Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise.

Exhaustive exercise is associated with an increase in circulating glucocorticoids (GCs), lymphocyte apoptosis, and a reduction in intestinal lymphocyte number. The present study examined the role of GCs on the numerical changes seen in intestinal lymphocytes after exercise. Female C57BL/6 mice were bilaterally adrenalectomized (ADX; n = 18) or given sham surgery (Sham; n = 18) and assigned to one of three exercise conditions: treadmill running (28 m/min, 90 min, 2 degrees slope) and killed immediately or after 24 h recovery, or not exercised and killed immediately after 90-min exposure to the treadmill environment. Lymphocytes were isolated from the intestines with CD45(+) cells collected by positive selection using magnetic bead separation columns, and lymphocyte subpopulations were analyzed by flow cytometry for CD45(+), CD3alphabeta(+), CD3gammadelta(+), CD8beta(+), CD8alpha(+), CD4(+), and NK(+) phenotypic markers. ADX mice had significantly more intestinal CD45(+) leukocytes (P < 0.05) and CD3alphabeta(+) (P < 0.05), CD3gammadelta(+) (P < 0.01), CD8alpha(+) (P < 0.001), and NK(+) (P < 0.05) intestinal lymphocytes than Sham mice. There was a significant effect of exercise condition on total intestinal CD45(+) leukocytes (P < 0.01) and CD3alphabeta(+) (P < 0.05), CD8alpha(+) (P < 0.001), and CD4(+) (P < 0.05) intestinal lymphocytes, with fewer cells at 24 h postexercise compared with the other treatment conditions. There were no surgical x exercise interaction effects on the CD3 and CD8 phenotype numbers. Plasma corticosterone was virtually nil in ADX mice regardless of exercise condition but was significantly elevated in Sham mice immediately postexercise (P < 0.001). The data indicate that ADX does not prevent the loss of lymphocytes from the intestinal mucosa 24 h after strenuous exercise and GCs are not directly causal in the leukopenia of exercise.     

A modified assay of 3':5'-cyclic-AMP phosphodiesterase.

A modification of the assay of cyclic nucleotide phosphodiesterase involving batch use of Dowex 1 anion exchange resin is described which allows for quantitative recovery of adenosine, guanosine, and their metabolites from the resin slurry. The assay described is suitable for use in crude preparations containing purine catabolizing enzymes. A standardized procedure for determining kinetic parameters of cyclic AMP hydrolysis is also discussed. This procedure was used in the partial characterization of the kinetics of cyclic AMP hydrolysis by rat and rabbit heart supernatant fractions.     

Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

Variants of the <Emphasis Type="Italic">EAAT2</Emphasis> Glutamate Transporter Gene Promoter Are Associated with Cerebral Palsy in Preterm Infants

Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns’ dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181 bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies.     

Testate amoeba records indicate regional 20th‐century lowering of water tables in ombrotrophic peatlands in central‐northern Alberta,Canada

Testate amoebae are abundant in the surface layers of northern peatlands. Analysis of their fossilized shell (test) assemblages allows for reconstructions of local water‐table depths (WTD). We have reconstructed WTD dynamics for five peat cores from peatlands ranging in distance from the Athabasca bituminous sands (ABS) region in western Canada. Amoeba assemblages were combined with plant macrofossil records, acid‐insoluble ash (AIA) fluxes and instrumental climate data to identify drivers for environmental change. Two functional traits of testate amoebae, mixotrophy and the tendency to integrate xenogenic mineral matter in test construction, were quantified to infer possible effects of AIA flux on testate amoeba presence. Age–depth models showed the cores each covered at least the last ~315 years, with some spanning the last millennium. Testate amoeba assemblages were likely affected by permafrost development in two of the peatlands, yet the most important shift in assemblages was detected after 1960 CE. This shift represents a significant apparent lowering of water tables in four out of five cores, with a mean drop of ~15 cm. Over the last 50 years, assemblages shifted towards more xerophilous taxa, a trend which was best explained by increasing Sphagnum s. Acutifolia and, to a lesser extent, mean summer temperature. This trend was most evident in the two cores from the sites located farthest away from the ABS region. AIA flux variations did not show a clear effect on mineral‐agglutinating taxa, nor on S. s. Acutifolia presence. We therefore suggest the drying trend was forced by the establishment of S. s. Acutifolia, driven by enhanced productivity following regional warming. Such recent apparent drying of peatlands, which may only be reconstructed by appropriate indicators combined with high chronological control, may affect vulnerability to future burning and promote emissions of CO₂.     

New antitumor leads from a peptidomimetic library

A parallel combinatorial library of over 1600 compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads. These peptidomimetic molecules are aimed at intervening with the substrate binding site of the pp60^c-src enzyme. The new structures were based on known PTK inhibitors with at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl-type inhibitory compounds were found in the range of 18–100 μM IC₅₀ concentrations from combinations of 12 different substituents. Molecular modeling of the active compounds showed a characteristic distance of 12–14 Å between the farthest sp² carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60^c-src PTK showed that the energy-minimized conformers had the same distance between the two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis-inducing effect on HT-29 human colon tumor cells.     

Expression of functionally distinct variants of the beta(4)A integrin subunit in relation to the differentiation state in human intestinal cells.

Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin alpha(6)beta(4), which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the beta(4) subunit expression with different antibodies revealed that two forms of beta(4) exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa beta(4)A subunit, whereas undifferentiated crypt cells express a novel beta(4)A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (beta(4)A(ctd-)). This new form was not found to arise from alternative beta(4) mRNA splicing. Moreover, we found that these two beta(4)A forms can associate into alpha(6)beta(4)A complexes; however, the beta(4)A(ctd-) integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel alpha(6)beta(4)A(ctd-) integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.     

Subcellular Distribution of Cadmium and Nickel in Chronically Exposed Wild Fish: Inferences Regarding Metal Detoxification Strategies and Implications for Setting Water Quality Guidelines for Dissolved Metals

The objective of this study was to investigate metal detoxification in chronically exposed juvenile yellow perch (YP: Perca flavescens) and to field test the commonly assumed threshold toxicity model. Fish were collected from lakes located along a cadmium (Cd) and nickel (Ni) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Hepatic metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of YP liver homogenates. Major proportions of hepatic Cd were found in the heat-stable cytosolic peptides and proteins fraction (HSP; including metallothioneins), whereas Ni was mainly found in the potentially metal-sensitive heat-denaturable proteins fraction (HDP). For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction or the organelle fraction did not occur. Metal detoxification was clearly incomplete and P. flavescens was subject to some metal-related stress, as evidenced notably by endocrine perturbations. Similar subcellular partitioning results were obtained when juvenile yellow perch were transferred from a reference lake to a Cd-contaminated lake and Cd accumulation was followed over time; there was no accumulation threshold below which Cd binding to the putative metal-sensitive fractions (HDP and organelles) did not occur. The presence of Cd and Ni in these fractions, even for low exposure concentrations and low hepatic accumulation, contradicts the threshold toxicity model that underpins metal toxicology theory and that is implicitly used in setting water quality guidelines for metals. Chronically exposed YP appear to have settled for a tradeoff between the cost of turning on their detoxification apparatus at full capacity, to completely suppress metal binding to metal-sensitive sites, and the alternative cost of allowing some binding of inappropriate metals to metal-sensitive sites.     

New cyclic somatostatin analogues containing a pyrazinone ring: importance of Tyr for antiproliferative activity

Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antiproliferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-D-Trp-Lys, with more hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicating that Tyr residue was an essential residue.