MosA, a protein implicated in rhizopine biosynthesis in Sinorhizobium meliloti L5-30, is a dihydrodipicolinate synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3-O-methyl-scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure-function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate beta-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by L-lysine are consistent with the enzyme being a DHDPS. E.coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Cx26 Affects the in Vitro Reconstruction of Human Epidermis

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.     

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

LINC01342 promotes the progression of ovarian cancer by absorbing microRNA-30c-2-3p to upregulate HIF3A

Ovarian cancer (OC) is a highly prevalent gynecologic malignancy and its mortality is extremely high. Therefore, the development of novel therapeutic approaches for OC is of great significance. In this study, LINC01342 was upregulated in OC tissue in the GSE38666 microarray and in tumor tissue samples collected in our center. The silencing of LINC01342 suppressed the proliferative and metastatic capacities of A2780 and HO8910 cells. Subcellular distribution assays showed that LINC01342 was mainly enriched in the cytoplasm. Subsequently, the downregulation of microRNA-30c-2-3p was proven to be the target of LINC01342. The silencing of microRNA-30c-2-3p enhanced the clonality and migratory capacity of OC cells. Moreover, the silencing of microRNA-30c-2-3p could reverse the inhibited migration and clonality in OC cells caused by LINC01342 knockdown. In addition, hypoxia-inducible factor 3 subunit α (HIF3A) was proven to be the target gene of microRNA-30c-2-3p, which was upregulated. HIF3A was negatively regulated by microRNA-30c-2-3p but positively regulated by LINC01342 in OC cells. An RNA binding protein immunoprecipitation assay showed that microRNA-30c-2-3p, LINC01342, and HIF3A could bind to argonaute RISC catalytic component 2. The overexpression of HIF3A reversed the inhibited migration and clonality in OC cells with LINC01342 knockdown. By analyzing the follow-up data from the enrolled OC patients, the LINC01342 and HIF3A levels were negatively correlated with prognosis, while the microRNA-30c-2-3p level was positively correlated with the same. In short, the upregulated LINC01342 in OC absorbs microRNA-30c-2-3p to release HIF3A. Thus, upregulated HIF3A expression accelerates the progression of OC.     

末次冰期以来北海道、萨哈林岛及亚洲东北部落叶松林演变史

萨哈林岛（库叶岛）和北海道的末次冰期沉积丰富的落叶松花粉,为了搞清楚落叶松花粉百分比和实际森林密度的关系,我们系统地分析了从苔原,森林苔原以及泰加林的表土样品,根据孢粉资料恢复了两个岛屿3万年以来的植被和气候,两个岛上3万年前后 以云杉与落叶松共生的森林为主,松和冷杉现在比现代相对较冷和干的气候条件下,萨哈林在末次冰盛期的草原和泰加林主要由落叶松和松组成,和现在西伯利亚东北部的泰加林相似,在北海道末次冰盛期的草原和泰加林主要由落叶松,松以及云杉组成,气候更加干冷,在新仙女木期森林苔原分布到中萨哈林岛,而北海道的草原和泰加林以落叶松为主,气候可能比末次冰盛期还要冷,在1万年前两个岛的落叶松含量都开始下降,6000年前在北海道消失,萨哈林岛的落叶松则残存至今。     

Formulation and optimization of mouth dissolve tablets containing rofecoxib solid dispersion

The purpose of the present investigation was to increase the solubility and dissolution rate of rofecoxib by the preparation of its solid dispersion with polyvinyl pyrrolidone K30 (PVP K30) using solvent evaporation method. Drug-polymer interactions were investigated using differential scanning calorimetry (DSC), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). For the preparation of rofecoxib mouth dissolve tablets, its 1∶9 solid dispersion with PVP K30 was used with various disintegrants and sublimable materials. In an attempt to construct a statistical model for the prediction of disintegration time and percentage friability, a 3² randomized full and reduced factorial design was used to optimize the influence of the amounts of superdisintegrant and subliming agent. The obtained results showed that dispersion of the drug in the polymer considerably enhanced the dissolution rate. The drug-to-carrier ratio was the controlling factor for dissolution improvement. FTIR spectra revealed no chemical incompatibility between the drug and PVP K30. As indicated from XRD and DSC data, rofecoxib was in the amorphous form, which explains the better dissolution rate of the drug from its solid dispersions. Concerning the optimization study, the multiple regression analysis revealed that an optimum concentration of camphor and a higher percentage of crospovidone are required for obtaining rapidly disintegrating tablets. In conclusion, this investigation demonstrated the potential of experimental design in understanding the effect of the formulation variables on the quality of mouth dissolve tablets containing solid dispersion of a hydrophobic drug.     

Modulation of synaptic plasticity by brain estrogen in the hippocampus

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention.