Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Effects of 50 Hz low voltage electric stimulation on angiogenesis and the expression of VEGF in the ischemic myocardium / 中华物理医学与康复杂志

Objective To study the effects of 50 Hz low voltage electric stimulation on angiogenesis and the expression of VEGF in the ischemic myoeardium in rats. Methods Twelve Wistar rats with experimentally induced myocardial ischemia were randomly divided into an electric stimulation (ES) group and a control group. The animals in the electric stimulation group were electrically stimulated at 50 Hz and 0.3 V through electrodes implanted in the epicardium of the anterior wall of the left artrium. Those in the control group were delivered sham electrical stimula- tion through similar implanted electrodes. Immunohistochemistry was used to count endothelial cells (ECs) and measure the capillary density (CD). A reverse transcription polymerase chain reaction (RT-PCR) was employed to measure the expression of VEGF protein and VEGF mRNA. Results The number of ECs, and the CDs in the ES group were significantly greater than in the control group, and the expression of VEGF protein and VEGF mRNA was also significantly higher. Conclusions Low voltage electrical stimulation at 50 Hz can promote angiogenesis and the expression of VEGF in the ischemic myocardium in rats.