Regulation of amylin release from cultured rabbit gastric fundic mucosal cells

Background  

Amylin (islet amyloid polypeptide) is a hormone with suggested roles in the regulation of glucose homeostasis, gastric motor and secretory function and gastroprotection. In the gastric mucosa amylin is found co-localised with somatostatin in D-cells. The factors regulating gastric amylin release are unknown. In this study we have investigated the regulation of amylin release from gastric mucosal cells in primary culture. Rabbit fundic mucosal cells enriched for D-cells by counterflow elutriation were cultured for 40 hours. Amylin and somatostatin release over 2 hours in response to agonists were assessed.     

Visualization of glycosaminoglycans in rat incisor extracellular matrix using a hyaluronidase-gold complex

Summary The enzyme-gold technique was used on dental tissues. Hyaluronidase was complexed with gold, and ultrathin sections of rat incisors were incubated with the hyaluronidase-gold complex to localize chondroitin-sulphate and hyaluronic acid at the ultrastructural level. The hyaluronidase-gold complex was, found in predentine and dentine, especially at the mineralization front, in interglobular spaces and around dentinal canaficuli. The very young enamel was labelled, but not the later stages of formation. This method allowed a very precise localization of hyaluronic acid and/or chondroitin sulphate in rat incisors extracellular matrices. These findings support the important role of glycosaminoglycans in dentine mineralization.     

Heroin addiction in African Americans: a hypothesis-driven association study

Heroin addiction is a chronic complex disease with a substantial genetic contribution. This study was designed to identify gene variants associated with heroin addiction in African Americans. The emphasis was on genes involved in reward modulation, behavioral control, cognitive function, signal transduction and stress response. We have performed a case–control association analysis by screening with 1350 variants of 130 genes. The sample consisted of 202 former severe heroin addicts in methadone treatment and 167 healthy controls with no history of drug abuse. Single nucleotide polymorphism (SNP), haplotype and multi-SNP genotype pattern analyses were performed. Seventeen SNPs showed point-wise significant association with heroin addiction (nominal P < 0.01). These SNPs are from genes encoding several receptors: adrenergic ( ADRA1A ), arginine vasopressin ( AVPR1A ), cholinergic ( CHRM2 ), dopamine (DRD1 ), GABA-A ( GABRB3 ), glutamate ( GRIN2A ) and serotonin ( HTR3A ) as well as alcohol dehydrogenase ( ADH7 ), glutamic acid decarboxylase ( GAD1 and GAD2 ), the nucleoside transporter ( SLC29A1 ) and diazepam-binding inhibitor ( DBI ). The most significant result of the analyses was obtained for the GRIN2A haplotype G-A-T (rs4587976-rs1071502-rs1366076) with protective effect ( P _uncorrected = 9.6E- 05, P _corrected = 0.058). This study corroborates several reported associations with alcohol and drug addiction as well as other related disorders and extends the list of variants that may affect the development of heroin addiction. Further studies will be necessary to replicate these associations and to elucidate the roles of these variants in drug addiction vulnerability.     

Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N′-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC₅₀ = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.     

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication

The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.     

Mitochondrial uncoupling protein‐2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age‐associated lung fibrosis

Mitochondrial dysfunction has been associated with age‐related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein‐2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro‐oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro‐fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age‐related diseases associated with impaired tissue regeneration and organ fibrosis.     

Imaging of an Inflammatory Injury in the Newborn Rat Brain with Photoacoustic Tomography

Background

The precise assessment of cerebral saturation changes during an inflammatory injury in the developing brain, such as seen in periventricular leukomalacia, is not well defined. This study investigated the impact of inflammation on locoregional cerebral oxygen saturation in a newborn rodent model using photoacoustic imaging.

Methods

1 mg/kg of lipopolysaccharide(LPS) diluted in saline or saline alone was injected under ultrasound guidance directly in the corpus callosum of P3 rat pups. Coronal photoacoustic images were carried out 24 h after LPS exposure. Locoregional oxygen saturation (SO₂) and resting state connectivity were assessed in the cortex and the corpus callosum. Microvasculature was then evaluated on cryosection slices by lectin histochemistry.

Results

Significant reduction of SO₂ was found in the corpus callosum; reduced SO₂ was also found in the cortex ipsilateral to the injection site. Seed-based functional connectivity analysis showed that bilateral connectivity was not affected by LPS exposure. Changes in locoregional oxygen saturation were accompanied by a significant reduction in the average length of microvessels in the left cortex but no differences were observed in the corpus callosum.

Conclusion

Inflammation in the developing brain induces marked reduction of locoregional oxygen saturation, predominantly in the white matter not explained by microvascular degeneration. The ability to examine regional saturation offers a new way to monitor injury and understand physiological disturbance non-invasively.     

Evidence for expression of a single distinct form of mammalian cysteine dioxygenase

Summary. Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO (23kDa, 25kDa, and 68kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his₆-tagged CDO (r-his₆-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the 25kDa band, but never detected a 68kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his₆-CDO yielded a molecular mass estimate of 25.7kDa and no evidence of dimerization. Mass spectrometry of r-his₆-CDO yielded two peaks with molecular masses of 24.1kDa and 24.3kDa. Anion-exchange FPLC of r-his₆-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the 23kDa band. We conclude that the physiologically active form of CDO is the 25kDa (i.e., 23.5kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23kDa gene product.     

Linkage drag constrains the roots of modern wheat

Roots, the hidden half of crop plants, are essential for resource acquisition. However, knowledge about the genetic control of below‐ground plant development in wheat, one of the most important small‐grain crops in the world, is very limited. The molecular interactions connecting root and shoot development and growth, and thus modulating the plant's demand for water and nutrients along with its ability to access them, are largely unexplored. Here, we demonstrate that linkage drag in European bread wheat, driven by strong selection for a haplotype variant controlling heading date, has eliminated a specific combination of two flanking, highly conserved haplotype variants whose interaction confers increased root biomass. Reversing this inadvertent consequence of selection could recover root diversity that may prove essential for future food production in fluctuating environments. Highly conserved synteny to rice across this chromosome segment suggests that adaptive selection has shaped the diversity landscape of this locus across different, globally important cereal crops. By mining wheat gene expression data, we identified root‐expressed genes within the region of interest that could help breeders to select positive variants adapted to specific target soil environments.