Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences

Mandibular osteoblasts originate from the neural crest and deposit bone intramembranously, mesoderm derived tibial osteoblasts by endochondral mechanisms. Bone synthesized by both cell types is identical in structure, yet functional differences between the two cell types may exist. Thus, both matched juvenile and adult mandibular and tibial osteoblasts were studied regarding their proliferative capacity, their osteogenic potential and the expression of osteogenic and origin related marker genes. Juvenile tibial cells proliferated at the highest rate while juvenile mandibular cells exhibited higher ALP activity depositing more mineralized matrix. Expression of Hoxa4 in tibial cells verified their mesodermal origin, whereas very low levels in mandibular cells confirmed their ectodermal descent. Distinct differences in the expression pattern of bone development related genes (collagen type I, osteonectin, osteocalcin, Runx2, MSX1/2, TGF-β₁, BAMBI, TWIST1, β-catenin) were found between the different cell types. The distinct dissimilarities in proliferation, alkaline phosphatase activity, the expression of characteristic genes, and mineralization may aid to explain the differences in bone healing time observed in mandibular bone when compared to long bones of the extremities.     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.     

Eimeria stiedae: experimental infection in rabbits and the effect of treatment with toltrazuril and ivermectin

The aim of this study was to investigate the clinical, haematological, biochemical, lipid peroxidation, ultrasonographic and pathologic findings in hepatic coccidiosis induced by Eimeria stiedae in rabbits, and also to compare the treatment effects of both toltrazuril and ivermectin separately and in combination. In this study, 56 rabbits were divided into eight groups. The first group was designated as healthy control group. Rabbits were infected with 40.000 sporulated oocysts of E. stiedae. Groups 2, 3, 4, 5, 6, 7 and 8 were allocated as the infected control group, infected+toltrazuril-treated group, infected+ivermectin-treated group, infected+toltrazuril+ivermectin-treated group, non-infected+toltrazuril-treated group, non-infected+ivermectin-treated group, non-infected+toltrazuril+ivermectin-treated group, respectively. Haematocrit, Haemoglobin and MCV values as well as percentage of lymphocyte decreased in Groups 2 and 4 whereas leucocyte counts and percentage of granulocyte leucocyte increased. Serum GGT, ALT and AST activities increased but albumin value decreased. Plasma MDA concentrations increased whereas erythrocyte CAT, GSH-Px, and SOD activities decreased. Mean oocyst numbers in per gram faeces (epg values) increased in both groups during the study. Ultrasonographic examination revealed that the liver was enlarged and had hyperechogenic parenchyma. Bile ducts were dilated and hyperechogenic and the gall bladder was dilated. The livers of these animals were enlarged and typical macroscopic and microscopic findings of coccidiosis were present. Treatment with toltrazuril and toltrazuril+ivermectin combination were highly effective in reducing faecal oocyst output in infected rabbits. Haematological, biochemical and lipid peroxidation parameters and, ultrasonographic findings of the liver were close to control values for Groups 3 and 5. Necropsy of these animals showed no visible lesions related to hepatic coccidiosis although a few oocysts were detected in the bile duct epithelial cells.     

人牙龈成纤维细胞与牙周膜细胞的生物活性

采用组织块法分离培养牙周膜细胞和牙龈成纤维细胞,测定二者的增殖特性和ALP活性,利用免疫组化和FCM方法比较Ⅰ、Ⅲ型胶原、BMP的表达情况,以观察对比两种细胞的生物学特性的异同。找出牙龈成纤维细胞和牙周膜细胞在胶原基质合成方面存在差异,发现Ⅰ、Ⅲ型胶原可作为鉴别两种细胞的标志物,ALP与BMP可作为鉴别两种细胞的标志,牙周膜细胞比牙龈成纤维细胞具有较强的成骨能力。从而为今后改良两种细胞成为牙周组织工程的种子细胞奠定基础。     

Discovery,synthesis and molecular substantiation of N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides as anticancer agents

The effort was taken to develop a series of benzothiazole and quinoline fused bioactive compounds obtained through a four-step synthetic route using a range of substituted acetoacetanilides. Achieved N-(benzo[d]thiazol-2-yl)-2-hydroxyquinoline-4-carboxamides (6a-l) were produced up to 96% of yield while the eco-friendly p-TSA used as a catalyst. Further, the anticancer activity of these compounds was determined using a range of cancer cell lines starting from MCF-7 (Breast cancer), HCT-116 (Colon cancer), PC-3 & LNCaP (Prostate) and SK-HEP-1 (Liver cancer). Present study compounds were also testified for antioxidant properties prior to anticancer studies since the Reactive Oxygen Species (ROS) being vital in cancer development. To determine the cell membrane stability effects of the compounds, human red blood cells (HRBC) based membrane protection assay was determined. In the results, compounds 6a-l were able to produce a dominated result values over PC3 cell lines (Prostate cancer) than the other cell lines used in this study. Since the connectivity of human germ cell alkaline phosphatase (hGC-ALP) in the development of prostate cancer is known, the most active compounds were evaluated for the hGC-ALP inhibition in order to ensure a mechanism of anticancer action of these compounds. The mode of interaction and binding affinity of these compounds was also investigated by a molecular docking study. In the results, 6d, 6i, 6k, and 6l were found with least IC₅₀ values <0.075 µM and highest relative activity of 92%, 90%, and 96% respectively. The need for further animal model evaluation and pre-clinical studies recognized.     

MicroRNA-27 promotes the differentiation of odontoblastic cell by targeting APC and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/β-catenin signaling pathway has miR-27 binding site in the its 3′ UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b

Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-β (TGF-β)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-β/activin signaling pathway through inhibition of AcvR1b.