Abstract:Lysyl oxidases (LOXs) are a family of anabolic enzymes that function to maintain, heal, and remodel tissue architecture by cross linking the extracellular matrix proteins, collagens and elastins. In a series of studies, LOXs have been considered as a family of multi-functional enzymes, which play an essential role in cell proliferation, cell chemotactical responses and tumor genesis. LOXs have now been implicated in several pathological conditions including connective tissue disease, exfoliation syndrome, disorders of copper metabolism, pelvic organ prolapse and bone disorders, so on. Its biosynthesis, structure characteristics, multi-function and relationship between LOXs and human diseases were summarized.

Abstract:Silicon exerts beneficial effects on plant growth and development by alleviating biotic and abiotic stresses. The uptake and translocation of Si in plants is mediated by Si transporters, recently, several genes encoding Si transporters have been identified from rice, barley and maize. OsLsi shows polar localization in root tissues, OsLsi1 is localized at the plasma membrane of both exodermis and endodermis on the distal side in rice roots, where Casparian strips exist; OsLsi2 is localized on the proximal side of the same cells. Therefore, OsLsi1 is responsible for transport of Si from the external solution to the root cells, whereas OsLsi2 is an efflux transporter responsible for the transport of Si from the exodermal cells to the apoplast of aerenchyma; Si is transported into the stele by OsLsi1 and OsLsi2 coordination and then translocated to the shoot by transpirational flow through the xylem. OsLsi6 was observed in the xylem parenchyma cells that were adjacent to vessels in both leaf sheaths and leaf blades besides roots, which is responsible for the export of Si from the xylem and for the subsequent distribution of Si. In maize and barley, Si is taken up from the external solution by the influx transporter (ZmLsi1/HvLsi1) localized on the distal side of cells in the epidermis and cortex layer, and then transferred to the endodermis through the symplastic pathway. At the endodermis, Si is released by an active Si efflux transporter (ZmLsi2/HvLsi2) to the stele. In addition, ZmLsi6 has the similar the localization and transport activity with OsLsi6 and might have similar functions, however, the Lsi6 in barley is not identified until now. More researches in the mechanism of Si transport in plant are needed further.

Abstract:Ryanodine receptors (RyR), as intracellular Ca²⁺ release channels, mediate Ca²⁺ release from intracellular stores in the sarcoplasmic reticulum and play essential roles in some excitable cells such as cardiac and skeletal cells. Cyclic adenosine diphosphate ribose (cADPR) and FK 506 binding proteins(FKBP) are tightly associated with RyR in these cells. A summary on latest research progress in RyR and some channel modulators for RyR2 was given based on authors′ research.

Abstract:As described in enzymology, "induced fit" and "lock and key" models are used to explain the enzymic specificity of substrate. Previously, authors have studied the substrate specificity of Eisenia fetida proteaseⅠ(EfP-Ⅰ), showing that the interaction between this protease and its substrates underwent an "induced fit" followed by "lock and key" model. It needs further investigating whether this model is suitable for other enzymes. Here, the reactions of substrate-induced Eisenia fetida proteaseⅡ(EfP-Ⅱ), subtilisin (Sub) and lactate dehydrogenase (LDH) with their substrates were shown. EfP-Ⅱ and Sub could not recognize chromozym U (CU) (P < 0.05) after incubated with chromozym TH (CTH) although the two proteases are natively able to react with both CTH and CU. The reaction followed an "induced fit-lock and key" pattern. In contrast, the two proteases were still able to react with CTH even though they have been incubated with CU. But neither earthworm protease nor subtilisin could recognize CU after CU and CTH treatment in turn, still suggesting that the reactions followed an "induced fit and then lock and key" procedure. Furthermore, the activity of LDH with lactate significantly decreased (P < 0.05) after the enzyme had been incubated with pyruvate. The activity on the conversion of pyruvate into lactate was not significantly affected by a prior incubation with lactate. This suggests that the pyruvate-induced complementary conformation of LDH is more stable than lactate-induced conformation.

