TMPyP4-regulated cell proliferation and apoptosis through the Wnt/ß-catenin signaling pathway in SW480 cells.

BACKGROUND: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. METHODS: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3ß, ß-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. RESULTS: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, ß-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. CONCLUSION: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/ß-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.

[Cloning and expression of actin gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene (Accession No. XM_002369622.1). The RT-PCR product was cloned into the prokaryotic expression pET-30a (+) vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5alpha. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was performed with anti-poly-histidine tag (anti-His) antibody or rabbit anti-T. gondii serum. RESULTS: The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a-TgACT was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21(DE3) in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T. gondii serum. CONCLUSION: The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.

Gene-cloning, expression and antigenicity analysis of rhoptry protein 17 of Toxoplasma gondii.

OBJECTIVE: To clone and express the rhoptry protein 17 (ROP17) gene of RH strain of Toxoplasma gondii, analyze the antigenicity of recombinant protein. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgROP17 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgROP17 gene (GenBank accession No. AM075203.1), the product of RT-PCR was digested with double restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-TgROP17 plasmid was transferred into E. coli DH5alpha and the positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The constructed pGEX-6P-1-TgROP17 was transformed into E. coli Rosetta (DE3) and induced with IPTG for expression. The expression products were analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with GST primary antibody and rabbit anti-T. gondii serum was used to confirm the expression of GST-ROP17 and analyze its antigenic properties. RESULTS: The product of RT-PCR was with 1 850 bp. The recombinant plasmid pGEX-6P-1-TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. A soluble recombinant protein with relative molecular weight of 96,000 was analyzed by SDS-PAGE followed by Coomassie blue staining. The GST tag in GST-ROP17 and the antigenicity of ROP17 were detected efficiently by Western blotting with the GST primary antibody and with the prepared antiserum against T. gondii, respectively. CONCLUSION: The recombinant GST-ROP17 protein has been produced in E. coli and shows specific antigenicity.

[Cloning, expression and antigenicity analysis of phosphoglycerate mutase 2 gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein. METHODS: Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5alpha first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its antigenicity. RESULTS: The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum. CONCLUSION: The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.

[Effect of oral administration of tribendimidine at different dosages against Trichinella spiralis encapsulated larvae in mice].

OBJECTIVE: To observe the efficacy of oral administration of tribendimidine (TBD) at different dosages against Trichinella spiralis encapsulated larvae in murine striated muscle. METHODS: A total of 88 BALB/c mice were divided equally into 11 groups. Each mouse was infected orally with 50 T spiralis encapsulated larvae. At day 29 after infection, TBD was each orally administered to mice of the 11 groups with doses of 0 (control group), 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mg/(kg x d), respectively. All mice were administered once a day and lasted for 6d, and untoward drug reactions for mice were observed. Mice were sacrificed at the 7th day after administration of TBD, the encapsulated larvae in diaphragmatic muscle, jugomaxillary muscle, pectoral muscle and gastrocnemius muscle were examined by pellet method, and the total, survival and dead worms were counted. The therapeutic effect was estimated on the basis of average quantity of encapsulated larvae per gram muscle. RESULTS: During the administration period, no untoward reaction were observed in mice of 50-300 mg/(kg x d) groups. Mice in 350 and 400 mg/(kg x d) groups showed body hair dishevelment, emaciation and food-intake decrease, death rates were 25% and 50%, respectively. All mice in 450 and 500 mg/(kg x d) groups died on day 4 and 5 after TBD administration, respectively. In control group, the highest total burden (per gram) was found in diaphragmatic muscle, followed by jugomaxillary muscle, gastrocnemius muscles and pectoral muscles. TBD at dose of 50 mg/(kg x d) was unable to kill encapsulated larvae. In the rest groups, with the increase of drug dose, the total worm burden and the number of survival worms showed a decreasing trend in four kinds of muscles, and were significantly lower than that of the control group (P < 0.05 or P < 0.01). In 300 mg/(kg x d) group the number of survival worms in diaphragmatic muscle, jugomaxillary muscle, pectoral muscle and gastrocnemius muscle [8.6 +/- 1.7, 2.8 +/- 0.7, 3.9 +/- 0.8, and 0, respectively] were significantly lower than that of the control group [3648.1 +/- 989.2, 1266.4 +/- 812.3, 701.9 +/- 196.4, and 711.6 +/- 34.6] (P < 0.01). All encapsulated larvae in the four kinds of muscle died in 350 and 400 mg/(kg x d) groups. With the increase of TBD dosage, the mortality of encapsulated larvae increased in the muscles, reached up to 98.6%--100% in 300 m (kg x d) group (P < 0.01), and 100% in 350 and 400 mg/(kg x d) groups (P < .01). CONCLUSION: Oral tribendimidine administered at 50 mg/(kg x d) to mice for 6 d is unable to reduce worm burden in muscle. Tribendimidine 300 mg/(kg x d) effectively kill encapsulated larvae and is a suitable dose against encapsulated larva stage. However, tribendimidine at doses of 350 mg/(kg x d) and above for 6d is toxic to mice and even causing death.

