Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Resolvin D1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an M2-like phenotype

We recently demonstrated that ω-3-polyunsaturated fatty acids ameliorate obesity-induced adipose tissue inflammation and insulin resistance. In this study, we report novel mechanisms underlying ω-3-polyunsaturated fatty acid actions on adipose tissue, adipocytes, and stromal vascular cells (SVC). Inflamed adipose tissue from high-fat diet-induced obese mice showed increased F4/80 and CD11b double-positive macrophage staining and elevated IL-6 and MCP-1 levels. Docosahexaenoic acid (DHA; 4 μg/g) did not change the total number of macrophages but significantly reduced the percentage of high CD11b/high F4/80-expressing cells in parallel with the emergence of low-expressing CD11b/F4/80 macrophages in the adipose tissue. This effect was associated with downregulation of proinflammatory adipokines in parallel with increased expression of IL-10, CD206, arginase 1, resistin-like molecule α, and chitinase-3 like protein, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. This shift was confined to the SVC fraction, in which secretion of Th1 cytokines (IL-6, MCP-1, and TNF-α) was blocked by DHA. Notably, resolvin D1, an anti-inflammatory and proresolving mediator biosynthesized from DHA, markedly attenuated IFN-γ/LPS-induced Th1 cytokines while upregulating arginase 1 expression in a concentration-dependent manner. Resolvin D1 also stimulated nonphlogistic phagocytosis in adipose SVC macrophages by increasing both the number of macrophages containing ingested particles and the number of phagocytosed particles and by reducing macrophage reactive oxygen species production. No changes in adipocyte area and the phosphorylation of hormone-sensitive lipase, a rate-limiting enzyme regulating adipocyte lipolysis, were observed. These findings illustrate novel mechanisms through which resolvin D1 and its precursor DHA confer anti-inflammatory and proresolving actions in inflamed adipose tissue.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

The AtRbx1 protein is part of plant SCF complexes,and its down-regulation causes severe growth and developmental defects

Recently in yeast and animal cells, one particular class of ubiquitin ligase (E3), called the SCF, was demonstrated to regulate diverse processes including cell cycle and development. In plants SCF-dependent proteolysis is also involved in different developmental and hormonal regulations. To further investigate the function of SCF, we characterized at the molecular level the Arabidopsis RING-H2 finger protein AtRbx1. We demonstrated that the plant gene is able to functionally complement a yeast knockout mutant strain and showed that AtRbx1 protein interacts physically with at least two members of the Arabidopsis cullin family (AtCul1 and AtCul4). AtRbx1 also associates with AtCul1 and the Arabidopsis SKP1-related proteins in planta, indicating that it is part of plant SCF complexes. AtRbx1 mRNAs accumulate in various tissues of the plant, but at higher levels in tissues containing actively dividing cells. Finally to study the function of the gene in planta, we either overexpressed AtRbx1 or reduced its expression by a dsRNA strategy. Down-regulation of AtRbx1 impaired seedling growth and development, indicating that the gene is essential in plants. Furthermore, the AtRbx1-silenced plants showed a reduced level of AtCul1 protein, but accumulated higher level of cyclin D3.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

High-fat diet induced adiposity and insulin resistance in mice lacking the myotonic dystrophy protein kinase

Myotonic dystrophy 1 (MD1) is caused by a CTG expansion in the 3′-unstranslated region of the myotonic dystrophy protein kinase (DMPK) gene. MD1 patients frequently present insulin resistance and increased visceral adiposity. We examined whether DMPK deficiency is a genetic risk factor for high-fat diet-induced adiposity and insulin resistance using the DMPK knockout mouse model. We found that high-fat fed DMPK knockout mice had significantly increased body weights, hypertrophic adipocytes and whole-body insulin resistance compared with wild-type mice. This nutrient-genome interaction should be considered by physicians given the cardiometabolic risks and sedentary lifestyle associated with MD1 patients.     

Differences in N-glycan structures foundon recombinant IgA1 and IgA2 producedin murine myeloma and CHO cell lines

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. the choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. these glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHo cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galα(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHo cells contained glycans that were more similar to those found on human IgA. expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHo cells was significantly lower than that previously reported for serum IgA1. these data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.Key words: recombinant antibody, IgA, glycosylation, expression system, mass spectrometry