Enzyme Electrophoresis Analysis Research Articles

Specific cytotoxicity of epidermal groth factor variantIII oriented chimeric antigen receptor-engineered T cells on SMMC-7721

Objective To observe the specific killing effect of anti-epidermal groth factor variantⅢ (EGFRvⅢ) chimeric antigen receptor modified T cells (EGFRvⅢ/2CAR-T) on liver cancer cell SMMC-7721. Methods Expression cassette of EGFRvⅢ/2CAR (1 374 bp) was inserted into XbaⅠ and BamHⅠ sites of piggybac transposon vector PB513B-1 (PB-EGFRvⅢ/2CAR), the latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation (1×20 ms, 840 V). The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry. The expression of EGFRvⅢ in SMMC-7721 was detected by Western blotting. After EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721, the killing effect was detected by lactate dehydrogenase (LDH) release test and cytokine interferon (IFN)-γ secretion. Results The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaⅠ and BamHⅠ enzyme digestion, electrophoresis and DNA sequencing analysis. The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry (55.5%). Western blotting analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells, which corresponds to the calculated molecular weight of EGFRvⅢ. EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24 h, LDH release test showed that tumor specific killing was positively correlated with E∶T ratio (E∶T=1∶1, 5∶1, 10∶1, 15∶1, 20∶1); enzyme linked immunosorbent assay (ELISA) showed that cytokine IFN-γ secretion was (1 060.1±89.0) pg/ml, which was significantly higher than control group (P=0.000). Conclusion Anti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ+ liver cancer cell SMMC-7721, providing research basis for immunotherapy of hepatic cancer. Key words: Epidermal groth factor variantⅢ; Chimeric antigen receptor; Immunotherapy; PiggyBac transposon

Characterization of CSF2A fusion gene and its effect on Epstein-Barr virus-positive tumor cells.

We build the latent membrane protein gene latent membrane protein 2A (LMP2A) and the granulocyte-macrophase colony-stimulating factor (GM-CSF) gene fusion gene (CSF2A) and discuss how the CSF2A fusion protein influenced the proliferation and apoptosis of Epstein-Barr virus-positive (EBV+ ) tumor cells. Reverse-transcription polymerase chain reaction (RT-PCR) method was used to amplify the LMP2A gene and GM-CSF gene fragments, respectively, according to the principle of overlap extension in the coding (Gly4Ser)3 polypeptide gene fragments of DNA restructured under the connection. The CSF2A gene could be connected with the pIRES2-enhanced green fluorescent protein vector by recombinant DNA technology and identified by enzyme electrophoresis analysis and DNA sequencing. Then, the recombinant vector was transfected into dendritic cells (DCs); RT-PCR and Western blot analysis were used for testing the CSF2A gene messenger RNA and protein expression. The impacts of CSF2A on the proliferation and apoptosis of EBV+ tumor cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hochest 33342 staining. We successfully obtained the recombinant vector named pIRES2-CSF2A. The expression of CSF2A could be detected by transfecting pIRES2-CSF2A into DCs. The DCs were cocultured with T lymphocytes and then acted on the EBV+ CNE2 nasopharyngeal carcinoma cells. MTT assay showed that the inhibiting effect of CSF2A obviously increased and the time dependency (**P < 0.01, *P < 0.05) also existed. Hochest 33342 staining showedapoptosis morphological changes of cells in nucleus staining and generated the apoptotic body. Apoptosis cells of the pIRES2-CSF2A group increased significantly at 48 hours. The results showed that the pIRES2-CSF2A recombinant vector was effectively transfected into DCs and the fusion gene CSF2A could promote EBV+ CNE2 cell apoptosis, laying the foundation for the specificity of EBV+ tumor targeting immune gene therapy in the future.

Multilocus enzyme electrophoresis typing of Candida albicans populations isolated from healthy children according to socioeconomic background

The aim of this research was to evaluate the genetic diversity within and between C. albicans populations isolated from the oral cavity of healthy Brazilian children classified into five socioeconomic categories (A to E). Multilocus Enzyme Electrophoresis (MLEE) analysis was the method used to assess genetic diversity. High genetic diversity was observed in all populations that showed predominance of some C. albicans subtypes (Electrophoretic Types - ETs). However, no correlation was observed between a specific ET and a specific population of children. Clustering analysis showed one or more highly related ET clusters, suggesting the existence of indirect and direct propagation routes of C. albicans among healthy children. Microevolutionary changes were observed in some C. albicans populations isolated from children with the same or very similar socioeconomic condition. Furthermore, low transition of C. albicans subtypes can be occurring among certain populations of children coming from high and medium/high, or high and medium/low, or medium/high and medium/low socioeconomic categories, which can also be explained by their own socioeconomic and cultural characteristics.

Epidemiology of meningococcal disease in Denmark 1974–1999: contribution of the laboratory surveillance system

The Danish meningococcal disease laboratory surveillance system was established in 1974, based on close collaboration between local Departments of Clinical Microbiology and the Reference Laboratory at Statens Serum Institut. The completeness of the clinical notification system integrated with the laboratory surveillance system has been estimated to be more than 95%. Overall 4257 (79%) of 5356 cases of meningococcal disease notified during 1974-1999 were confirmed by culture of Neisseria meningitidis. The proportion of culture-confirmed cases ranged from 70% in 1989 to 89% in 1980. Only 26 patients (0.6%) with culture-confirmed meningococcal disease were not notified. Serological phenotype and susceptibility to penicillin and sulphonamide were determined for all isolates. Multilocus enzyme electrophoresis and/or DNA-based analyses were used for the assessment of clusters and outbreaks. Meningococcal antibody tests and counter-immunoelectrophoresis were used for the ascertainment of suspected cases. These combined systems allowed timely and reliable management of outbreaks and identification of clusters.