[Early kinetics of Toxoplasma gondii infection in mice infected intragastrically with tachyzoites by chromogenic in situ hybridization targeting SAG2 mRNA].

OBJECTIVE: To observe the early kinetics of Toxoplasma gondii infection in mice inoculated with tachyzoites of RH strain. METHODS: Twenty BALB/c mice were administered intragastrically with tachyzoites of RH strain (2 x 10(4)/mice). Parasite burdens in mesenteric lymph node (MLN), liver, spleen, lung and brain were determined by chromogenic in situ hybridization targeting SAG2 mRNA at 1, 2, 4, 6 and 8 days postinfection. Five mice were inoculated with PBS as blank control. RESULTS: The MLN, liver and spleen were the first organs where tachyzoites were found on the first day after infection, followed by the lungs on the 4th day and the brain on the 6th day. On days 6 to 8 after infection, there was a significant difference on parasite load among the tissues (P < 0.05), and the parasite load in MLN was highest, followed by that of liver, spleen, lungs and brain. The number of tachyzoites in various tissues was time-dependent. CONCLUSION: T. gondii tachyzoites were first detected in MLN, liver and spleen, then in the lungs, and finally in the brain. The number of tachyzoites in the MLNs increased more rapidly.

[Kinetics of IgA secreting cells and IgA in small intestine of mice induced by Toxoplasma gondii tachyzoite infection].

OBJECTIVE: To investigate the kinetics of IgA secreting cells (IgASCs) in small intestine and the specific antibody level induced by Toxoplasma gondii tachyzoite infection in mice. METHODS: Ninety-six BALB/c mice were randomly divided into 2 groups, 12 were intragastrically given 0.5 ml PBS as control, the rest were each intragastrically infected with 1 x 10(4) tachyzoites of the virulent RH strain Toxoplasma gondii. On the day 2, 4, 6, 8, 10, 12, and 14 post infection, 12 mice were sacrificed respectively. The quantity of IgASCs in mucosa of duodenum, jejunum and ileum was detected by immunohistochemistry analysis. IgA in sera and in intestinal washes was determined by ELISA. RESULTS: The IgASCs were found in lamina propria of the small intestine mucosa. The amount of IgASCs in duodenal mucosa increased gradually with the time after infection, while in jejunal mucous membrane it increased from the day 2 to 8, and then decreased to the level of before infection on day 14. The amount of IgASCs in ileal mucous membrane also increased from day 2 to 6, then descended gradually and on the day 12 to a level lower than that of before infection. IgA level in the intestinal washes increased continually but there was no significant change in serum samples. The correlation between IgA level in intestinal washes and the quantitative change of IgASCs in mucosa of duodenum, jejunum and ileum was r=0.732 (P<0.01), r=0.116 (P>0.05) and r=-0.429 (P<0.01), respectively. CONCLUSION: Toxoplasma gondii infection induces a high level expression of IgASCs in duodenum and an increase of IgA antibody in the intestinal washes, showing a positive correlation. The high level IgA in the intestinal washes is mainly from IgASCs of the duodenal mucosa.

[Kinetics of IgA secreting cells and sIgA in small intestine of mice induced by intranasal immunization with Toxoplasma gondii STAg].

