Label-free analysis of membrane protein binding kinetics and cell adhesions using evanescent scattering microscopy.

Measuring ligand interactions with membrane proteins in single live cells is critical for understanding many cellular processes and screening drugs. However, developing such a capability has been a difficult challenge. Here, we employ evanescent scattering microscopy (ESM) to show that ligand binding to membrane proteins can change the cell adhesion properties, which are intrinsic cell properties and independent of random cell micromotions and ligand mass, thus allowing the kinetics analyses of both proteins and small molecules binding to membrane proteins in both single fixed and live cells. In addition, utilizing the high spatiotemporal resolution of ESM, the positions of cell adhesion sites can be tracked in real-time to analyze the cell deformations and migrations, thus providing a potential approach for understanding the cell activity during the ligand binding process in detail. The presented method may pave the road for developing a versatile and easy-to-use label-free detection strategy for in situ analysis of molecular interaction dynamics in living biosystems with single-cell resolution.

[Cloning and expressing cagT gene of type IV secretion system in Helicobacter pylori and influence of cytokine secretion and cell proliferation on SGC-7901 cell].

OBJECTIVE: To detect the influence of the CagT protein on interleukin-8 secretion and proliferation in SGC-7901 cells. METHODS: Helicobacter pylori cagT gene was amplified by PCR with the genomic DNA of H. pylori NCTC 11637 as template, then it was inserted into an expression vector pQE30. The recombinant plasmid was transformed into E. coli M15. Recombinant protein was expressed by Isopropylthio-beta-D-Galacgoside (IPTG) induction and confirmed by Western blot. Fusion protein with 6xHis tag was purified using Ni(2+)-NTA agarose. Interleukin-8 mRNA expression of SGC7901 cells was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). RESULTS: The GenBank accession number of the amplified sequence is EF114758. The sequence analysis for cagT showed that it shares 97%-99% homology with other strains of H. pylori in Gene bank. The molecular mass of the product is 32kDa, and its purity is 98% analyzed by SDS-PAGE. After the protein was dialyzed, it can stimulate SGC7901 cells to express interleukin-8 and the growth of cells was inhibited in a dose- and time-dependent manner. CONCLUSION: It is indicated that we have obtained the correct cagT gene and expressed in E. coli M15. The protein can stimulate the cells to express cytokine interleukin-8 and inhibit proliferation of cells, which posed a basis for further research on its biological function.

Mutations in Helicobacter pylori porD and oorD genes may contribute to furazolidone resistance.

AIM: To determine the rate of furazolidone resistance of Helicobacter pylori (H. pylori) isolated from gastric biopsy specimens and to explore the relationship between genetic mutations in porD and oorD genes of H. pylori and its resistance to the antibiotic. METHODS: Gastric biopsy was performed in 83 adult patients aged 31-77 years with gastric complaints. H. pylori was isolated from biopsy specimens of 46 patients. E-test and 2-fold agar dilution method were used to determine the rate of H. pylori resistance to furazolidone. The genes porD and oorD from susceptible and resistant isolates were amplified by polymerase chain reaction (PCR), and their PCR products were sequenced. RESULTS: Resistance to furazolidone was found in 8.7% of H. pylori isolates and 6 mutations were detected in porD and oorD genes of the resistant isolates. Three mutations--G353A, A356G, and C357T--occurred in porD and the other mutations--A041G, A122G, C349A(G)--occurred in oorD genes. CONCLUSIONS: Changes in 6 amino acids may be associated with the resistance of H. pylori to furazolidone.