[Fret-based single-molecule probes for monitoring induced activation of Rac, Cdc42 signaling pathways in living cells].

Rho GTPases, including Rac1, Cdc42, play a critical role in the regulation of a variety of cellular processes such as cell morphology, cell migration, transcriptional activation and gene expression. We constructed several FRET-based single-molecule probes containing red fluorescent protein dsRed1, cyan fluorescent protein ECFP, the GTPase binding domain of the effector, Pak1 or N-WASP, and Rac1 or Cdc42. Rac1 and Cdc42 signaling pathways were activated in transfected cells by the inducer, insulin or bradykinin respectively. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in transfected NIH3T3 and Hela cells. For all 3 signaling pathways in NIH3T3 cells, the values of FRET efficiency reached the highest after induction for 5 min, but the increasing extents of the values of FRET efficiency varied in 3 signaling pathways. The values of FRET efficiency decreased with the extention of the induction time, but differed significantly in the decreasing speed for the signaling pathways. Rac1 and Cdc42 activation assays indicated that Rac1 and Cdc42 were in the activated state (GTP-bound) in the induced cells. Their relative activated activities in the cells induced for different time were consistent with the values of FRET efficiency. The activated Rac1, Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively. The results showed that these single-molecule probes could be used to directly monitor the spatial and temporal imaging of the induced activation of the signaling pathways in living cells. With these single-molecule probes, the GEF or GAP activities of putative regulatory proteins for Rac1 and Cdc42 were analyzed and judged, thus greatly simplifying the currently-used methods for identifying the regulatory proteins for Rho GTPases.

Comparison of mitochondrial cytochrome oxidase 1 DNA sequences from Necator americanus hookworms maintained for 100 generations in golden hamsters (Mesocricetus auratus) and hookworms from natural human infections.

The human hookworm Necator americanus was maintained through one hundred generations in the golden hamsters. The strain is now routinely maintained in laboratory hamsters through serial passage, and is the laboratory strain of choice for vaccine studies. Comparison of the mitochondrial cytochrome oxidase 1 (cox-1) sequences was shown previously to be useful for comparing the genetic structure of populations of N. americanus in China. Cytochrome oxidase 1 genes were amplified by the polymerase chain reaction, and the sequences compared to those of N. americanus recovered from infected humans from several regions in China. Sequence comparison revealed little difference between the laboratory strain and the field isolates at the cox-1 locus, but also indicated that the laboratory strain is represented by a single cox-1 haplotype. These results suggest that the laboratory strain of N. americanus has undergone a severe genetic bottleneck, and that the genetic diversity in other genes, including potential vaccine antigens, could be similarly limited.

[Polymorphism analysis of cytochrome C oxidase 1 (COI) gene in Necator americanus collected from 5 provinces in China].

OBJECTIVE: To detect and compare the COI gene sequences of Necator americanus collected from the provinces of Sichuan, Hainan, Yunnan, Hubei and Jiangsu, and to analyze the genetic diversity of the geographic isolates. METHODS: COI genes of N. americanus were amplified from the genomic DNA by PCR and sequenced. RESULTS: The COI gene sequences of N. americanus from five provinces were 97%-99% identical over 595 bp, and base variation occurred in 19 nucleotide sites in which the transition was more frequent than the transversion. The difference between the sequences ranged from 1.34% to 2.18%. CONCLUSION: The COI gene sequences show high identity among the geographic isolates of N. americanus with some difference at specific nucleotide sites.

[Cloning and expression of specific antigen genes of Ancylostoma caninum].

OBJECTIVE: To search for the gene encoding specific antigen of Ancylostoma caninum that induces host's protective immunity. METHODS: A lambda ZAP II cDNA library of A. caninum was screened with sera from dogs immunized subcutaneously with hookworm larvae(L3). After sequencing, insert gene (AcAg) from positive clones was ligated into PUC18 and PET28C. Recombinant pET28C plasmid was induced to expressed protein in the E. coli BL21. The characteristic of recombinant protein is analyzed by SDS-PAGE and Western blotting assay. RESULTS: Five positive clones were obtained, and proved to be the same. The insert gene (AcAg) in pET28C vector expressed a recombinant protein of about 43 kDa. Using Western blotting assay, this protein was recognized by the sera from dog immunized with Ancylostoma caninum third-stage infected larvae and used for screening library. CONCLUSION: The AcAg, which exhibits 35% homologous to Caenorhabditis elegans gene unc-89, is a novel specific antigen of A. caninum. Its ability to elicit the protective immunity and the probability of the recombinant protein as a vaccine need to be further evaluated.

[Reexamination of specific antibodies in sera of cystic echinococccosis patients with IgG negative seroresponse].

OBJECTIVE: To explore the factors of false negative antibody response in patients with echinococcosis granulosus (Eg) for improving immunodiagnosis. METHODS: Indirect ELISA and sandwich ELISA were used to detect the specific antibody of IgG subclass (IgG1, IgG2, IgG3, IgG4) and IgA, IgM, IgE, as well as circulating antigen (CAg) and immunocomplex (CIC) in sera of Eg patients with negative response of total IgG. RESULTS: Among 42 sera with IgG negative seroresponse, 32 were positive with IgG subclass, IgA, IgM or IgE antibody, 10 were negative in all 7 kinds of antibody response. The detection rate of specific IgG1, IgG4, IgA, IgM and IgE was 42.9%, 11.9%, 28.6%, 26.2% and 21.4% respectively, being significantly higher than in sera of the control. IgM level in children was higher than that in adults. IgG subclass in patients with liver Eg was higher than that of pulmonary hydatidosis, when testing with IgG1 combined one or two of the other six Ig antibodies. The highest positive rate (64.3%) was seen in IgG1 + IgA + IgM antibody system. The positive rate of CAg and CIC in IgG negative patients was 28.57% and 30.95%, respectively. CONCLUSION: The factors involved with seronegative response of total IgG in Eg patients might be low specific IgG, variant Ig antibody expression and formation of CIC. Combined detection of IgG1 + IgA + IgM could enhance the sensitivity of serological tests in Eg patients.