High level of genetic divergence between sympatric color morphs of the littoral sea anemone Anthopleura orientalis (Anthozoa: Actiniaria).

Using enzyme electrophoresis and nematocyst analysis, the sympatrically occurring "light" and "dark" color morphs of the sea anemone Anthopleura orientalis from the Sea of Japan were shown to be two valid species. The "light" morph was identified as A. orientalis (Averintsev, 1967 Issledovaniya fauny morei: Vyp. 5 (13). Nanka, Leningrad, pp. 62-77), while the "dark" morph was designated as Anthopleura sp. The analysis of 21 isozyme loci revealed high value of Nei's genetic distance (D=1.284) between the two species, which are indistinguishable in their external morphology. The mean values of observed and expected heterozygosities for A. orientalis and Anthopleura sp. are high (H(o)=0.252+/-0.061, H(e)=0.250+/-0.061 and H(o)=0.327+/-0.052, H(e)=0.351+/-0.054, respectively). The species differ significantly in the size of spirocysts and nematocysts, among which the atrichs from acrorhagi and the basitrichs from actinopharynx contribute most to the observed difference. Strong qualitative difference is revealed between distributions of nematocysts in mesenteric filaments of the two sea anemone species studied. The possible conspecificity of Anthopleura sp. with Anthopleura artemisia (Dana, 1848) is discussed and the conclusion made that these are two separate species.

CHARACTERIZATION OF INVASIVE CONYZA SPECIES (ASTERACEAE) IN EUROPE: QUANTITATIVE TRAIT AND ISOZYME ANALYSIS

The genus Conyza (Asteraceae) represents one of the foremost examples of intercontinental plant invasions from the New World to the Old World that has resulted in a number of taxonomic problems owing to the dearth of knowledge concerning the biosystematics of the genus. In this study, quantitative trait and enzyme electrophoresis analysis was used to assess the pattern of variation within and among five short‐lived taxa of Conyza (C. blakei, C. bonariensis, C. canadenis, C. floribunda, C. sumatrensis), introduced into Europe, and to test the hypothesis of a recent hybrid origin of C. floribunda. The five taxa exhibit marked differences in morphological and life‐history characteristics that are concordant with divergence in genes encoding isozymes. In addition, a recent hybrid origin of C. floribunda is clearly not supported by either quantitative or electrophoretic data. These findings suggest that the five taxa differentiated genetically in their native range prior to their introduction in Europe. As formerly suggested by Arthur Cronquist (Bulletin of the Torrey Botanical Club 70: 629–632, 1943), C. canadensis appears to be more closely related to the genus Erigeron than the other taxa. The electrophoretic results also confirm that this particular species is diploid, while the presence of multiple bands and fixed heterozygosity demonstrates that the other Conyza taxa are all genetic allopolyploids. Both quantitative and electrophoretic data suggest the presence of two groups among the polyploid taxa: one comprising C. bonariensis and C. sumatrensis, the other C. blakei and C. floribunda. The latter grouping is supported by the observation that C. blakei and C. floribunda, both native to Argentina, exhibit a striking ability to shift from semelparous to iteroparous reproduction, while the other taxa are strictly semelparous.

Characterization ofListeria strains from a foodborne listeriosis outbreak by rDNA gene restriction patterns compared to four other typing methods

The rDNA gene restriction patterns of 134 isolates of Listeria species were determined with pKK3535--a pBR322 derived plasmid containing an Escherichia coli rRNA operon--used as a probe following digestion of chromosomal DNA by EcoRI endonuclease. Nineteen reference and type strains representing all species and serotypes of Listeria showed 17 distinct ribotypes. One hundred and fifteen wild strains of Listeria monocytogenes were ribotyped and the results were compared to those of serotyping, phage typing, multilocus enzyme electrophoresis (MEE) and restriction endonuclease analysis (REA). Ninety-six Listeria monocytogenes serotype 4b wild strains displayed six distinct ribotypes (I-VI), 72% (69/96) of them clustering in two very close rDNA patterns (I and II) of eight and nine bands, respectively. The same 96 strains displayed six REA patterns and eight MEE electrotypes. Among the 96 Listeria monocytogenes 4b isolates, the 34 epidemic strains defined by phage typing and by epidemiological data all belonged to one ribotype (ribotype I) representing 56% of the strains belonging to this ribotype. These same 34 epidemic strains were also grouped by REA and MEE typing in a unique profile (REA-A) and MEE electrotype (ET 1). Twenty-two Listeria monocytogenes strains of serogroup 1/2 analyzed by rDNA typing showed nine distinct ribotypes. For the 96 Listeria monocytogenes 4b strains studied, the discriminatory index was highest for phage typing and for any combination including phage typing. Ribotyping appears to be a well reproducible molecular typing method and could be a useful complement to other typing methods for the epidemiological study of listeriosis.