Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.     

A comprehensive breath plume model for disease transmission via expiratory aerosols

The peak in influenza incidence during wintertime in temperate regions represents a longstanding, unresolved scientific question. One hypothesis is that the efficacy of airborne transmission via aerosols is increased at lower humidities and temperatures, conditions that prevail in wintertime. Recent work with a guinea pig model by Lowen et al. indicated that humidity and temperature do modulate airborne influenza virus transmission, and several investigators have interpreted the observed humidity dependence in terms of airborne virus survivability. This interpretation, however, neglects two key observations: the effect of ambient temperature on the viral growth kinetics within the animals, and the strong influence of the background airflow on transmission. Here we provide a comprehensive theoretical framework for assessing the probability of disease transmission via expiratory aerosols between test animals in laboratory conditions. The spread of aerosols emitted from an infected animal is modeled using dispersion theory for a homogeneous turbulent airflow. The concentration and size distribution of the evaporating droplets in the resulting "Gaussian breath plume" are calculated as functions of position, humidity, and temperature. The overall transmission probability is modeled with a combination of the time-dependent viral concentration in the infected animal and the probability of droplet inhalation by the exposed animal downstream. We demonstrate that the breath plume model is broadly consistent with the results of Lowen et al., without invoking airborne virus survivability. The results also suggest that, at least for guinea pigs, variation in viral kinetics within the infected animals is the dominant factor explaining the increased transmission probability observed at lower temperatures.     

Mass spectrometric characterization of the sarcoplasmic reticulum from rabbit skeletal muscle by on-membrane digestion

The sarcoplasmic reticulum from skeletal muscle constitutes an elaborate membrane system that contains a considerable number of integral and very large proteins that exist in highly complex supramolecular clusters. Conventional proteomics using two-dimensional gel electrophoresis greatly underestimates the presence of these proteins. Here, we have applied one-dimensional gradient gels and on-membrane digestion to overcome this technical problem. Mass spectrometric analysis has determined the presence of 31 distinct protein species in the sarcoplasmic reticulum, including key Ca2+-handling proteins such as the ryanodine receptor, Ca2+-ATPase, calsequestrin and sarcalumenin. Immunoblotting confirmed the relative position of these Ca2+-regulatory elements in analytical gel replicas. Interestingly, aldolase and phosphofructokinase were found to be present in the purified sarcoplasmic reticulum, supporting the idea of a close physical coupling between the glycolytic pathway and the energy-dependent sarcoplasmic reticulum. Hence, on-membrane digestion is highly suitable as the method of choice for studying integral and high-molecular-mass proteins in proteomic studies.     

Single dose oral dexamethasone versus multi-dose prednisolone in the treatment of acute exacerbations of asthma in children who attend the emergency department: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Asthma is a major cause of pediatric morbidity and mortality. In acute exacerbations of asthma, corticosteroids reduce relapses, subsequent hospital admission and the need for Ss2-agonist therapy. Prednisolone is relatively short-acting with a half-life of 12 to 36 hours, thereby requiring daily dosing. Prolonged treatment course, vomiting and a bitter taste may reduce patient compliance with prednisolone. Dexamethasone is a long-acting corticosteroid with a half-life of 36 to 72 hours. It is used frequently in children with croup and bacterial meningitis, and is well absorbed orally.The purpose of this trial is to examine whether a single dose of oral dexamethasone (0.3 mg/kg) is clinically non-inferior to prednisolone (1 mg/kg/day for three days) in the treatment of exacerbations of asthma in children who attend the Emergency Department.Methods/designThis is a randomized, non-inferiority, open-label clinical trial. After informed consent with or without assent, patients will be randomized to either oral dexamethasone 0.3 mg/kg stat or prednisolone 1 mg/kg/day for three days. The primary outcome measure is the comparison between the Pediatric Respiratory Assessment Measure (PRAM) across both groups on Day 4. The PRAM score, a validated, responsive and reliable tool to determine asthma severity in children aged 2 to 16 years, will be performed by a clinician blinded to treatment allocation. Secondary outcomes include relapse, hospital admission and requirement for further steroid therapy. Data will be analyzed on an intention-to-treat and a per protocol basis. With a sample size of 232 subjects (105 in each group with an estimated 10% loss to follow-up), we will be able to reject the null hypothesis - that the population means of the experimental and control groups are equal with a probability (power) of 0.9. The Type I error probability associated with this test (of the null hypothesis) is 0.05. DISCUSSION: This clinical trial may provide evidence that a shorter steroid course using dexamethasone can be used in the treatment of acute pediatric asthma, thus eliminating the issue of compliance to treatment.RegistrationISRCTN26944158 and EudraCT Number 2010-022001-18.     

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster ‘protects’ against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.     

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains ^4–5FnI and ³FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes ³FnIII and disrupts the observed ^4–5FnI-³FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.Fibronectin (FN),4 a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2–4). Although interactions between domains that are close in primary sequence have been demonstrated (3, 5), studies of multidomain fragments generally assume a beads-on-string model (2). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5–8), sites of protein interactions (9), and sites that control cellular activity (10, 11). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12–14); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15–17) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18) has also not been straightforward (2), possibly because of domain stabilization through nearest-neighbor effects (19, 20).A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10, 21) and breast carcinoma cells using the Boyden chamber (22). MSF comprises FN domains ¹FnI to ⁹FnI, a truncated ¹FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to ¹FnI-⁹FnI (Fn70kDa) displays the same activity (10). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains ⁶FnI-⁹FnI) (23) and partial activity to a shorter fragment spanning domains ^7–9FnI (24). Two IGD tripeptides of domains ⁷FnI and ⁹FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10, 24, 25); however, similar IGD tripeptides outside the GBD, on domains ³FnI and ⁵FnI, appear to have little effect (10, 23). Full-length adult FN does not affect cell migration in similar assays (10, 23); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement.Open in a separate windowFIGURE 1.Motogenic activity of FN fragments. a, schematic representation of the FN domain structure (top) and enlargement of the FN N terminus (bottom). Type I domains are shown as pentagons; type II domains as hexagons; and type III domains as ovals. b, comparison of motogenic activity versus protein concentration of wild-type Fn70kDa and Fn100kDa fragments. Error bars are derived from duplicate experiments, and a gray band denotes migration activity of media without additives. c, similar comparisons for mutant Fn100kDa fragments. d, analytical size exclusion chromatography of large FN fragments. The trace of UV absorbance at 280 nm versus elution volume shown here indicates a larger hydrodynamic radius for Fn100kDa R222A compared with the wild type, consistent with our model (Fig. 6a).Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26), as well as amphibians, birds, and mammals (27), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (^1–3FnIII), suggesting that these domains are responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains ^4–5FnI and ³FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences.     

Distinct subsets of dendritic cells regulate the pattern of acute xenograft rejection and susceptibility to cyclosporine therapy

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c(+)CD8alpha(+) splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c(+)CD8alpha(-) splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c(+)IL-12(+) splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8alpha(-) bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8alpha(+) bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 +/- 0.6 days to 14.2 +/- 0.8 days. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.     

scid cells efficiently integrate hairpin and linear DNA substrates.

The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.