OBJECTIVE: To investigate the kinetics of IgA secreting cells (IgASCs) and secretory IgA (sIgA) level in small intestine induced by intranasal immunization with Toxoplasma gondii soluble tachyzoite antigen (STAg) in mice. METHODS: Ninety-six 5 to 6-week old BALB/c mice were randomly divided into immunity and control groups. Mice of the immunity group were each intranasally immunized with STAg 20 microg in 20 microl PBS, twice at an interval of 2 weeks, while the control mice were each given 20 microl PBS. All mice were challenged intragastrically with 1 x 10(4) tachyzoites in 0.5 ml per mouse in 1 week after the last immunization. The body weight and infection incidence of mice were recorded. Eight mice of each group were sacrificed on the day 6, 7, 8, 9, 10 and 11 post infection, respectively. The quantity of IgASCs in mucosa of duodenum, jejunum and ileum was detected by immunohistochemistry. The sIgA in intestinal washes were determined by ELISA. RESULTS: All mice fell ill post infection, but the symptom of mice in the immunity group was milder, the increasing level of body weight of mice in the immunity group was higher considerably than that in the control group (P < 0.05). Two mice died in control group on the 7th day after infection. sIgA level in intestinal washes increased continually in two groups, but the increasing level in the immunity group was higher than that of the control (P < 0.05). The number of IgASCs in duodenum increased slightly in the control group, but increased continuously and maintained a high level after 9 d in the immunity group, for instance, 20.65 +/- 1.67 in the immunity group and 12.30 +/- 2.67 in the control. The correlation of the sIgA level in intestinal washes and the quantitative change of IgASCs in duodenum was positive in the immunity group (r = 0.566, P < 0.05) and the control (r = 0.378, P < 0.05). The number of IgASCs in jejunum decreased in the control group but increased then slightly decreased after 9 d in the immunity group. Positive correlation between the sIgA level in intestinal washes and the quantitative change of IgASCs in jejunum was found in the immunity group (r = 0.218, P > 0.05) but negative in the control (r = -0.557, P < 0.05). The number of IgASCs in ileum declined in the control group but maintained a high level in the immunity group. The correlation between the sIgA level in intestinal washes and the quantitative change of IgASCs in ileum was r = -0.053 (P > 0.05) in the immunity group and r = -0.685 (P < 0.05) in the control. CONCLUSION: Intranasal immunization with STAg in mice orally infected with Toxoplasma gondii can increase the number of IgASCs in jejunum and ileum, and enhance the immune barrier function of mucosa in small intestine of mice.

[Dynamic observation of attachment and invasion of Toxoplasma gondii tachyzoites to intestinal mucosa in BALB/c mice by chromogenic in situ hybridization targeting SAG2 mRNA].

OBJECTIVE: To observe dynamically the location and time of attachment and invasion of Toxoplasma gondii tachyzoites to murine intestinal mucosa by chromogenic in situ hybridization targeting SAG2 mRNA. METHODS: Thirty 7- to 8-week-old BALB/c mice were randomly divided into experiment group (24 mice) and control group (6 mice). Each animal in the experiment group was given 2 x 10(4) tachyzoites of RH stain in 0.2 ml PBS by intragastric administration and that in the control group was given 0.2 ml PBS. Four mice in the experiment group and one in the control group were sacrificed at 15 min, 30 min, 1 h, 2 h, 4 h and 8 h after infection, respectively, and paraffin sections of duodenum, jejunum and ileum were prepared to perform the in situ hybridization with Dig-labeled oligonucleotide probe complementary to SAG2 mRNA of T. gondii. RESULTS: Tachyzoites were found on the striated border of small intestine epithelial cells (absorptive cells, goblet cells and endocrine cells), in or between two absorptive cells or in the lamina propria. At 15 min-2 h after infection, there was significant difference in the number of attachment on jejunum and ileum (P<0.05); the number of invasion in jejunum was significantly higher than in duodenum and ileum at minute 15 and 30 after infection (P<0.05) . Following the lapse of time, the number of attaching tachyzoites gradually reduced, whereas the number of invading tachyzoites gradually increased. Compared with 15 min after infection, for all the intestinal sections, the number of attachment significantly reduced at 8 h after infection (P<0.05), in contrast, the number of invasion significantly increased at 4 h and 8 h after infection (P<0.05) . Between 4 h and 8 h after infection, a significant increase in the number of invasion was showed in jejunum and ileum (P<0.05). CONCLUSION: Although the cell selectivity of attachment has not been observed, the location selectivity of invasion is present, jejunum is more susceptible to the tachyzoite invasion.