Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister-daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

Requirement of the exopolyphosphatase gene for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.

Genetic and Modifiable Risk Factors Contributing to Cisplatin-induced Toxicities.

Effective administration of traditional cytotoxic chemotherapy is often limited by off-target toxicities. This clinical dilemma is epitomized by cisplatin, a platinating agent, which has potent antineoplastic activity due to its affinity for DNA and other intracellular nucleophiles. Despite its efficacy against many adult-onset and pediatric malignancies, cisplatin elicits multiple off-target toxicities that can not only severely impact a patient's quality of life but also lead to dose reductions or the selection of alternative therapies that can ultimately affect outcomes. Without an effective therapeutic measure by which to successfully mitigate many of these symptoms, there have been attempts to identify a priori those individuals who are more susceptible to developing these sequelae through studies of genetic and nongenetic risk factors. Older age is associated with cisplatin-induced ototoxicity, neurotoxicity, and nephrotoxicity. Traditional genome-wide association studies have identified single-nucleotide polymorphisms in ACYP2 and WFS1 associated with cisplatin-induced hearing loss. However, validating associations between specific genotypes and cisplatin-induced toxicities with enough stringency to warrant clinical application remains challenging. This review summarizes the current state of knowledge with regard to specific adverse sequelae following cisplatin-based therapy, with a focus on ototoxicity, neurotoxicity, nephrotoxicity, myelosuppression, and nausea/emesis. We discuss variables (genetic and nongenetic) contributing to these detrimental toxicities and currently available means to prevent or treat their occurrence.

In vivo identification of novel TGIF2LX target genes in colorectal adenocarcinoma using the cDNA-AFLP method.

BACKGROUND AND STUDY AIMS: Homeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene(s) of TGIF2LX in colorectal adenocarcinoma. METHODS: A human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice (n = 6 per group). The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. RESULTS: The real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 (Nir1) gene was suppressed whereas Nir2 and fragile histidine triad (FHIT) genes were upregulated followed by the overexpression of TGIF2LX gene. CONCLUSION: Downregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma.

Reduction in squamous cell carcinomas in mouse skin by dietary zinc supplementation.

Inadequate dietary Zn consumption increases susceptibility to esophageal and other cancers in humans and model organisms. Since Zn supplementation can prevent cancers in rodent squamous cell carcinoma (SCC) models, we were interested in determining if it could have a preventive effect in a rodent skin cancer model, as a preclinical basis for considering a role for Zn in prevention of human nonmelanoma skin cancers, the most frequent cancers in humans. We used the 7,12-dimethyl benzanthracene carcinogen/phorbol myristate acetate tumor promoter treatment method to induce skin tumors in Zn-sufficient wild-type and Fhit (human or mouse protein) knockout mice. Fhit protein expression is lost in >50% of human cancers, including skin SCCs, and Fhit-deficient mice show increased sensitivity to carcinogen induction of tumors. We hypothesized that: (1) the skin cancer burdens would be reduced by Zn supplementation; (2) Fhit(-/-) (Fhit, murine fragile histidine triad gene) mice would show increased susceptibility to skin tumor induction versus wild-type mice. 30 weeks after initiating treatment, the tumor burden was increased ~2-fold in Fhit(-/-) versus wild-type mice (16.2 versus 7.6 tumors, P < 0.001); Zn supplementation significantly reduced tumor burdens in Fhit(-/-) mice (males and females combined, 16.2 unsupplemented versus 10.3 supplemented, P = 0.001). Most importantly, the SCC burden was reduced after Zn supplementation in both strains and genders of mice, most significantly in the wild-type males (P = 0.035). Although the mechanism(s) of action of Zn supplementation in skin tumor prevention is not known in detail, the Zn-supplemented tumors showed evidence of reduced DNA damage and some cohorts showed reduced inflammation scores. The results suggest that mild Zn supplementation should be tested for prevention of skin cancer in high-risk human cohorts.

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway.

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640-1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins.

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Anti-carcinogenic effects of the phenolic-rich extract from abnormal Savda Munziq in association with its cytotoxicity, apoptosis-inducing properties and telomerase activity in human cervical cancer cells (SiHa).

