A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.     

Reproductive and Neurobehavioral Effects of Brilliant Blue FCF in Mice

Brilliant blue FCF of food color was given in the diets of mice at levels of 0% (control), 0.08, 0.24, and 0.72% from 5 weeks of age in the F₀ generation and continuing to 11 weeks of age in the F₁ generation and selected reproductive and neurobehavioral parameters were measured. Mice were mated at 9 weeks of age and dams were delivered offspring at 12 weeks of age. Offspring were weaned at 4 weeks of age. Regarding exploratory behavior at 8 weeks of age in the F₀ generation, movement time (sec) displayed a significant tendency to be increased and the average time of rearing (sec) displayed a significant tendency to be decreased in females in the treatment groups in a trend test (p = 0.019 and 0.027, respectively). In the F₁ generation, the development of surface righting at postnatal day 4 was delayed significantly in the high‐dose group (0.72%) in male and female offspring, and those effects were significantly related to dose in a trend test (p< 0.01 for both). Regarding exploratory behavior at 8 weeks of age in the F₁ generation, the number of horizontal activities exhibited a significant tendency to be decreased in females in the treatment groups in a trend test (p = 0.015). Regarding spontaneous behavior, average time of movement (sec) was significantly accelerated in females in the high‐dose group. The dose levels of brilliant blue FCF used in the present study produced a few significant effects on neurobehavioral parameters in multiple generations in mice.     

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.     

Inhibitor of IκB kinase activity,BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

Amino acids 356-372 constitute a Gi-activator sequence of the alpha 2-adrenergic receptor and have a Phe substitute in the G protein-activator sequence motif.

The human alpha 2-adrenergic receptor contains the sequence KASRWRGRQNREKRFTF (amino acids 356-372) at the C-terminal end of its third intracellular loop. This sequence satisfies the structural criteria for G protein-activating sequences [(1992) J. Biol. Chem. 267, 8342-8346] except that the C-terminal sequence is B-B-X-X-Phe instead of B-B-X-B or B-B-X-X-B (B: basic residue, X: non-basic residue). Nevertheless, the synthetic peptide corresponding to this sequence (peptide alpha 2-F) was found to activate Gi and Go strongly with a saturated effect at 1-3 microM. Furthermore, the substitution of the C-terminal Phe of peptide alpha 2-F with Arg, Trp, and Tyr (but not Ala or Asp) did not appreciably affect the Gi-activating potency. It is suggested that the C-terminal basic residue of the B-B-X-X-B motif in Gi-activating sequences can be replaced by an aromatic residue.     

Molecular phylogenetic relationships of pond frogs distributed in the Palearctic region inferred from DNA sequences of mitochondrial 12S ribosomal RNA and cytochrome b genes

The evolutionary relationships of pond frogs distributed in the Far East and Europe were investigated by analyses of nucleotide sequences of mitochondrial 12S ribosomal RNA (12S rRNA) and cytochrome b (cyt b) genes. The nucleotide sequences of a 412-bp segment of the 12S rRNA gene and a 534-bp segment of the cyt b gene were determined by the PCR-direct sequencing method using 19 frogs belonging to six species and one subspecies distributed in the Palearctic region. Phylogenetic trees were constructed by the neighbor-joining and maximum-likelihood methods using Rana catesbeiana or Xenopus laevis as an outgroup. The 412-bp segment of the 12S rRNA gene contained 65 variable sites including gap sites, and the 534-bp segment of the cyt b gene contained 160 variable sites. The nucleotide sequence divergences of the 12S rRNA gene were 0.25-4.83% within the Far Eastern frogs, 0.25-6.22% within the European frogs, and 8.74-11.24% between the Far Eastern and the European frogs, whereas those of the cyt b gene were 3.64-14.73% within the Far Eastern frogs, 0.38-14.42% within the European frogs, and 16.53-23.58% between the Far Eastern and the European frogs. Although most nucleotide substitutions were at the third codon position of the cyt b gene and were silent mutations, 4 amino acid replacements occurred within the Far Eastern frogs, 4 within the European frogs, and 11 between the Far Eastern and the European frogs. The phylogenetic trees constructed from the nucleotide sequence divergences showed slightly different topologies for the 12S rRNA and cyt b genes. R. esculenta from Ukraine was closely related to R. lessonae from Luxembourg in both the 12S rRNA and the cyt b gene sequences.