Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.     

Ultrastructural immunolocalization of dentin matrix protein 1 on Sharpey's fibers in monkey tooth cementum

Despite the importance of dentin matrix protein 1 (DMP1) in the formation of mineralized tissue, including dentinogenesis and osteogenesis, its precise role in cementogenesis remains to be clarified fully. The purpose of our study was to demonstrate the ultrastructural immunolocalization of DMP1 in monkey molar tooth cementum. Japanese Macaca fuscata monkeys were fixed by perfusion. The upper molar teeth and accompanying periodontium then were dissected and demineralized with EDTA. Cryosections were obtained, incubated in anti-DMP1 polyclonal antibody, and processed by immunoperoxidase and immunogold labeling. Intense immunoperoxidase staining for DMP1 was observed in acellular extrinsic fiber cementum, particularly in Sharpey's fibers. Cementocyte lacunae with canaliculi showed DMP1 staining in the apical region of the tooth root. Electron immunomicroscopy revealed the close proximity of DMP1 to collagen fibrils in Sharpey's fibers at the mineralization front. Intense immunogold labeling was localized on the walls of the cementocyte lacunae in cellular cementum. These results should contribute to better understanding of the role of DMP1, not only in Sharpey's fiber biomineralization, but also in the maintenance of the cementocyte lacunar space in cementum.     

Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture

Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O₂ (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm² flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O₂ in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O₂, respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O₂ and 500 μl (1,480-μm depth) at 5% O₂ in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.     

Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.     

N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.     

Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS), a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO) caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R) related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.     

Large-scale mapping of human protein interactome using structural complexes

Although the identification of protein interactions by high-throughput (HTP) methods progresses at a fast pace, 'interactome' data sets still suffer from high rates of false positives and low coverage. To map the human protein interactome, we describe a new framework that uses experimental evidence on structural complexes, the atomic details of binding interfaces and evolutionary conservation. The structurally inferred interaction network is highly modular and more functionally coherent compared with experimental interaction networks derived from multiple literature citations. Moreover, structurally inferred and high-confidence HTP networks complement each other well, allowing us to construct a merged network to generate testable hypotheses and provide valuable experimental leads.     

Tissue localization of maize acetylcholinesterase associated with heat tolerance in plants

Our recent study reported that maize acetylcholinesterase (AChE) activity in the coleoptile node is enhanced through a post-translational modification response to heat stress and transgenic plants overexpressing maize AChE gene had an elevated heat tolerance, which strongly suggests that maize AChE plays a positive, important role in maize heat tolerance. Here we present (1) maize AChE activity in the mesocotyl also enhances during heat stress and (2) maize AChE mainly localizes in vascular bundles including endodermis and epidermis in coleoptile nodes and mesocotyls of maize seedlings.     

BmEts upregulates promoter activity of lebocin in Bombyx mori

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D, attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two κB elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two κB elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal κB element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates.