The chromogranin A peptide vasostatin-I inhibits gap formation and signal transduction mediated by inflammatory agents in cultured bovine pulmonary and coronary arterial endothelial cells

The proinflammatory agent tumour necrosis factor alpha (TNFalpha) is one of several agents causing vascular leakage. The N-terminal domain of CgA, vasostatin-I (CgA1-76), has recently been reported to inhibit TNFalpha induced gap formation in human umbilical venous endothelial cells. Here we report on the effect of recombinant human CgA1-78, vasostatin-I, on TNFalpha induced gap formation in two model systems of vascular leakage in arterial endothelial cells of bovine pulmonary (BPAEC) and coronary (BCAEC) origin. Vasostatin-I inhibited the TNFalpha induced gap formation in both models, being inactive in the unstimulated cells. The phosphorylation of p38MAP kinase in TNFalpha activated BPAEC was markedly attenuated in the presence of vasostatin-I and the inhibitory effect corresponded to that of the specific p38MAPK inhibitor SB203580. Vasostatin-I also inhibited the phosphorylation of p38MAPK induced by both thrombin and pertussis toxin in these cells. The results demonstrate that vasostatin-I has inhibitory effects on TNFalpha-induced disruption of confluent layers of cultured pulmonary and coronary arterial endothelial cells. This suggests that vasostatin-I may affect endothelial barrier dysfunction also in arterial vascular beds. Furthermore, the inhibitory activity of vasostatin-I may be associated with the p38MAPK signalling cascade via a pertussis toxin sensitive, presumably Galphai coupled mechanism.     

Phosphoproteomic Analysis of Neurotrophin Receptor TrkB Signaling Pathways in Mouse Brain

SUMMARY 1. The signaling pathways activated by trkB neurotrophin receptor have been studied in detail in cultured neurons, but little is known about the pathways activated by trkB in intact brain. TrkB is a tyrosine kinase and protein phosphorylation is a key regulatory process in the neuronal signal transduction pathways.2. We have investigated trkB signaling in the transgenic mice overexpressing trkB in postnatal neurons (trkB.TK) using phosphoproteomics.3. We found that several proteins are overphosphorylated on tyrosine residues in the brain of trkB.TK mice and identified some of these proteins.4. We demonstrate that the well characterized signaling molecules mitogen-activated protein kinase (MAPK) and cyclic AMP responsive element binding protein (CREB) were phosphorylated at a higher level in the brain of trkB.TK mice when compared to the wild type littermates. Furthermore, we found that β-actin was tyrosine phosphorylated in the brain of the transgenic mice.5. Our results demonstrate that phosphoproteomics is a sensitive approach to investigate signaling pathways activated in mouse brain.     

Molecular genotype identification of the Gallus gallus major histocompatibility complex

The chicken major histocompatibility complex (MHC) is commonly defined by serologic reactions of erythrocytes with antibodies specific to the highly polymorphic MHC class I (BF) and MHC class IV (BG) antigens. The microsatellite marker LEI0258 is known to be physically located within the MHC, between the BG and BF regions. DNA from various serologically defined MHC haplotypes was amplified by polymerase chain reaction with primers surrounding this marker. Twenty-six distinctive allele sizes were identified. Some serologically well-defined MHC haplotypes shared a common LEI0258 allele size but could be distinguished either by the addition of information from another nearby marker (MCW0371) or by small indels or single nucleotide polymorphism (SNP) differences between the alleles. The association between LEI0258 allele and serologically defined MHC haplotype was very consistent for the same haplotype from multiple sources. Sequence information for the region defined by LEI0258 was obtained for 51 different haplotypes. Two internal repeats whose lengths were 13 and 12 bp, respectively, are the primary basis for allelic variability. Allele size variation ranges from 182 to 552 bp. Four indels and five SNPs in the surrounding sequence provide additional means for distinguishing alleles. Typing with LEI0258 and MCW0371 will be useful in identifying MHC haplotypes in outbred populations of chickens particularly for the initial development of serological reagents.     

REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.     

Chromatin signatures of pluripotent cell lines

Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.     

Mapping of the Interaction Site between Sortilin and the p75 Neurotrophin Receptor Reveals a Regulatory Role for the Sortilin Intracellular Domain in p75 Neurotrophin Receptor Shedding and Apoptosis

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75^NTR). To identify the interaction site between sortilin and p75^NTR, we analyzed binding between chimeric receptor constructs and truncated p75^NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75^NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75^NTR. Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75^NTR-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75^NTR binding, it does regulate the rates of p75^NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K_d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC₅₀ values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.     

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-13C]glucose was used to monitor metabolism. Brain levels of 13C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived 13C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.