Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53^(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.     

Stimulation of NEIL2-mediated oxidized base excision repair via YB-1 interaction during oxidative stress

The recently characterized enzyme NEIL2 (Nei-like-2), one of the four oxidized base-specific DNA glycosylases (OGG1, NTH1, NEIL1, and NEIL2) in mammalian cells, has poor base excision activity from duplex DNA. To test the possibility that one or more proteins modulate its activity in vivo, we performed mass spectrometric analysis of the NEIL2 immunocomplex and identified Y box-binding (YB-1) protein as a stably interacting partner of NEIL2. We show here that YB-1 not only interacts physically with NEIL2, but it also cooperates functionally by stimulating its base excision activity by 7-fold. Moreover, YB-1 interacts with the other NEIL2-associated BER proteins, namely, DNA ligase III alpha and DNA polymerase beta and thus could form a large multiprotein complex. YB-1, normally present in the cytoplasm, translocates to the nucleus during UVA-induced oxidative stress, concomitant with its increased association with and activation of NEIL2. NEIL2-initiated base excision activity is significantly reduced in YB-1-depleted cells. YB-1 thus appears to have a novel regulatory role in NEIL2-mediated repair under oxidative stress.     

Impaired fat-induced thermogenesis in obese subjects: the NUGENOB study

Objectives: To study energy expenditure before and 3 hours after a high‐fat load in a large cohort of obese subjects (n = 701) and a lean reference group (n = 113). Research Methods and Procedures: Subjects from seven European countries underwent a 1‐day clinical study with a liquid test meal challenge containing 95% fat (energy content was 50% of estimated resting energy expenditure). Fasting and 3‐hour postprandial energy expenditures, as well as metabolites and hormones, were determined. Results: Obese subjects had a reduced postprandial energy expenditure after the high‐fat load, independent of body composition, age, sex, research center, and resting energy expenditure, whereas within the obese group, thermogenesis increased again with increasing BMI category. Additionally, insulin resistance, habitual physical activity, postprandial plasma triacylglycerols, and insulin were all independently positively related to the postprandial energy expenditure. Resting energy expenditure, adjusted for fat‐free mass, increased with degree of obesity, a difference that disappeared after adjustment for fat mass. Furthermore, insulin resistance, fasting plasma free fatty acids, and cortisol were positively associated, whereas fasting plasma leptin and insulin‐like growth factor‐1 were negatively associated, with resting energy expenditure. Discussion: The 3‐hour fat‐induced thermogenic response is reduced in obesity. It remains to be determined whether this blunted thermogenic response is a contributory factor or an adaptive response to the obese state.     

The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

Effect of areca nut on salivary copper concentration in chronic chewers

The chewing of areca nut is associated with the development of oral submucous fibrosis (OSF), a condition predominantly encountered in Asians indulging in the habit. The pathogenesis of this condition is however, unclear, though several mechanisms have been proposed. Copper has previously been implicated as a possible aetiological factor. In this study, total copper concentration was measured via atomic absorption spectrophotometry in whole mouth saliva of 15 volunteers who were regular chewers, before and after their habitual chew. An aliquot of the latter was also analysed for copper. Six non-chewing volunteers acted as controls. Salivary copper concentrations were corrected for protein content. Over 50% of the subjects had basal salivary copper concentration higher than the range seen in normal controls. All but two subjects demonstrated an increase in the salivary [Cu] following their habitual chew. Marked changes were seen in those with low basal salivary concentrations. These data indicate that soluble copper found in areca nut is released into the oral environment of habitual chewers. Its buccal absorption may contribute to the oral fibrosis in Asians who regularly chew this nut.     

Recent advances in proton pump inhibitors and management of acid-peptic disorders

Acid-peptic ulcers and diseases have been increasingly on rise in today's era of globalization, which is characterized by hurry, worry, and curry. This review summarizes various disorders associated with increased gastric acid secretion and various therapeutic strategies to control them. The emphasis has been laid, in particular, on the role of proton pump inhibitors (PPIs) widely used nowadays for the treatment of gastric acid diseases. The medicinal chemistry aspects and mechanism of action of irreversible PPIs and APAs have been discussed at molecular levels. The ongoing research status in this field has also been covered. Further, biological evaluation methods that can be used for screening of PPIs are also discussed in short.     

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Microwave-Assisted Development of Orally Disintegrating Tablets by Direct Compression

Orally disintegrating tablets (ODTs) are challenged by the need for simple technology to ensure good mechanical strength coupled with rapid disintegration. The objective of this work was to evaluate microwave-assisted development of ODTs based on simple direct compression tableting technology. Placebo ODTs comprising directly compressible mannitol and lactose as diluents, super disintegrants, and lubricants were prepared by direct compression followed by exposure to >97% relative humidity and then microwave irradiation for 5 min at 490 W. Placebo ODTs with hardness (>5 kg/cm²) and disintegration time (<60 s) were optimized. Palatable ODTs of Lamotrigine (LMG), which exhibited rapid dissolution of LMG, were then developed. The stability of LMG to microwave irradiation (MWI) was confirmed. Solubilization was achieved by complexation with beta-cyclodextrin (β-CD). LMG ODTs with optimal hardness and disintegration time (DT) were optimized by a 2³ factorial design using Design Expert software. Taste masking using sweeteners and flavors was confirmed using a potentiometric multisensor-based electronic tongue, coupled with principal component analysis. Placebo ODTs with crospovidone as a superdisintegrant revealed a significant increase in hardness from ～3 to ～5 kg/cm² and a decrease in disintegration time (<60 s) following microwave irradiation. LMG ODTs had hardness >5 kg/cm², DT?<?30s, and rapid dissolution of LMG, and good stability was optimized by DOE and the design space derived. While β-CD complexation enabled rapid dissolution and moderate taste masking, palatability, which was achieved including flavors, was confirmed using an electronic tongue. A simple step of humidification enabled MWI-facilitated development of ODTs by direct compression presenting a practical and scalable advancement in ODT technology.