kappaB and serine/threonine kinase Akt pathways by curcumin. An electrophoretic mobility shift assay revealed that activation of NF-kappaB induced by Taxol is down-regulated by curcumin. We also noted that curcumin-down-regulated Taxol induced phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-kappaB. Interestingly, tubulin polymerization and cyclin-dependent kinase Cdc2 activation induced by Taxol was not affected by curcumin. Altogether, our observations indicate that Taxol in combination with curcumin may provide a superior therapeutic index and advantage in the clinic for the treatment of refractory tumors.