Hypoxia rescues early mortality conferred by superoxide dismutase deficiency

Oxidative stress is widely associated with disease and aging but the underlying mechanisms are incompletely understood. Here we show that the premature mortality of Drosophila deficient in superoxide scavengers, superoxide dismutase (SOD) 1 or SOD2, is rescued by chronic hypoxia. Strikingly, switching moribund SOD2-deficient adults from normoxia into hypoxia abruptly arrests their impending premature mortality and endows the survivors with a near-normal life span. This finding challenges the notion that irreversible oxidative damage initiated by unscavenged superoxide in the mitochondrial matrix underpins the premature mortality of SOD2-deficient adults. In contrast, switching moribund SOD1-deficient flies from normoxia into hypoxia fails to alter their mortality trajectory, suggesting that the deleterious effects of unscavenged superoxide in the cytoplasm/intermembrane space compartment are cumulative and largely irreversible. We conclude that cellular responses to superoxide-initiated oxidative stress are mediated through different compartment-specific pathways. Elucidating these pathways should provide novel insights into how aerobic cells manage oxidative stress in health, aging, and disease.     

Novel role of Wag31 in protection of mycobacteria under oxidative stress

Wag31 of Mycobacterium tuberculosis belongs to the DivIVA family of proteins known to regulate cell morphology in Gram-positive bacteria. Here we demonstrate an unrecognized, novel role of Wag31 in oxidatively stressed mycobacteria. We report the cleavage of penicillin-binding protein 3 (PBP3) by the intramembrane metalloprotease Rv2869c (MSMEG_2579) in oxidatively stressed cells. Amino acids ¹⁰²A and ¹⁰³A of PBP3 are required for Rv2869c-mediated cleavage. Wag31_MTB, by virtue of its interaction with PBP3 through amino acid residues ⁴⁶NSD⁴⁸, protects it from oxidative stress-induced cleavage. PBP3 undergoes cleavage in Mycobacterium smegmatis (strain PM2) harbouring wag31 (Δ⁴⁶NSD⁴⁸) instead of the wild type, with concomitant reduction in ability to withstand oxidative stress. Overexpression of Wag31(Δ⁴⁶NSD⁴⁸) attenuates the survival of M. tuberculosis in macrophages with concomitant cleavage of PBP3, and renders the organism more susceptible towards hydrogen peroxide as well as drugs which generate reactive oxygen species, namely isoniazid and ofloxacin. We propose that targeting Wag31 could enhance the activity of mycobactericidal drugs which are known to generate reactive oxygen species.     

Synthesis, pulse radiolysis, and in vitro radioprotection studies of melatoninolipoamide, a novel conjugate of melatonin and alpha-lipoic acid

A novel conjugate of melatonin 2 and alpha-lipoic acid 4 has been prepared using DCC mediated coupling. The conjugate named melatoninolipoamide has been assigned its structure 1 on the basis of spectral analysis (UV, IR, NMR, and EI-MS). Pulse radiolysis studies of the conjugate were carried out in aqueous solutions with both oxidizing and reducing radicals. The results indicate that the melatonin moiety of the conjugate reacts preferably with oxidizing radicals and the lipoic acid moiety exhibits preferential reaction with reducing radicals. The in vitro radioprotection ability of 1 was examined by gamma-radiation induced lipid peroxidation in liposomes and hemolysis of erythrocytes, and compared the results with those of melatonin and alpha-lipoic acid. The studies suggest that the conjugate can be explored as a probable radioprotector.     

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.     

The essential requirement of an animal heme peroxidase protein during the wing maturation process in Drosophila

Background

Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools we found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults.

Results

The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well.

Conclusion

Cysu, a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H₂O₂, which plays an essential role in post-eclosion wing morphogenesis.

     

Complex formation of rutin and quercetin with copper alters the mode of inhibition of ribonuclease A

Rutin and quercetin, both minor components of green tea and their Cu(II) complexes interact with Ribonuclease A (RNase A) in a novel way. The effects of rutin, quercetin and their copper complexes on the catalytic activity of the protein were investigated. Rutin shows an enhancement in the ribonucleolytic activity whereas the copper complexes and quercetin behave as non-competitive type inhibitors with K(i) values in the μM range. The secondary structural changes of RNase A in presence of the ligands were measured by circular dichroism and Fourier transform infrared spectroscopy. The binding parameters were obtained using a fluorescence quenching analysis.     

Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.     

Hydration of protein–RNA recognition sites

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces.