The study was purposed to investigate the expression and function of non-muscle myosin heavy chain-IIA (NMMHC-IIA) in Fechtner syndrome in order to explore the pathologic changes of kindy disease and the mechanism of granulocyte inclusion body formation. NMMHC-IIA levels in granulocytes were analyzed by Western-blot, the expressions of NMMHC-IIA, IIB in HEK-293 cells were detected by RT-PCR and were analyzed by co-immunoprecipitation. The results indicated that the IIA/beta-actin ratio for Fechtner syndrome granulocytes was (0.35 +/- 0.12), and obviously decreased as compared with that of normal control (0.87 +/- 0.18) (p < 0.01). The IIA and IIB expressed higher in HEK-293 cells. The interaction of IIA and IIB was confirmed by co-immunoprecipitation in HEK-293 cells. It is concluded that dominant-negative effect of NMMHC-IIA is involved in the formation of inclusion bodies. IIA and IIB show obvious interaction, IIB partly compensates the IIA defect derived from MYH9 mutations, and may delay or prevent the development of clinically relevant abnormalities.
Blood Platelet Disorders ; genetics ; metabolism ; pathology ; Cell Line ; Granulocytes ; pathology ; Humans ; Inclusion Bodies ; pathology ; Kidney ; cytology ; embryology ; metabolism ; Mutation ; Nonmuscle Myosin Type IIA ; genetics ; metabolism ; physiology ; Nonmuscle Myosin Type IIB ; genetics ; metabolism ; physiology ; Syndrome ; Thrombocytopenia ; genetics ; metabolism ; pathology

The mutation of the PKD1 gene causes autosomal dominant polycystic kidney disease (ADPKD), and the PKD1 gene encodes polycystin-1 (PC-1). PC-1 is thought to be a cell-cell/matrix adhesion receptor molecule at the cell surface that is widely expressed in the kidney. However, there are controversies about the role of PC-1 protein and its expression when using different antibodies to detect it. We used two PC-1 antibodies; C-20 (Santa Cruz, sc-10372) as the C-terminal antibody, and P-15 (Santa Cruz, sc-10307) as the N-terminal antibody. We evaluated the PC-1 expression by performing immunoblotting on the human embryonic kidney (HEK) 293 cells and the renal proximal tubular epithelial cell (RPTEC) lysates. We characterized the expression of PC-1 in the fetal, adult and polycystic kidneys tissues by performing immunohistochemistry. We confirmed the PC-1 expression in the HEK 293 cells and the RPTEC lysates, but the expression was very low. The PC-1 proteins were diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult kidneys, and the PC-1 expression was more prominent in the proximal tubules of the fetal kidney. In the ADPKD kidney, the PC-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells. Our data suggests that the development of the kidney may regulate the expression of PC-1, and an altered PC-1 expression may contribute to cyst formation in ADPKD.
TRPP Cation Channels/chemistry/*metabolism ; Protein Structure, Tertiary ; Polycystic Kidney, Autosomal Dominant/*metabolism ; Middle Aged ; Male ; Kidney/*embryology/metabolism/*pathology ; Immunohistochemistry ; Humans ; *Gene Expression Regulation, Developmental ; *Gene Expression Regulation ; Cytoplasm/metabolism ; Cell Line

The purpose of this study was to determine if mild hypoxia alters the responsiveness to vasoactive agents in the renal and the femoral arteries in the fetal sheep. Ten pregnant sheep were operated under halothane anesthesia at 116 to 124 days' gestation. A maternal tracheal catheter was placed for infusing compressed air (control group, n=5) or nitrogen (hypoxia group, n=5) starting on post operative day 6 and maintained for 5 days. Femoral and renal arteries were harvested from the fetus to study the constriction response to phenylephrine (PE 10(-9) to 10(-5) mol/L). To determine the involvement of nitric oxide as a modulator of vessel constriction, N-nitro-Larginine methyl ester (L-NAME) was used at a concentration of 10(-4) mol/L in parallel chambers. In the hypoxia group, maternal Pao2 significantly decreased from a base-line of 110.4 +/-1.4 to 80.5 +/-1.6 (mmHg, p <0.01), fetal Pao2 significantly decreased from a baseline of 20.9 +/-0.3 to 15.5 +/-0.1 (mmHg, p <0.01). Hypoxia was associated with a significant increase in PE maximal response in the absence (184.5 +/-6.6 vs. 146.2 +/-4.3) and presence (166.9 +/-6.3 vs. 145.0 +/-4.5) of L-NAME, and a decrease in EC50 in the absence (6.0 +/-1.1 vs. 27.0 +/-4.1) of L-NAME of femoral arteries. However, there were no significant differences in PE maximal response and EC50 in the absence and presence of L-NAME of renal arteries. We concluded that mild chronic hypoxia seems to increase the fetal femoral artery response to PE, but not in the fetal renal artery. This observation is consistent with a redistribution of cardiac output away from the carcass.
Animals ; *Anoxia ; Blood Glucose/metabolism ; Dose-Response Relationship, Drug ; Enzyme Inhibitors/pharmacology ; Femoral Artery/*embryology/*pathology ; Hematocrit ; Hydrogen-Ion Concentration ; Kidney/blood supply ; Lactates/blood/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitric Oxide/metabolism ; Nitric-Oxide Synthase/antagonists & inhibitors ; Phenylephrine/chemistry/metabolism/pharmacology ; Renal Artery/pathology ; Sheep/*embryology ; Time Factors ; Vasoconstrictor Agents/*pharmacology

OBJECTIVETo construct a lentiviral vector carrying human NESG1-EGFP gene and observe its expression in 293FT cells.

METHODSThe CDS region of NESG1 gene was amplified from a plasmid containing the full-length NESG1 sequence and cloned into the lentiviral vector pGC-FU-EGFP by restriction endonuclease AgeI digestion and T(4) DNA ligase ligation. After transformation into competent E. coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293FT cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. The 293FT cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. Western blotting was used to detect the expression of NESG1 protein in the transfected cells.

RESULTSThe lentiviral vector pGC-FU-NESG1-EGFP for NESG1 gene was constructed successfully. Strong green fluorescence was observed in 293FT cells under fluorescent microscope after co-transfection of the cells with the 3 plasmids of lentiviral vector. The virus in the supernatant reached a titer of 2×10(7) TU/ml. The transfection efficiency of the collected virus exceeded 90% in 293FT cells with a multiplicity of infection of 1. Western blotting identified the presence of NESG1 expression in the transfected 293FT cells.

CONCLUSIONThe lentiviral vector for NESG1 has been successfully constructed with a high yield of lentivirus, which facilitate further investigation of the roles of NESG1 gene in the development and progression of nasopharyngeal carcinoma.

Cell Line ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Kidney ; cytology ; embryology ; Lentivirus ; genetics ; metabolism ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection