Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori

To investigate the pathogenicity of Pseudomonas aeruginosa in insects, a gacA mutant of P. aeruginosa PA01 was constructed by site-directed mutagenesis. The mutant was designated as C1. C1 was less virulent to Bombyx mori than the parent strain. To complement the gacA gene, P. aeruginosa C1 was transformed with the broad host range plasmid pJB3Km1 carrying a 3.9-kbp gacA fragment. The expression of the gacA mRNA in C1 (pgacA) was detected. In addition, the complemented mutant restored the level and timing of pyocyanin production, indicating that functional GacA is produced in the complemented strain. However, no significant difference was observed between C1 and C1 (pgacA) with respect to the killing of B. mori larvae.     

Chondromodulin-I and tenomodulin: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues

In tissues and/or organs of mesenchymal origin, the vasculature is usually well developed. However, there are certain hypovascular tissues that exhibit powerful anti-angiogenic resistance, implying the presence of tissue-type specific inhibitors of angiogenesis. Hyaline cartilage is one example, and several anti-angiogenic factors have been purified from cartilage. We previously identified chondromodulin-I (ChM-I) as a tissue-specific inhibitor of angiogenesis in fetal bovine cartilage. ChM-I is specifically expressed in the avascular regions of the growth-plate and cartilaginous bone rudiments in embryos. Recently, we cloned a novel type II transmembrane protein, tenomodulin (TeM), having a domain homologous to ChM-I at its C-terminus. TeM turned out to be expressed specifically in other hypovascular structures in the mesenchyme, such as the epimysium, tendon, and ligaments. In this overview, we discuss the structural characteristics of this class of anti-angiogenic molecules and their pathophysiological role in the control of vascularity.     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.     

Chondrogenesis,joint formation,and articular cartilage regeneration

The repair of joint surface defects remains a clinical challenge, as articular cartilage has a limited healing response. Despite this, articular cartilage does have the capacity to grow and remodel extensively during pre‐ and post‐natal development. As such, the elucidation of developmental mechanisms, particularly those in post‐natal animals, may shed valuable light on processes that could be harnessed to develop novel approaches for articular cartilage tissue engineering and/or regeneration to treat injuries or degeneration in adult joints. Much has been learned through mouse genetics regarding the embryonic development of joints. This knowledge, as well as the less extensive available information regarding post‐natal joint development is reviewed here and discussed in relation to their possible relevance to future directions in cartilage tissue repair and regeneration. J. Cell. Biochem. 107: 383–392, 2009. © 2009 Wiley‐Liss, Inc.     

A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer

We have identified an enhancer responsible for induction by 3-methylcholanthrene in the upstream region of the CYP1A2 gene. The enhancer does not contain the invariant core sequence of XREs that are binding sites for the Ah receptor (AhR) and Arnt heterodimer. The enhancer did not show any inducible expression in Hepa-1-derived cell lines, C4 and C12, deficient of Arnt and AhR, respectively. On the other hand, bacterially expressed AhR-Arnt heterodimer could not bind to the enhancer. Mutational analysis of the enhancer revealed that a repeated sequence separated by six nucleotides is important for expression. A factor binding specifically to the enhancer was found by using gel shift assays. Bacterially expressed AhR-Arnt heterodimer interacted with the factor. A dominant negative mutant of the AhR to XRE activated the enhancer. Collectively, these results demonstrate that a novel induction mechanism is present in which the AhR-Arnt heterodimer functions as a coactivator.     

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

Upregulation of transient receptor potential melastatin 6 channel expression by rosiglitazone and all-trans-retinoic acid in erlotinib-treated renal tubular epithelial cells

Anti-epidermal growth factor receptor (EGFR) drugs including erlotinib cause a side effect of hypomagnesemia. In lung adenocarcinoma A549 cells, anticancer agents such as cisplatin and doxorubicin dose-dependently increased toxicity, but the effects were significantly suppressed by culturing the cells in low Mg²⁺-containing media. To obtain the maximum effect in cancer chemotherapy, it should be necessary to prevent the reduction of body Mg ²⁺ content. Anti-EGFR drugs inhibit EGF-induced elevation of transient receptor potential melastatin 6 (TRPM6) Mg ²⁺ channel in renal tubular epithelial NRK-52E cells. Here, we found that rosiglitazone, an antidiabetic drug, and all- trans-retinoic acid (ATRA), a vitamin A derivative, increase the messenger RNA (mRNA) level of TRPM6 in the presence of erlotinib. The rosiglitazone- and ATRA-induced elevation of mRNA level, Mg ²⁺ influx, and promoter activity of TRPM6 were inhibited by GW-9662, a potent antagonist of peroxisome proliferator-activated receptor (PPAR)γ, and LE135, a retinoic acid receptor (RAR) antagonist, respectively. Rosiglitazone increased the phosphorylation and nuclear localization levels of PPARγ, which were inhibited by GW-9662. In contrast, RAR was mainly distributed in the nuclei under control conditions, which was unchanged by ATRA and LE135. The promoter activity of TRPM6 was inhibited by a mutation in the peroxisome proliferator hormone response element (PPRE). A chromatin immunoprecipitation assay revealed that PPARγ and RAR bind to the PPRE, which was blocked by GW-9662 and LE135, respectively. These results suggest that rosiglitazone and ATRA reverse the reduction in Mg ²⁺ reabsorption caused by anti-EGFR drugs.     

Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein

Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.