Overexpression of spermidine/spermine N 1-acetyltransferase or treatment with N 1-N 11-diethylnorspermine attenuates the severity of zinc-induced pancreatitis in mouse

Depletion of pancreatic intracellular polyamine pools has been observed in acute pancreatitis both in the animal models and in humans. In this study, the wild-type mice, polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase overexpressing (SSAT mice) and SSAT-deficient mice were used to characterize the new zinc-induced acute pancreatitis mouse model and study the role of polyamines and polyamine catabolism in this model. Intraperitoneal zinc injection induced acute necrotizing pancreatitis in wild-type mice as well as in SSAT-overexpressing and SSAT-deficient mice. Serum α-amylase activity was significantly increased in all zinc-treated mice compared with the untreated controls. However, the α-amylase activities in SSAT mice were constantly lower than those in the other groups. Histopathological examination of pancreatic tissue revealed edema, acinar cell necrosis and necrotizing inflammation, typical for acute pancreatitis. Compared with the other zinc-treated mice less damage according to the histopathological analysis was observed in the pancreatic tissue of SSAT mice. Levels of intracellular spermidine, and occasionally spermine, were significantly decreased in pancreases of all zinc-treated animals and SSAT enzyme activity was enhanced both in wild-type and SSAT mice. Interestingly, a spermine analog, N(1), N(11)-diethylnorspermine (DENSpm), enhanced the proliferation of pancreatic cells and reduced the severity of zinc-induced pancreatitis in wild-type mice. The results show that in mice a single intraperitoneal zinc injection causes acute necrotizing pancreatitis accompanied by decrease of intracellular polyamine pools. The study supports the important role of polyamines for the integrity and function of the pancreas. In addition, the study suggests that whole body overexpression of SSAT obtained in SSAT mice reduces inflammatory pancreatic cell injury.     

Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.     

Reliability of genetic bottleneck tests for detecting recent population declines

The identification of population bottlenecks is critical in conservation because populations that have experienced significant reductions in abundance are subject to a variety of genetic and demographic processes that can hasten extinction. Genetic bottleneck tests constitute an appealing and popular approach for determining if a population decline has occurred because they only require sampling at a single point in time, yet reflect demographic history over multiple generations. However, a review of the published literature indicates that, as typically applied, microsatellite-based bottleneck tests often do not detect bottlenecks in vertebrate populations known to have experienced declines. This observation was supported by simulations that revealed that bottleneck tests can have limited statistical power to detect bottlenecks largely as a result of limited sample sizes typically used in published studies. Moreover, commonly assumed values for mutation model parameters do not appear to encompass variation in microsatellite evolution observed in vertebrates and, on average, the proportion of multi-step mutations is underestimated by a factor of approximately two. As a result, bottleneck tests can have a higher probability of 'detecting' bottlenecks in stable populations than expected based on the nominal significance level. We provide recommendations that could add rigor to inferences drawn from future bottleneck tests and highlight new directions for the characterization of demographic history.     

Phylodynamics of H5N1 avian influenza virus in Indonesia

Understanding how pathogens invade and become established in novel host populations is central to the ecology and evolution of infectious disease. Influenza viruses provide unique opportunities to study these processes in nature because of their rapid evolution, extensive surveillance, large data sets and propensity to jump species boundaries. H5N1 highly pathogenic avian influenza virus (HPAIV) is a major animal pathogen and public health threat. The virus is of particular importance in Indonesia, causing severe outbreaks among poultry and sporadic human infections since 2003. However, little is known about how H5N1 HPAIV emerged and established in Indonesia. To address these questions, we analysed Indonesian H5N1 HPAIV gene sequences isolated during 2003-2007. We find that the virus originated from a single introduction into East Java between November 2002 and October 2003. This invasion was characterized by an initially rapid burst of viral genetic diversity followed by a steady rate of lineage replacement and the maintenance of genetic diversity. Several antigenic sites in the haemagglutinin gene were subject to positive selection during the early phase, suggesting that host-immune-driven selection played a role in host adaptation and expansion. Phylogeographic analyses show that after the initial invasion of H5N1, genetic variants moved both eastwards and westwards across Java, possibly involving long-distance transportation by humans. The phylodynamics we uncover share similarities with other recently studied viral invasions, thereby shedding light on the ecological and evolutionary processes that determine disease emergence in a new geographical region.     

Joint analysis of demography and selection in population genetics: where do we stand and where could we go?

Teasing apart the effects of selection and demography on genetic polymorphism remains one of the major challenges in the analysis of population genomic data. The traditional approach has been to assume that demography would leave a genome-wide signature, whereas the effect of selection would be local. In the light of recent genomic surveys of sequence polymorphism, several authors have argued that this approach is questionable based on the evidence of the pervasive role of positive selection and that new approaches are needed. In the first part of this review, we give a few empirical and theoretical examples illustrating the difficulty in teasing apart the effects of selection and demography on genomic polymorphism patterns. In the second part, we review recent efforts to detect recent positive selection. Most available methods still rely on an a priori classification of sites in the genome but there are many promising new approaches. These new methods make use of the latest developments in statistics, explore aspects of the data that had been neglected hitherto or take advantage of the emerging population genomic data. A current and promising approach is based on first estimating demographic and genetic parameters, using, e.g., a likelihood or approximate Bayesian computation framework, focusing on extreme outlier regions, and then using an independent method to confirm these. Finally, especially for species where evidence of natural selection has been limited, more experimental and versatile approaches that contrast populations under varied environmental constraints might be more successful compared with species-wide genome scans in search of specific signatures.     

Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis

Sudden exposure of plants to high light (HL) leads to metabolic and physiological disruption of the photosynthetic cells. Changes in ROS content, adjustment of photosynthetic processes and the antioxidant pools and, ultimately, gene induction are essential components for a successful acclimation to the new light conditions. The influence of salicylic acid (SA) on plant growth, short-term acclimation to HL, and on the redox homeostasis of Arabidopsis thaliana leaves was assessed here. The dwarf phenotype displayed by mutants with high SA content (cpr1-1, cpr5-1, cpr6-1, and dnd1-1) was less pronounced when these plants were grown in HL, suggesting that the inhibitory effect of SA on growth was partly overcome at higher light intensities. Moreover, higher SA content affected energy conversion processes in low light, but did not impair short-term acclimation to HL. On the other hand, mutants with low foliar SA content (NahG and sid2-2) were impaired in acclimation to transient exposure to HL and thus predisposed to oxidative stress. Low and high SA levels were strictly correlated to a lower and higher foliar H(2)O(2) content, respectively. Furthermore high SA was also associated with higher GSH contents, suggesting a tight correlation between SA, H(2)O(2) and GSH contents in plants. These observations implied an essential role of SA in the acclimation processes and in regulating the redox homeostasis of the cell. Implications for the role of SA in pathogen defence signalling are also discussed.     

Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris

Although an increasing number of studies show that many plant species have the capacity to take up amino acids from exogenous sources, the importance of such uptake for plant nitrogen nutrition is largely unknown. Moreover, little is known regarding metabolism and distribution of amino acid-N following uptake or of the regulation of these processes in response to plant nitrogen status. Here results are presented from a study following uptake, metabolism, and distribution of nitrogen from NO(3)(-) NH(4)(+), Glu, or Ala in Scots pine (Pinus sylvestris L). In a parallel experiment, Ala uptake, processing, and shoot allocation were also monitored following a range of pretreatments intended to alter plant C- and N-status. Uptake data, metabolite profiles, N fluxes through metabolite pools and tissues, as well as alanine aminotransferase activity are presented. The results show that uptake of the organic N sources was equal to or larger than NH(4)(+) uptake, while NO(3)(-) uptake was comparatively low. Down-regulation of Ala uptake in response to pretreatments with NH(4)NO(3) or methionine sulphoximine (MSX) indicates similarities between amino acid and inorganic N uptake regulation. N derived from amino acid uptake exhibited a rapid flux through the amino acid pool following uptake. Relative shoot allocation of amino acid-N was equal to that of NH(4)(+) but smaller than for NO(3)(-) Increased N status as well as MSX treatment significantly increased relative shoot allocation of Ala-N suggesting that NH(4)(+) may have a role in the regulation of shoot allocation of amino acid-N.     

A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA

We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.     

Antigen-specific accumulation of naïve, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy

Uveitis is an immune-mediated ocular disease and a leading cause of blindness. We characterized a novel model of uveitis with intravital microscopy. Transfer of ovalbumin-specific T cells from DO11.10 spleen to BALB/c recipients and subsequent challenge with ovalbumin in the anterior chamber of the eye resulted in anterior uveitis. Antigen-specificity was verified by injection of irrelevant antigen and transfer of T cells with a different specificity. Subsets of CD4 T cells, including naive (DO11.10 RAG(-/-)) and in vitro-activated Th2 effector CD4 T cells, infiltrated anterior segment tissues early in the inflammation. Memory-like CD44(high) CD4 T cells from unprimed transgenic mice and in vitro-activated Th1 effector CD4 T cells accumulated to larger numbers than naive or Th2 effector cells at 48 and 72 h. Of these, the alpha(2)-integrin+CD4 unprimed T cells entered the eye more efficiently, and antibody to alpha(2)-integrin markedly inhibited the inflammatory response. Intravital microscopy revealed the early arrival and antigen-specific accumulation of CD4 T cells in inflamed tissue and should be helpful in understanding T cell migration to other organs.     

Insulin-facilitated increase of muscle protein synthesis after resistance exercise involves a MAP kinase pathway

Recent studies have implicated the mTOR-signaling pathway as a primary component for muscle growth in mammals. The purpose of this investigation was to examine signaling pathways for muscle protein synthesis after resistance exercise. Sprague-Dawley rats (male, 6 mo old) were assigned to either resistance exercise or control groups. Resistance exercise was accomplished in operantly conditioned animals using a specially designed flywheel apparatus. Rats performed two sessions of resistance exercise, separated by 48 h, each consisting of 2 sets of 25 repetitions. Sixteen hours after the second session, animals were killed, and soleus muscles were examined for rates of protein synthesis with and without insulin and/or rapamycin (mTOR inhibitor) and/or PD-098059 (PD; MEK kinase inhibitor). Results of this study demonstrated that rates of synthesis were higher (P < 0.05) with insulin after exercise compared with without insulin, or to control muscles, regardless of insulin. Rapamycin lowered (P < 0.05) rates of synthesis in controls, with or without insulin, and after exercise without insulin. However, insulin was able to overcome the inhibition of rapamycin after exercise (P < 0.05). PD had no effect on protein synthesis in control rats, but the addition of PD to exercised muscle resulted in lower (P < 0.05) rates of synthesis, and this inhibition was not rescued by insulin. Western blot analyses demonstrated that the inhibitors used in the present study were selective and effective for preventing activation of specific signaling proteins. Together, these results suggest that the insulin-facilitated increase of muscle protein synthesis after resistance exercise requires multiple signaling pathways.