A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275–3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.     

Knockdown of RAGE Inhibits Growth and Invasion of Gastric Cancer Cells

The receptor for advanced glycation end-products (RAGE) is an oncogenic trans-membranous receptor, which is overexpressed in multiple human cancers. However, the role of RAGE in gastric cancer is still elusive. In this study, we investigated the expression and molecular mechanisms of RAGE in gastric cancer cells. Forty cases of gastric cancer and corresponding adjacent non-cancerous tissues (ANCT) were collected, and the expression of RAGE was assessed using immunohistochemistry (IHC) in biopsy samples. Furthermore, RAGE signaling was blocked by constructed recombinant small hairpin RNA lentiviral vector (Lv-shRAGE) used to transfect into human gastric cancer SGC-7901 cells. The expression of AKT, proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase-2 (MMP-2) was detected by Real-time PCR and Western blot assays. Cell proliferative activities and invasive capability were respectively determined by MTT and Transwell assays. Cell apoptosis and cycle distribution were analyzed by flow cytometry. As a consequence, RAGE was found highly expressed in cancer tissues compared with the ANCT (70.0% vs 45.0%, P=0.039), and correlated with lymph node metastases (P=0.026). Knockdown of RAGE reduced cell proliferation and invasion of gastric cancer with decreased expression of AKT, PCNA and MMP-2, and induced cell apoptosis and cycle arrest. Altogether, upregulation of RAGE expression is associated with lymph node metastases of gastric cancer, and blockade of RAGE signaling suppresses growth and invasion of gastric cancer cells through AKT pathway, suggesting that RAGE may represent a potential therapeutic target for this aggressive malignancy.Key words: RAGE, gastric cancer, growth, invasion     

Active Role of the Predecidual-Like Zone in Endometrial Shedding in a Mouse Menstrual-Like Model

Cyclic shedding of the endometrium is unique to menstruating species. The status of the decidua in mouse menstrual-like models seems to differ from that of the predecidua in humans before endometrial breakdown. The aim of this study was to determine how this difference in decidual status is related to endometrial breakdown. A mouse menstruallike model was generated by pharmacological progesterone withdrawal. Histomorphological analysis and reticular fiber staining were used to evaluate endometrial status. In situ zymography was used to determine the localization of active collagenase and gelatinase. The functional endometrial layer containing the mature decidual-like zone (MDZ) and predecidual-like zone (PZ) underwent breakdown. The reticular fibers underwent disruption and fragmentation and became loose or disappeared at 12 h in the PZ, where active collagenase and gelatinase were limited. The reticular fibers were visibly reduced at 24 h in the MDZ, where active collagenase was detected. A few reticular fibers remained; however, the functional layer had sloughed into the lumen of the uterus. The results showed that reticular fibers of the PZ are actively degraded during endometrial shedding.Key words: mouse menstrual-like model, predecidual-like zone, reticular fiber, gelatinase, collagenase     

Simulated microgravity alters the metastatic potential of a human lung adenocarcinoma cell line

Simulated microgravity (SM) has been implicated in affecting diverse cellular pathways. Although there is emerging evidence that SM can alter cellular functions, its effect in cancer metastasis has not been addressed. Here, we demonstrate that SM inhibits migration, gelatinolytic activity, and cell proliferation of an A549 human lung adenocarcinoma cell line in vitro. Expression of antigen MKI67 and matrix metalloproteinase-2 (MMP2) was reduced in A549 cells stimulated by clinorotation when compared with the 1×g control condition, while overexpression of each gene improves ability of proliferation and migration, respectively, under SM conditions. These findings suggest that SM reduced the metastatic potential of human lung adenocarcinoma cells by altering the expression of MKI67 and MMP2, thereby inhibiting cell proliferation, migration, and invasion, which may provide some clues to study cancer metastasis in the future.     

Developmental changes of BKCa channels depend on differentiation status in cultured podocytes

The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Podocytes are of keen interests because of their key roles in kidney development and disease. Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca channels) are important ion channels located in podocytes and play the essential role in regulating calcium homeostasis cell signaling. In this research, we studied the undergoing developmental changes of BK_Ca channels and their contribution to functional maturation of podocytes. Our results showed that the distribution of BK_Ca channels changed with the maturity of differentiation in a conditionally immortalized mouse podocyte cell line. Additionally, the increase of BK_Ca channel protein expression was detected by immunofluorescence staining with confocal microscopy in podocytes, which was consistent with the increase in the current density of BK_Ca channels examined by whole-cell patch-clamp technique. Our results suggested that the developmental changes of BK_Ca channels may help podocytes adapt to changes in pressure gradients occurring in physiological conditions. Those findings may have implications for understanding the physiology and development of kidney and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in podocytes.     

Role of Complement 3 in TNF-α-Induced Mesenchymal Transition of Renal Tubular Epithelial Cells In Vitro

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.     

Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.     

The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein

Abstract

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903?U/mg. Moreover, the denaturation temperature of proApp was 4.1?°C higher than App’s. The proApp showed 104?U/mg and 252?U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.