Abstract:Studies of nucleosome positioning around the sites of nucleotide polymorphism are important for analysis of mechanism of genome variation. The distributions of sites single nucleotide polymorphism (SNP), simple insertion, insertion-deletion, and deletion are analyzed for human genome. Characteristics of nucleosomes in the vicinity of the polymorphism sites are also studied. The results indicated polymorphism sites downstream of transcription start sites (TSSs) occurs with an ～211 bp periodicities. For single nucleotide polymorphism, there is also a periodicity with 146 bp. The periodicity with ～211 bp is very close to the periodicity (204 bp) of nucleosome distribution downstream of TSS. The 146 bp periodicity is just the length of linker DNA sequence of a nucleosome. The results indicate that the distribution of polymorphism sites has an intimate relationship with nucleosome positioning. Further studies suggest that most of single nucleotide polymorphism sites are at two ends of core DNA, while sites of insertion, deletion, and insertion and deletion (in-del) tend to be in nucleosome-depleted region. The equally-spaced configuration of nucleosomes downstream of TSS causes the periodic distribution of polymorphism sites. The studies suggest genome variations occur in different regions relatively to nucleosomes, and nucleosome positioning has a role in forming nucleotide polymorphism.

Abstract:To investigate the effects of 8-prenylnaringenin on osteoblasts in vitro under the conditions of the ability to differentiate and biomineralization. Neonatal SD rat skull was segregated, bone cells were obtained by enzyme digestion and cultured in MEM containing 10% FBS. Three days later the culture medium was changed at the first time, serial subcultivation was proceeded until cells covered with 90% culture dish. The 8-prenylnaringenin final concentration is 1×10-6 mol/L. Under the induce condition, the Alkaline phosphatase activity, osteocalcin and calcium salt sediment yield were measured at the 3rd, 6th, 9th, 12th day. At the 12th day, histochemistry dyeing was carried for calcified tubercle and ALP. Total RNA was isolated and the gene expression of bFGF, IGF-1, osterix, BMP-2 and Runx-2 were investgated by RT-real time PCR. 8-Prenylnaringenin significantly advanced osteoblasts to differentiate and biomineralization. Raised the ALP activity and calcium salt sediment yield and osteocalcin and COL-Ⅰ, increased calcified tubercle amount were manifested. Besides, 8-Prenylnaringenin also can enhance the mRNA level of bFGF, IGF-1, osterix, BMP-2 and Runx-2. The 8-prenylnaringenin with final concentration 1×10^-6 mol/L can predominantly promote ROB differentiation and biomineralization.

Abstract:In order to elucidate the mechanisms of let-7a down-regulation in pathogenesy of gastric carcinoma, proteins associated with the function of let-7a were detected in high throughout screening. The cell line of SGC-7901 stablely overexpressing let-7a was successfully established by gene clone. Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of SGC-7901/let-7a, SGC-7901/EV and SGC-7901, and PDQuest software was applied to analyze 2-DE images. Ten differential protein spots were identified by MALDI-TOF-MS, and they may be the proteins associated with let-7a function. The overexpressed proteins included antioxidant protein 2, insulin-like growth factor binding protein 2, protein disulfide isomerase A2, C-1-tetrahydrofolate synthase, cyclin-dependent kinase inhibitor1 and Rho-GTPase activating protein 4. The underexpressed proteins were consisted of S-phase kinase-associated protein 2, platelet membrane glycoprotein, fibronectin and Cks1 protein. Furthermore, the differential expression levels of the partial proteins (CDKN1, Spk2 and fibronectin) were confirmed by Western blot analysis. The 10 differentially expressed proteins could be divided into groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence，cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of let-7a in gastric carcinoma, and will be valuable for further to study the mechanisms of let-7a down-regulation in pathogenesy of gastric carcinoma.

Abstract:To detect the expression of ADAM19 (a disintegrin metalloproteinase 19) in the placenta and peripheral blood and study the correlation between ADAM19 and the pathology of preeclampsia. The patients were divided into the study group (preeclampsia group) and the control group (normal group), and there were 50 cases in the early-onset preeclampsia group, 44 cases in the late-onset group and 50 cases in control group. Venous blood was obtained before childbirth, placental tissues were obtained after delivery. The expression of ADAM19 protein in the placental tissues was detected by immunohistochemistry and Western blot, and that of the plasma was detected by ELISA. ADAM19 protein was detected in the cytotrophoblast cells, syncytiotrophoblast cells, villous stromal cells and capillaries, and the positive signals were localized in the cell membrane and cytoplasm. The expression of ADAM19 protein in the normal placenta was 0.34 ± 0.03, while that of the late-onset preeclampsia group was 0.53 ± 0.02, and that of the early-onset group was 0.82 ± 0.03. And there were significant differences among the three groups (P <0.01). The expression of ADAM19 protein in the plasma of the normal group was (4.52 ± 0.10) μg/L, while that of the late-onset preeclampsia group was (4.32± 0.11) μg/L and that of the early-onset group was (3.78 ± 0.10) μg/L. There was significant difference between the early-onset group and the control group (P < 0.001), also between the early-onset group and the late-onset group (P < 0.001), while there was no significant difference between the late-onset and the control group (P > 0.05). The over-expression of ADAM19 in the placenta of preeclampsia patients may be associated with the occurrence and development of preeclampsia, and ADAM19 may be a marker used for predicting preeclampsia.