BACKGROUND: Abnormal Savda Munziq (ASMq) is a herbal preparation used in Traditional Uighur Medicine for the treatment cancer. The polyphenol is main compounds contained in ASMq preparation responsible for anticancer effect of ASMq. METHODS: In this study,Real-time quantitative Polymerase Chain Reaction (RT-PCR) assay, MTT assay and flow cytometry were used to investigate the effect of polyphenol of ASMq on cell viability and the potential of the phenolic rich extracts of ASMq to induce apoptosis in human cervical cancer cells SiHa and its effects on telomerase activity were investigated. Cellular morphological change was observed by phase contrast microscopy. The MTT cell viability data revealed that treatment with phenolic rich extracts at 75 ~ 175 µg/ml significantly inhibited the viability and proliferation of cells, and these effects occurred in a concentration-dependent manner and time dependent manner (P < 0.01). RESULTS: The phenolic rich extracts can induce apoptosis of SiHa cells, can increase the apoptosis rate in a concentration-dependent manner and time dependent manner (P < 0.01). Growth inhibition and apoptosis induction by phenolic rich extracts treatment on SiHa cells was associated with down-regulation of anti-apoptotic Bcl-2 expression and telomerase (P < 0.05) and Survivin expression. In addition, phenolic rich extracts exerted a dose-dependent induction of FHIT expression. CONCLUSION: These results suggest that phenolic rich extracts may have anti-tumor effects in human cervical cancer through cytotoxicity, apoptosis-inducing properties and telomerase activity.

A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Temperature influence on biological phosphorus removal induced by aerobic/extended-idle regime.

Previous researches have demonstrated that biological phosphorus removal (BPR) from wastewater could be driven by the aerobic/extended-idle (A/EI) regime. This study further investigated temperature effects on phosphorus removal performance in six A/EI sequencing batch reactors (SBRs) operated at temperatures ranging from 5 to 30 °C. The results showed that phosphorus removal efficiency increased with temperature increasing from 5 to 20 °C but slightly decreased when temperature continually increased to 30 °C. The highest phosphorus removal rate of 97.1 % was obtained at 20 °C. The biomass cultured at 20 °C contained more polyphosphate accumulating organisms (PAO) and less glycogen accumulating organisms (GAO) than that cultured at any other temperatures investigated. The mechanism studies revealed that temperature affected the transformations of glycogen and polyhydroxyalkanoates, and the activities of exopolyphosphatase and polyphosphate kinase activities. In addition, phosphorus removal performances of the A/EI and traditional anaerobic/oxic (A/O) SBRs were compared at 5 and 20 °C, respectively. The results showed the A/EI regime drove better phosphorus removal than the A/O regime at both 5 and 20 °C, and more PAO and less GAO abundances in the biomass might be the principal reason for the higher BPR in the A/EI SBRs as compared with the A/O SBRs.

Topical hypochlorite ameliorates NF-κB-mediated skin diseases in mice.

Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB-dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB-driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB-dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB-mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims.

Synergistic anticancer activity of arsenic trioxide with erlotinib is based on inhibition of EGFR-mediated DNA double-strand break repair.

Arsenic trioxide (ATO), one of the oldest remedies used in traditional medicine, was recently rediscovered as an anticancer drug and approved for treatment of relapsed acute promyelocytic leukemia. However, its activity against nonhematologic cancers is rather limited so far. Here, we show that inhibition of ATO-mediated EGF receptor (EGFR) activation can be used to potently sensitize diverse solid cancer types against ATO. Thus, combination of ATO and the EGFR inhibitor erlotinib exerted synergistic activity against multiple cancer cell lines. Subsequent analyses revealed that this effect was based on the blockade of ATO-induced EGFR phosphorylation leading to more pronounced G2-M arrest as well as enhanced and more rapid induction of apoptosis. Comparable ATO-sensitizing effects were also found with PI3K/AKT and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, suggesting an essential role of the EGFR-mediated downstream signaling pathway in cancer cell protection against ATO. H2AX staining and comet assay revealed that erlotinib significantly increases ATO-induced DNA double-strand breaks (DSB) well in accordance with a role of the EGFR signaling axis in DNA damage repair. Indeed, EGFR inhibition led to downregulation of several DNA DSB repair proteins such as Rad51 and Rad50 as well as reduced phosphorylation of BRCA1. Finally, the combination treatment of ATO and erlotinib was also distinctly superior to both monotreatments against the notoriously therapy-resistant human A549 lung cancer and the orthotopic p31 mesothelioma xenograft model in vivo. In conclusion, this study suggests that combination of ATO and EGFR inhibitors is a promising therapeutic strategy against various solid tumors harboring wild-type EGFR.

