Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase

Covalent and site-specific protein immobilization catalyzed by microbial transglutaminase (MTG) was investigated using recombinant Escherichia coli alkaline phosphatase (AP) tagged with a glutamyl donor substrate peptide (MLAQGS) of MTG. A polystyrene surface physically coated with beta-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic varepsilon-(gamma-glutamyl)lysine bond formation between the peptide tag of recombinant APs and beta-casein- or BSA-coated surface was verified by the detection of AP activity on the surface. It was found that the length and the insertion position of the peptide tag did not significantly affect the efficacy of enzymatic immobilization of the recombinant APs. On the other hand, pH and ionic strength in the reaction media had crucial effects on the immobilization yields. Interestingly, the optimum pH range of MTG-mediated protein immobilization differed markedly from that for an MTG-catalyzed reaction in aqueous solution. The results suggest that the concentration of reactive species due to electrostatic interaction between the enzyme-substrate intermediate and the protein-adsorbed surface is a key factor governing MTG catalysis at a solid surface.     

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms

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Gingival and dermal fibroblasts: their similarities and differences revealed from gene expression

Gene expression profiles in normal human gingival and dermal fibroblasts were investigated using DNA microarrays. Their fundamental characteristics were almost identical, but 5% of their genes were uniquely expressed. These results help us to choose an optimal cell source for effective fibroblast-based cell therapy that is dependent on differential gene expression profiles.     

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.     

The Potential of Using an Autogenous Tendon Graft by Injecting Bone Marrow Aspirate in a Rabbit Meniscectomy Model

Bone marrow aspirate (BMA) is an excellent source of cells and growth factors and has been used successfully for bone, cartilage, and soft-tissue healing. This study aimed to investigate the histological and biomechanical properties of autogenous tendon graft by injecting BMA and its protective effect against degenerative changes in a rabbit model of meniscal defects. Adult white rabbits were divided into untreated, tendon, and tendon + BMA groups, and meniscal defects were created in the knees. The tendon graft and articular cartilage status were evaluated by macroscopic and histological analysis at 4, 12, and 24 weeks postoperatively among the three groups. The tendon graft in the tendon and tendon + BMA groups were used for biomechanical evaluation at 4, 12, and 24 weeks postoperatively. The meniscal covering ratio in the tendon + BMA group was better than that in the tendon and untreated groups at 12 and 24 weeks postoperatively. The matrix around the central portion of cells in the tendon + BMA group was positively stained by safranin O and toluidine blue staining with metachromasia at 24 weeks. The histological score of the tendon graft in the tendon + BMA group was significantly higher than that in the untreated and tendon groups at 12 and 24 weeks postoperatively. In the tendon + BMA group, cartilage erosion was not shown at 4 weeks, developed slowly, and was better preserved at 12 and 24 weeks compared to the untreated and tendon groups. Histological scores for the articular cartilage were significantly better in the tendon + BMA group at 24 weeks. The compressive stress on the tendon graft in the tendon + BMA group was significantly higher than that in the tendon group at 12 weeks postoperatively. Transplantation of autogenous tendon grafts by injecting BMA improved the histologic score of the regenerated meniscal tissue and was more effective than the tendon and untreated group for preventing cartilage degeneration in a rabbit model of massive meniscal defects.     

Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.     

Pig weight prediction system using RGB-D sensor and AR glasses: analysis method with free camera capture direction

Artificial Life and Robotics - Pig weights are important indicator for the healthcare and the economic operation of pig farms, and the development of a system to easily estimate these weights is...