Abstract:Rubisco (Ribulose 1, 5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) is crucial in biological circumstance fluctuation. Although disassembly of Rubisco after chill treatment has been reported in previous studies, there is only little known data on Rubisco interactive proteins involved in the disassembly process of Rubisco. Both repression of net photosynthesis rate and disassembly of Rubisco large subunits (Ru-L) have been investigated in the wild type, Arabidopsis thaliana (Col-0), treated at 4℃ for 4 h and 24 h together with their 24 h recoveries at 20℃. Co-immunoprecipitation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and MALDI-TOF MS identification was used to explore Rubisco-interacting proteins. Five protein candidates were profiled. The identified AAA-type ATPase family and glycosyltransferase were determined crucial for Rubisco activity. It is also strongly correlated to cold acclimation. Results suggest that the disassembly of Rubisco might have been the main cause of photosynthesis rate reduction under chill conditions, rather than photosystem or biogenesis involvement.

Abstract:Syntenin and Pick1 (PDZ-domain proteins) have been reported to bind to ephrinB ligands. However, there is no data related to whether ephrinB ligands, located on the membrance of osteoclasts, regulate expression levels of syntenin and Pick1, following activation by EphB receptors. RAW264.7 cell line was used as osteoclast precursors, which can differentiate into osteoclast induced by RANKL. Western blot analysis and/or immunofluorescence staining revealed that not only syntenin and Pick1, but also ephrinB2 were prominently expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, thus furtherly illustrating that the model of RANKL-induced osteoclast differentiation of RAW264.7 cells can be used for further investigation. In order to study the effects of reverse signaling on the expression levels of PDZ-domain proteins, soluble EphB4-Fc protein was used to stimulate ephrinB2 because EphB4 exclusively interacts with ephrinB2. In contrast to the similar expression level of syntenin between EphB4-Fc and Fc treated group, the protein and mRNA expression levels of Pick1 were obviously enhanced in EphB4-Fc treated group compared with Fc treated group. However, co-immunoprecipitation results showed that there were no direct interactions between ephrinB2 and endogenously expressed syntenin and Pick1 in the RANKL-induced osteoclasts of RAW264.7 cells in vitro. In summary, it was demonstrated that both syntenin and Pick1 were expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, and EphB4/ephrinB2 reverse signaling regulates the expression levels of Pick1, but not of syntenin. These data help to preliminarily explore the potential PDZ-domain proteins involved in the downstream of ephrinB2 during the osteoclast differentiation of RAW264.7 cells in vitro.

Abstract:Data normalization plays a crucial role in the interpretation of experimental result. House-keeping genes were utilized as internal controls to accurately determine the gene expression in quantitative PCR. However, significant expression variation of these internal controls was revealed recently. A novel normalization approach (per cell normalization, percellome), which is based on DNA and RNA normalization, is developed to calibrate miRNA expression in quantitative PCR. In which, a cocktail of three external RNAs with different copy numbers were spiked, so as be able to normalize miRNA expression against cell number. Gene expression of 14 miRNAs, as well as commonly used internal controls (U6 ncRNA and 5S rRNA), were examined in the brain samples of 8 and 40 week-old mice. By using "per cell normalization" method, the expression level of theses miRNAs varied from 2- to 26-fold, while the absolute miRNA copy number per cell were from 2.0×10⁵ to 4.3×10⁵ copies per cell. Interestingly, the fold-change of U6 ncRNA and 5S rRNA were found to be 1.5- and 4.8-fold (based on DNA normalization), and 5.8- and 3.8-fold (based on RNA normalization), indicating significant expression variations of these two house-keeping genes. The study provides an novel approach to reliably normalize miRNA expression in quantitative PCR.