Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells.

Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production--an important step that mediates membrane raft clustering--was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation.

SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium.

The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91(phox), and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function.

Oral vaccination of mice against rodent malaria with recombinant Lactococcus lactis expressing MSP-1(19).

AIM: To construct the recombinant Lactococcus lactis as oral delivery vaccination against malaria. METHODS: The C-terminal 19-ku fragments of MSP1 (MSP-1(19)) of Plasmodium yoelii 265-BY was expressed in L. lactis and the recombinant L. lactis was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythrocytic stage. The protective efficacy of recombinant L. lactis was evaluated. RESULTS: The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8+/-0.4% and 20.8+/-26.5%, respectively, and those of their control groups were 12.0+/-0.8% and 60.8+/-9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION: The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.

Effect of F209S Mutation of Escherichia coli AroG on Resistance to Phenylalanine Feedback Inhibition.

In Escherichia coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase was encoded by aroG gene. The aroG gene was amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant strain resistant to phenylalanine analogues. The PCR products were cloned and subject to DNA sequence analysis. A single base mutation of Tright curved arrow C was detected at nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene product. The gene was expressed on pTrc99A in E.coli strain JM105. Under the induction of IPTG, distinct band with the expected molecule weight was detected on SDS-polyacrylamide gel electrophoresis and the specific activity of DAHP of the crude extract of the transformed cells increased by 1.8-fold. Enzyme activity inhibition analysis revealed the high resistance of mutant AroG to feedback inhibition by phenylalanine. JM105 cells harboring with mutant aroG gene showed were able to grow on medium containing higher concentration of analogues than that carrying normal aroG gene. Discussion was focused on the varieties of mutations contributing to desensitization of feedback inhibition.