The protective role of vitamin C on intestinal damage induced by high-dose glycinin in juvenile Rhynchocypris lagowskii Dybowski.

This study was to evaluate the mitigative effects of vitamin C (VC) on growth inhibition and intestinal damage induced by glycinin in juvenile Rhynchocypris lagowskii Dybowski. 270 healthy juvenile Rhynchocypris lagowskii Dybowski (4.65 ± 0.04 g) were randomly divided into 3 treatments, and fed with control diet, 80 g/kg glycinin diet and 80 g/kg glycinin+200 mg/kg VC diet respectively for 8 weeks. The results showed that glycinin significantly decreased the weight gain rate, specific growth rate, protein efficiency rate, feed efficiency rate and feeding rate of fish compared with the control group (P < 0.05), while VC supplementation improved the growth performance and feed utilization efficiency, and reached a level similar to the control group. Similarly, VC significantly increased the crude protein content of muscle and whole-body, and hepatopancreas and intestinal protease activities of fish fed with glycinin diet (P < 0.05). The distal intestine of fish in glycinin group showed typical damage characteristics, including breakage and atrophy of intestinal mucosal fold, and increased intestinal mucosal permeability. However, fish fed the glycinin + VC diet showed an unimpaired normal intestinal morphology. Usefully, VC supplementation could also restore impaired immune function and antioxidant capacity. VC down-regulated the mRNA levels of pro-inflammatory cytokines TNF-α and IL-1ß, and up-regulated the mRNA levels of anti-inflammatory cytokines IL-10 and TGF-ß in the distal intestine of fish fed with glycinin. Furthermore, glycinin exposure could reduce the mRNA levels of HO-1, CAT and GPx by inhibiting the activation of Nrf2-Keap1 signaling pathway, while VC supplementation reversed this phenomenon and maintained the homeostasis of antioxidant defense system. Concluded, glycinin causes growth inhibition, digestive dysfunction and intestinal damage of Rhynchocypris lagowskii Dybowski, while sufficient VC intake is beneficial for fish to resist the adverse effects of glycinin.

Monitoring oxidative stress, immune response, Nrf2/NF-κB signaling molecules of Rhynchocypris lagowski living in BFT system and exposed to waterborne ammonia.

The present study was implemented to evaluate oxidative stress, immune response, Nrf2 and NF-κB signaling molecules related genes expression of Rhynchocypris lagowski living in biofloc technology (BFT) system and exposed to waterborne ammonia. According to the differences of C:N ratios, the experiment was divided into four groups: C:N 10.8:1 (control group), C:N 15:1, C:N 20: 1 and C:N 25:1. The results demonstrated that BFT can effectively regulate water quality and promote growth, and the C:N 20:1 group has the most significant effect (P < 0.05). Besides, significant increases in immune enzymes (lysozyme, complement C3, C4, immunoglobulin M and nitric oxide synthase) and anti-inflammatory factor (IL-2) activity of R. lagowski were emerged in the treatment C:N 20:1 after the 56-d growth experiment and the challenging trial (P < 0.05). Comparing the antioxidant status of R. lagowski in liver and gut before and after ammonia stress: superoxide dismutase, total antioxidant capacity and catalase activity in treatments C:N 20:1 were significant increased (P < 0.05), while the level of malondialdehyde was marked lower than that in control. Meanwhile, treatment C:N 20:1 considerably upregulated Nrf2 signaling molecules related genes and significantly down-regulated the expression of pro-inflammatory factor gene in NF-κB signaling pathway compared with the control (P < 0.05). These results indicated that BFT could enhance growth, antioxidant and immune response and regulate Nrf2 and NF-κB related genes expression in R. lagowski, with most excellent effects in fish given C:N 20:1 group.

[Regulatory Role of Nitric Oxide in Development and Hatching of Mouse Blastocysts].

OBJECTIVE: To determine the regulatory role and mechanism of nitric oxide (NO) in the development and hatching of mouse blastocysts. METHODS: The Kunming female mice were superovulated and then mated with mature male mice. On the day 2.5 of their pregnancy, morulae were flushed from their uterine horns with culture media. Morulae were cultured in different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), or the combination of L-NAME and SNP in culture media for 48 hours. The development and hatching of blastocysts were examined on day 4 and day 5 and the total numbers of blastocyst cells and cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope. RESULTS: With the increase of the concentration of L-NAME or SNP, the hatching rate of blastocysts and the total number of blastocyst cells were significantly reduced. The addition of 10 nmol/L SNP in culture media with 5 mmol/L L-NAME significantly increased the development of blastocysts and promoted hatching of blastocysts. However, with increase of SNP concentration in culture media with 5 mmol/L L-NAME, the development and hatching rates of blastocysts were significantly decreased. L-NAME had no obvious effect on the expression of active caspase 3 in blastocyst cells. However,when being above 500 nmol/L,SNP significantly increased the expression of caspase 3 in blastocyst cells. CONCLUSIONS: NO plays an important role in development and hatching of mouse blastocysts. Excessively high or low NO can damage the division of blastomeres, resulting in the failure of the blastocyst development and hatching. Also, excessively high NO can lead to the apoptosis of the blastocyst cells.