The impacts of silver nanoparticles and silver ions on wastewater biological phosphorous removal and the mechanisms.

The effects of AgNPs and Ag+ (0-5 mg/L) on wastewater enhanced biological phosphorus removal (EBPR) were investigated. The phosphorus removal efficiency was maintained at 99% no matter what the concentrations of AgNPs were, but it was decreased to 48.8% at Ag+ of 1 mg/L, and no net phosphorus was removed at Ag+ greater than 2 mg/L in the batch tests. The uptake of wastewater carbon source and the anaerobic and aerobic transformations of phosphorus, polyhydoxyalkanoates and glycogen were inhibited by Ag+ other than AgNPs. Ag+ showed stronger toxicity on polyphosphate accumulating organisms (PAOs) than glycogen accumulating organisms (GAOs). Further investigation revealed that the decrease in anaerobic phosphorus release by Ag+ was caused by the inhibition on the activities of adenylate kinase and expolyphosphatase, whereas the decrease in aerobic phosphorus uptake was due to the suppression in energy generation for phosphorus uptake. However, the presence of either AgNPs or Ag+ did not lead to cell leakage and membrane damage.

Heat induces gene amplification in cancer cells.

BACKGROUND: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. MATERIALS AND METHODS: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44°C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) γH2AX immunostaining to detect γH2AX foci as an indication for DNA double strand breaks. RESULTS: (1) Heat exposure at 42 or 44°C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44°C for 30 min induces DNA double strand breaks in HCT116 cells as shown by γH2AX immunostaining. CONCLUSION: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the ß-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

Effect of zinc supplementation on N-nitrosomethylbenzylamine-induced forestomach tumor development and progression in tumor suppressor-deficient mouse strains.

Zinc deficiency is associated with high incidences of esophageal and other cancers in humans and leads to a highly proliferative hyperplastic condition in the upper gastrointestinal tract in laboratory rodents. Zn replenishment reduces the incidence of lingual, esophageal and forestomach tumors in Zn-deficient rats and mice. While previous animal studies focused on Zn deficiency, we have investigated the effect of Zn supplementation on carcinogenesis in Zn-sufficient mice of wild-type and tumor suppressor-deficient mouse strains. All mice received N-nitrosomethylbenzylamine and half the mice of each strain then received Zn supplementation. At killing, mice without Zn supplementation had developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice developed an average of 7.0 versus 5.0 tumors for Zn supplemented (P < 0.05); Zn-supplemented Fhit-/- mice averaged 5.7 versus 8.0 for control mice (P < 0.01); Zn-supplemented Fhit-/-Nit1-/- mice averaged 5.4 versus 9.2 for control mice (P < 0.01) and Zn-supplemented Fhit-/-Rassf1a-/- (the murine gene) mice averaged 5.9 versus 9.1 for control mice (P < 0.01). Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit-/-Nit1-/-). Histological analysis of forestomach tissues also showed significant decreases in severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse strain. Thus, Zn supplementation significantly reduced tumor burdens in mice with multiple tumor suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final carcinogen dose, the reduction in tumor burden was the same as observed when supplementation began immediately after carcinogen dosing, suggesting that Zn supplementation may affect tumor progression rather than tumor initiation.

Development and validation of a high-density fluorescence polarization-based assay for the trypanosoma RNA triphosphatase TbCet1.

RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.

Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity.

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM. Diadenosine tetraphosphate (Ap(4)A) and diethenoadenosine tetraphosphate, epsilon-(Ap(4)A), displaced the radioligand, indicating dinucleotide binding to ADPbetaS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap(4)A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap(4)A signalling mediated by P2Y(1,12,13) receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain.