OBJECTIVETo construct a recombinant adenovirus vector containing bone morphogenic protein-7 (BMP-7) gene and to identify its biological activities in proximal tubule epithelial cells (PTECs).

METHODSForty-six fragments of BMP-7 gene were obtained by PCR method and then were ligated to the full-length gene. The full length sequence of BMP-7 was subcloned into pBluescript II(+) vectors, and confirmed by sequencing. Double digested with Not I and Hind III, BMP-7 gene was inserted into pShuttle-CMV. EcoR I pre-linearized pShuttle plasmid was transformed into competence bacterium BJ5183 to obtain the recombinant adenovirus-BMP-7 by efficient homologous recombination. Then the AdBMP-7 was obtained by packaging Pac I linearized in 293 cells. Adenoviral titer was determined by adenovirus fluorescent detection kit. The protein expression of BMP-7 in PTECs was respectively detected by ELISA and Western blot. RT-PCR method was used for analyzing the alpha-smooth muscle action (alpha-SMA) expression in PTECs, which was treated consecutively with TGF-beta and AdBMP-7.

RESULTSThe recombinant plasmid AdBMP-7 was successfully generated, which increased BMP-7 protein expression levels in PTECs, and down-regulated TGF-beta-induced alpha-SMA expression.

CONCLUSIONThe bioactive recombinant adenovirus AdBMP-7 has been successfully constructed, which may be effective in inhibition of chronic renal fibrosis.

Adenoviridae ; genetics ; metabolism ; Animals ; Base Sequence ; Bone Morphogenetic Protein 7 ; biosynthesis ; genetics ; Cells, Cultured ; Epithelial Cells ; metabolism ; Genetic Vectors ; genetics ; Kidney Tubules, Proximal ; cytology ; metabolism ; Molecular Sequence Data ; Rats ; Recombinant Proteins ; biosynthesis ; genetics

To investigate the function of exogenous unsaturated fatty acids in hyposmotic membrane stretch enhancement of muscarinic current (ICCh) in antral circular smooth muscle cells of guinea pig, we recorded the membrane current with the conventional whole cell patch-clamp technique. I(CCh) elicited by 50 micromol/L carbachol (CCh) at the holding potential of 20 mV under isosmotic condition was taken as control. Hyposmotic membrane stretch increased I(CCh) to 226.0+/-21.0%. When the cells were pretreated with 5 micromol/L arachidonic acid (AA), linoleic acid (LA) or oleic acid (OA), I(CCh)was inhibited to 3.8+/-0.6%, 35.2+/-0.8% and 66.6+/-0.6% respectively. Hyposmotic membrane stretch increased I(CCh) to 106.0+/-2.5%, 173.2+/-6.8% and 222.1+/-11.0% of the control respectively. Five micromol/L AA inhibited hyposmotic membrane stretch-enhanced I(CCh) by 51.2+/-3.8%, while the control I(CCh) under isosmotic condition was inhibited by 96.2+/-1.6%. The results suggest that unsaturated fatty acids inhibited I(CCh) and the inhibitory effect is more significant when the unsaturation degree is increased. However, the unsaturated fatty acids are not involved in the increase of I(CCh) induced by hyposmotic membrane stretch.
Animals ; Fatty Acids, Unsaturated ; pharmacology ; Guinea Pigs ; Membrane Potentials ; drug effects ; Myocytes, Smooth Muscle ; cytology ; physiology ; Osmotic Pressure ; Patch-Clamp Techniques ; Pyloric Antrum ; cytology ; physiology ; Receptors, Muscarinic ; physiology ; Sodium Chloride

To investigate the relationship between cytoskeleton and hyposmotic membrane stretch-induced increase in muscarinic current, the role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current was studied with the whole-cell patch clamp technique in guinea-pig gastric myocytes. In this study, the muscarinic current was induced by carbachol (50 micromol/L) or GTPgammaS (0.5 mmol/L). The results showed that hyposmotic superfusate (202 mOsmol/L) increased carbachol-induced current (I(CCh)) by 145+/-27% and increased GTPgammaS-induced current by 183+/-30%; but in the presence of cytochalasin-B (Cyt-B, 20 micromol/L), an actin cytoskeleton disruptor, hyposmotic membrane stretch increased I(CCh) by 70+/-6%. However, hyposmotic membrane stretch induced increase in I(CCh) was potentiated to 545+/-81% by phalloidin (20 micromol/L), an actin microfilament stabilizer. The results demonstrated that hyposmotic membrane stretch increased the muscarinic currents induced by carbachol or GTPgammaS and that the actin microfilament is involved in the process in guinea-pig gastric myocytes.
Actin Cytoskeleton ; physiology ; Animals ; Carbachol ; pharmacology ; Female ; Guinea Pigs ; Male ; Membrane Potentials ; drug effects ; Myocytes, Smooth Muscle ; physiology ; Osmotic Pressure ; Patch-Clamp Techniques ; Pyloric Antrum ; cytology ; Receptors, Muscarinic ; physiology

Periplaneta americana is an important medicinal insect. A series of new drugs developed from it have remarkable clinical effects and are in great demand in the market. Because of unclear biology, the quality and yield of P. americana are affected. Understanding the developmental threshold temperature and effective accumulated temperature of P. americana can provide theoretical basis for standardized culture of P.americana. Under climate chamber, the threshold temperature and effective accumulated temperature for egg development of P. americana to were determined through effective accumulated temperature law. The threshold temperature was （15.8±0.71）°C, the effective accumulated temperature was 415.8±38.05 degree days. A model of the relationship between temperature and developmental rates was established.
Animals ; Ovum ; physiology ; Periplaneta ; physiology ; Temperature