[Effects of bisphenol-A on blastocyst development and implantation].

OBJECTIVE: To determine the effects of bisphenol-A (BPA) on blastocyst development and implantation. METHODS: According to completely randomized grouping method, 90 pregnant mice were divided into 100, 300, and 600 mg/(kg·d)BPA groups and control group. BPA-treated pregnant mice were orally administered with BPA at concentrations of 100, 300 and 600 mg/(kg·d) from day 0.5 to day 3.5 of their pregnancy. Blastocyst implantation and development were studied. RESULTS: In the 300 mg/(kg·d) BPA group, the number of implantation sites and implantation rate were significantly decreased. In the 600 mg/(kg·d) group, no implantation sites were observed among pregnant mice and BPA inhibited embryo implantation. Blastocyst development on day 4 was examined, and findings showed that the development rate and total numbers of blastocysts in BPA treatment groups had no significant difference from the control group. However, BPA at 300 and 600 mg/(kg·d) significantly reduced blastocyst hatching rate and dramatically increased the number of blastocyst apoptotic cells when compared with those in the control group. CONCLUSION: BPA at a high concentration damages the blastocyst development before implantation and inhibits embryo implantation.

V-val subtype of Epstein-Barr virus nuclear antigen 1 preferentially exists in biopsies of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) has been suggested to be involved in pathogenesis of nasopharyngeal carcinoma (NPC). However, EBV infection is ubiquitous, whereas NPC occurs with strong geographic and racial distribution. Whether a substrain of EBV contributes to this phenomenon remains uncertain. Epstein-Barr virus nuclear antigen 1 (EBNA-1) is one of the most frequently detected EBV proteins in NPC tissues. Based on the polymorphism of amino acids at position 487, EBNA-1 is classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. To examine the relationship between subtypes of EBNA-1 and NPC, we determined the subtypes of EBNA-1 in biopsies of NPC, peripheral blood lymphocytes (PBL), and throat washings (TWs) obtained in endemic and non-endemic areas of NPC within China. The results revealed that V-val was the only subtype detected in NPC tissue, whereas three subtypes of EBNA-1, V-val, P-ala, and P-thr, were detected in PBL and TWs irrespective of origin, and mixed infection of V-val and P-ala was also observed. In addition, the variations of V-val derived from biopsies of NPC were identical to those derived from PBL and TWs in the context of N-terminus and C-terminus of EBNA-1. These facts indicate that a substrain of EBV with V-val subtype of EBNA-1 infects NPC preferentially and a susceptibility to a particular EBV isolate in the nasopharynx may exist during development of NPC.

[Inducement effect of Meisoindigo on apoptosis of leukemia cell line HL-60 and its mechanism].

BACKGROUND & OBJECTIVE: Meisoindigo is a powerful drug used in treating chronic myeloid leukemia (CML), but little is known about the mechanisms. This study was to investigate the inducement effect of meisoindigo on apoptosis of myelocytic leukemia cell line HL-60, and explore the possible mechanisms. METHODS: After treatment of meisoindigo, the proliferation of HL-60 cells was detected by trypan blue exclusion assay, and DNA fragmentation by agarose electrophoresis; cell morphology was observed under fluorescent microscope. Cell apoptosis and the expression of Fas were detected by flow cytometry. The expression of Caspase-3, Caspase-8, Caspase-9, PARP, Bcl-2, Bax and the concentration of cytochrome c in cytosol were analyzed by Western blot. RESULTS: Meisoindigo inhibited proliferation and induced apoptosis in HL-60 cells. When treated with 20 micromol/L meisoindigo for 12-48 h, the proliferation of HL-60 cells was significantly inhibited. When treated for 1 h, the apoptosis rate of HL-60 cells was (3.70+/-0.56)%; the apoptosis rate was significantly higher in HL-60 cells treated for 3, 6, and 12 h than in control cells [(19.80+/-1.13)%, (29.20+/-2.69)%, and (47.05+/-7.70)% vs. (2.65+/-0.78)%, P<0.05]. When treated with meisoindigo for 3 h, typical changes of apoptosis, such as chromatin condensation and DNA ladder, were detected in HL-60 cells. The positive rate of Fas was significantly higher in cells treated with 20 micromol/L meisoindigo for 1 h than in control cells [(21.30+/-1.27)% vs. (9.35+/-0.21)%, P<0.05]. Meisoindigo activated Caspase-3, Caspase-8, Caspase-9 and PARP, down-regulated the expression of Bcl-2, up-regulated the expression of Bax and the concentration of cytochrome c. Furthermore, pretreatment of caspase-3 inhibitor z-DEVD-fmk partially reversed the inhibitory effect of meisoindigo on cell proliferation, and decreased cell apoptosis; when treated with meisoindigo for 5 h, the apoptosis rate was significantly higher in pretreated cells than in cells without pretreatment [(29.8+/-5.4)% vs. (16.5+/-5.5)%, P<0.05]; when treated with meisoindigo for 12 h, the alive cell number was significantly lower in pretreated cells than in cells without pretreatment [(1.80+/-0.14) x 10(5)/ml vs. (3.57+/-0.18) x 10(5)/ml, P<0.05]. CONCLUSION: Meisoindigo induces apoptosis of HL-60 cells which may relate to regulation of caspases pathway and bcl-2 family proteins.

Transcriptional expression of RPMS1 in nasopharyngeal carcinoma and its oncogenic potential.

The Epstein-Barr virus (EBV) BamHI A rightward transcripts (BARTs) were originally identified in C15 xenograft of nasopharyngeal carcinoma (NPC) and easily detected in a wide variety of EBV latent infection and EBV-associated tumors. It had been reported that p31 cosmid containing BARTs immortalized monkey epithelial cells, but which particular gene among BARTs family participates in the transformation procedure remains to be identified. RPMS1 is the only full-length cDNA confirmed so far and one of the most abundant spliced forms in BARTs family. To investigate the involvement of RPMS1 gene in NPC, we examined the expression of RPMS1 transcripts in NPC biopsies from Guangdong and its oncogenic potential. Our results revealed that RPMS1 mRNA preferentially expressed in primary NPC to non-carcinoma tissue of nasopharynx and peripheral blood lymphocytes (PBLs) of NPC patients. Furthermore, by introducing RPMS1 ORF into HEK293 cells, these transfectants enhanced the anchorage-independent growth and produced tumors in nude mice. These data imply that RPMS1 gene might play an important role in the development of NPC.

[Expression of Epstein-Barr virus A73 gene in nasopharyngeal carcinoma tissues and its subcellular location].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) A73 gene encodes for a main mRNA of BamHI A rightward transcripts (BARTs) family which is widely transcribed in EBV-carrying cells and tumors. This study was designed to investigate the expression of A73 mRNA in nasopharyngeal carcinoma(NPC) from Guangdong region,from which A73 coding sequence(CDS) was cloned and introduced into epithelial cell line to observe the subcellular fraction of its encoding protein. METHODS: DNA and total RNA were isolated from NPC and noncarcinoma nasopharyngeal tissues,peripheral blood lymphocyte of NPC, SUNE1, B95.8, Raji, and BJAB cells; Nested PCR was designed to identify the EBV infection by amplifying the W sequence of EBV DNA in above- mentioned samples, and RT-PCR was used to analyze the transcription of A73 gene in the same samples. A73 CDS amplifying from a NPC tissue was cloned into pEGFP-C2 on special direction. After being transfected with this recombinant plasmid by lipofectin mediation, human embryo kidney(HEK293) cells were analyzed by RT-PCR and laser confocal microscope to observe the expression of A73 gene and its subcellular fraction. RESULTS: Except for BJAB cells, EBV W sequence existed in all samples. A73 transcribed in 79.63%(43/54)of NPC and 4%(1/25)of noncarcinoma nasopharynx biopsies between which the difference was significant, and no A73 mRNA was detected in all peripheral blood lymphocyte of NPC.A73 expressed at lower levels in B95.8 and Raji than in SUNE1. The constructed recombinant plasmid expressed stably in HEK293 cells, in which the fusion protein was found to be mainly cytoplasmic. CONCLUSION: EBV-A73 gene expresses widely in NPC tissue from Guangdong region and its encoding protein can stably express in cytoplasm of epithelial cell in vitro,suggesting A73 is highly correlated with the occurrence and development of NPC, and A73 gene may take part in the signal conduction related to epithelial oncogenesis as a signal molecule.