Molecular and cellular remodeling of HepG2 cells upon treatment with antitubercular drugs

Drug-induced liver injury (DILI) is an adverse outcome of the currently used tuberculosis treatment regimen, which results in patient noncompliance, poor treatment outcomes, and the emergence of drug-resistant tuberculosis. DILI is primarily caused by the toxicity of the drugs and their metabolites, which affect liver cells, biliary epithelial cells, and liver vasculature. However, the precise mechanism behind the cellular damage attributable to first-line antitubercular drugs (ATDs), as well as the effect of toxicity on the cell survival strategies, is yet to be elucidated. In the current study, HepG2 cells upon treatment with a high concentration of ATDs showed increased perforation within the cell, cuboidal shape, and membrane blebbing as compared with control/untreated cells. It was observed that ATD-induced toxicity in HepG2 cells leads to altered mitochondrial membrane permeability, which was depicted by the decreased fluorescence intensity of the MitoRed tracker dye at higher drug concentrations. In addition, high doses of ATDs caused cell damage through an increase in reactive oxygen species production in HepG2 cells and a simultaneous reduction in glutathione levels. Further, high dose of isoniazid (50–200 mM), pyrazinamide (50–200 mM), and rifampicin (20–100 µM) causes cell apoptosis and affects cell survival during toxic conditions by decreasing the expression of potent autophagy markers Atg5, Atg7, and LC3B. Thus, ATD-mediated toxicity contributes to the reduced ability of hepatocytes to tolerate cellular damage caused by altered mitochondrial membrane permeability, increased apoptosis, and decreased autophagy. These findings further emphasize the need to develop adjuvant therapies that can mitigate ATD-induced toxicity for the effective treatment of tuberculosis.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Control of circadian rhythm in nitrate reductase activity in Wolffia microscopica Griff

In Wolffia microscopica, the activity of nitrate reductase showedstrong diurnal rhythmicity with a peak in the forenoon. Therhythmicity persists under extended dark conditions as wellas under continuous light. The phase of the rhythm shifts underinverted light-dark cycles. Studies with various inhibitorsof macromolecular synthesis suggest that the increase in enzymeactivity depends on the new synthesis of both RNA and protein. (Received February 14, 1979; )     

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress

Journal of Plant Research - The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II)...     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIα

Abstract

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIα to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIα. The results indicate that topo IIα binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIα displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIα to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIα. These results implicate a dual role for topo IIα in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.