Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.     

miR-34a is essential for p19Arf-driven cell cycle arrest

The Arf tumor suppressor gene product, p19^Arf, regulates cell proliferation in incipient cancer cells and during embryo development. Beyond its commonly accepted p53-dependent actions, p19^Arf also acts independently of p53 in both contexts. One such p53-independent effect with in vivo relevance includes its repression of Pdgfrβ, a process that is essential for vision in the mouse. We have utilized cell culture-based and mouse models to define a new role for miR-34a in this process. Ectopic expression of Arf in cultured cells enhanced the expression of several microRNAs predicted to target Pdgfrß synthesis, including the miR-34 family. Because miR-34a has been implicated as a p53-dependent effector, we investigated whether it also contributed to p53-independent effects of p19^Arf. Indeed, in mouse embryo fibroblasts (MEFs) lacking p53, Arf-driven repression of Pdgfrβ and its blockade of Pdgf-B stimulated DNA synthesis were both completely interrupted by anti-microRNA against miR-34a. Ectopic miR-34a directly targeted Pdgfrβ and a plasmid reporter containing wild-type Pdgfrβ 3′UTR sequence, but not one in which the miR-34a target sequence was mutated. Although miR-34a expression has been linked to p53—a well-known effector of p19^Arf—Arf expression and its knockdown correlated with miR-34a level in MEFs lacking p53. Finally, analysis of the mouse embryonic eye demonstrated that Arf controlled expression of miR-34a, and the related miR-34b and c, in vivo during normal mouse development. Our findings indicate that miR-34a provides an essential link between p19^Arf and its p53-independent capacity to block cell proliferation driven by Pdgfrβ. This has ramifications for developmental and tumor suppressor roles of Arf.     

Photocatalytic antimicrobial activity of thin surface films of TiO(2), CuO and TiO (2)/CuO dual layers on Escherichia coli and bacteriophage T4

TiO₂-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO₂, CuO and hybrid CuO/TiO₂ prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO₂ prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol–gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO₂ which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO₂/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries.     

Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.     

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.     

Neuroprotective Effects of Withania somnifera on 4-Hydroxynonenal Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells Through ROS Inhibition and Apoptotic Mitochondrial Pathway

Neurochemical Research - The antioxidant, anti-inflammatory, and anticancer activities of Withania somnifera (WS) are known for a long time. This study was aimed to examine whether WS also...     

Foliar application of selenium increases fertility and grain yield in bread wheat under contrasting water availability regimes

The study was conducted to assess whether selenium (Se) application modulates fertility to alter grain yield in bread wheat grown under different moisture regimes. Seeds of wheat cultivar Millat-2011 were sown in the plots using a randomized complete block design with three replicates per treatment. After germination, the plants were exposed to six moisture regimes, viz. no irrigation after germination, irrigation at boot stage, irrigations at boot and grain-filling stages, irrigations at crown root, boot and grain-filling stages, irrigations at crown root, boot, heading and grain-filling stages and irrigations at crown root, stem elongation, boot, heading and grain-filling stages. At the heading stage, foliar spray of sodium selenate (0, 2 and 4 mg Se L^?1) was done. Withholding water at early growth stages significantly increased oxidative stress and decreased growth and grain yield. Irrespective of moisture regimes, foliar application of Se (2 mg L^?1) decreased oxidative stress, modulated photosynthetic pigments and fertility and increased grain yield in wheat. The Se-mediated increase in grain yield was attributed to the increase in chlorophyll and ascorbic acid contents and fertility coupled with decrease of oxidative stress under different moisture regimes. The results could be helpful to manage wheat production in the semi-arid environments.     

Aggressiveness spectrum and genetic variability of Fusarium spp. on rice and wheat varieties

Abstract

A total of 106 Fusarium spp. were isolated from infected roots and soil samples of wheat and rice. Of the 106 isolates, 32 from wheat, and 74 from rice, were isolated. Six Fusarium spp. (F. oxysporum, F. moniliforme, F. poae, F. graminearum, F. tricinctum and F. equiseti) were identified at specie level. In aggressiveness tests Fusarium spp. root rot causing fungi were screened out into different aggressiveness classes according to disease severity scales. The aggressiveness of Fusarium spp. was studied on wheat varieties (Inqalab-91 and chakwal-86) and on rice varieties (Basmati-385 and IRRI-6) under controlled conditions. The overall total number of aggressive isolates was higher in rice than in wheat. However, the percentage of severely aggressive isolates was high in wheat, whereas the percentage of moderately and slightly aggressiveness isolates was high in rice. In rice, five isolates were non-aggressive and on wheat 17 were non-aggressive. Random Amplified Polymorphism DNAs (RAPDs) were used to study the polymorphism and genetic variations within the population of Fusarium spp. that established to study correlation between taxonomical and genetical characters of fungi. Five random primers were used P1 (5′-AGGAGGACCC-3′), P2 (5′-ACGAGGGACT-3′), PE7 (5′-AGATGCAGCC-3′), P14 (5′-CCACAGCACG-3′) and PE20 (5′-AACGGTGACC-3′). Each of the 10-mer primers produced results based on the respective banding patterns they generated in present investigations. Primers distinguished the F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti. All the tested primers yielded amplification products, and that were reproducible. Although there was some intraspecific variation with primers, some strains were similar and some were different in banding pattern. In F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti were seen clustered close to one another but each primer separated them unambiguously. All primer (P1, P2, P14, PE7 and PE20) combination produced 62 bands. All primers have shown interspecific and intraspecific variations in banding patterns.     

Targeted Delivery of Probiotics: Perspectives on Research and Commercialization

Probiotics and Antimicrobial Proteins - Considering the significance of the gut microbiota on human health, there has been ever-growing research and commercial interest in various aspects of...     

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.