Discovery and characterization of tRNA lysidine synthetase (TilS)

In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNA^Ile bearing lysidine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that determines both the codon and amino acid specificities of tRNA^Ile. We identified an enzyme named tRNA^Ile lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as substrates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists of two consecutive reactions involving the adenylated tRNA intermediate. In addition, we deciphered how Escherichia coli TilS specifically discriminates between tRNA^Ile and the structurally similar tRNA^Met, which bears the same anticodon loop. Recent structural studies unveiled tRNA recognition by TilS, and a molecular basis of lysidine formation at atomic resolution.     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.     

Time-dependent expression of renal vaso-regulatory molecules in LPS-induced endotoxemia in rat

To elucidate roles of microvascular factors in the pathogenesis of renal complications during endotoxemia, that is characterized by renal vasoconstriction and systemic hypotension/generalized non-renal vasodilation, we profile the expression pattern and time-course of three key vaso-regulators, namely endothelin (ET)-1, nitric oxide (NO), and angiotensin II (Ang II). We hypothesize that disruption of the overall balance between vasodilatation and vasoconstriction in the kidney, during the early phase of sepsis, contribute to its (kidney) predisposition to acute renal failure. Adult male Wistar rats were rendered endotoxemic at different time points (1, 3, 6 and 10 h) by a single i.p. injection of lipopolysaccharide (LPS) (15 mg/kg) dissolved in saline. Control group was injected vehicle only (saline). Both systolic and diastolic blood pressures significantly decreased at different time points after LPS administration. Surprisingly, renal histopathological evaluation showed no remarkable changes in LPS-induced endotoxemia. However, overall, levels of the vaso-regulators and, where applicable, their respective receptors were upregulated: (1) plasma ET-1 increased 25-fold and peaked, as renal ET-1 mRNA, at 3 h; renal ET-1 protein and its receptors, ET type A (ET(A)) receptor (vasoconstrictive) and ET type B (ET(B)) receptor (vasodilatatory) increased in a time-dependent fashion, (2) Ang II increased by 53% compared to control, peaking at 6 h. However, while levels of Ang II type 1 (AT1) receptor increased over time after LPS injection, those of Ang II type 2 (AT2) receptor were downregulated, (3) data of NO system (NO-NOS), the key vasodilator, were the most intriguing. Whereas levels of renal NO increased time-dependently following LPS administration, with a 2240-fold increase in renal iNOS expression, levels of eNOS, were almost unchanged. In conclusion, the present study overall reveals intriguing and complex dynamics between levels of vasoconstrictors and vasodilators during the early phase of LPS-induced endotoxemia. These shifts in molecular expressions are likely triggered by compensatory mechanisms aimed at counteracting the undesirable and dominant effects of one group of vaso-regulatory moiety over the other.     

Suppression of discoidin domain receptor 1 by RNA interference attenuates lung inflammation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1alpha, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-beta in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.     

Multiple activation of mitogen-activated protein kinases by purified independent CCN2 modules in vascular endothelial cells and chondrocytes in culture

CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2.     

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.     

Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages

Summary In order to investigate the mechanism of synthesis and secretion of lysozyme (LZ) by human mononuclear phagocytes, the ultrastructural localization of LZ was studied by a pre-embedding direct immunoperoxidase method. Blood monocytes showed a reaction product for LZ in cytoplasmic granules, whereas cultured monocytes showed the reaction product in phagosomes as well as granules at 5 h of culture and in numerous large granules at 3 days of culture. In Kupffer cells, LZ was present in cytoplasmic granules, vacuoles and phagosomes. Some Kupffer cells showed a positive reaction for LZ in the rough endoplasmic reticulum, perinuclear cisterna and Golgi apparatus. Macrophages in the lymph nodes contained LZ in cytoplasmic granules. Bone marrow macrophages contained numerous phagosomes with electron-dense degradation products of erythrocytes, but the reaction product for LZ could not be clearly identified. The present study demonstrated that LZ is present in the granules of human mononuclear phagocytes and released into phagosomes. An in-vitro culture study, furthermore, demonstrated that macrophages produce LZ-containing large granules distinct from those of monocytes. However, findings that indicate the synthesis and secretion of LZ by cultured monocytes, as suggested previously by other investigators, were not observed in this study.     

A new tactic for the treatment of jaundice: an injectable polymer-conjugated Bilirubin oxidase

Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.