Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion.

Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells.

BACKGROUND: GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection. RESULTS: In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells. CONCLUSIONS: The accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.

[Isolation, identification and lead adsorption study of lead-resistant Lactobacillus casei strains from feces of healthy newborns].

OBJECTIVE: To isolate and identify lead-resistant Lactobacillus casei strains with lead adsorption ability from the stool of healthy newborns as a new source of bacteria for developing lead-eliminating food products. METHODS: MRS was used to isolate lead-resistant bacteria from the feces of 30 healthy and full-term neonates. A phylogenetic tree was constructed based on the morphological characteristics and 16S rRNA sequences of the isolated bacteria. Physiological and biochemical characterizations of the bacteria were performed according to the Berger's Systematic Bacteriology Handbook, followed by antimicrobial susceptibility test and acid-tolerant bile salt test. The adsorption capacity of Pb2+ of the bacteria was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES). RESULTS: Three strains of Lactobacillus casei were isolated, which were resistant to penicillin and ceftriaxone and could tolerate the exposure to 500 mg/L Pb2+. Acid-tolerant bile salt test showed that the bacteria were resistant to culture in the presence of artificial gastric juice (pH 2.0) for 3 h, and their survival rate reached 62.5% following exposure to 0.3% bile salt for 8 h. The bacteria showed a Pb2+ adsorption rate of 90.4% at a low Pb2+ concentration (1 mg/L) and of 86.27% at a high Pb2+ concentration (50 mg/L). CONCLUSION: Three Lactobacillus casei strains lead adsorption ability were isolated from the feces of newborns. These bacterial strains provide a new solution to alleviate lead poisoning by probiotic dietary.

[Preparation and identification of egg yolk antibodies against HLA-A* 0201 heavy chain].

OBJECTIVE: To prepare a high-titer specific and efficient egg yolk immunoglobulin (IgY). METHODS: The antigen of HLA-A* 0201 heavy chain was used to immunize the hens together with Freud's adjuvant and the eggs these hens laid were collected. IgY was purified from the egg yolk by water extraction, salt precipitation with ammonium sulfate and dialysis. The immunoactivity of the IgY was measured by enzyme-linked immunosorbent assay (ELISA) and its purity was determined by SDS-PAGE. RESULTS: The titer of IgY was 1 10(6) as shown by ELISA, with protein concentration of 9.77 mg/ml. The purity of the resultant IgY was more than 90% as suggested by SDS-PAGE analysis. CONCLUSIONS: The antigen of HLA-A* 0201 heavy chain along with complete Freund adjuvant is effective to elicit an immune response of hens for producing IgY antibodies in high yields.

BNIP3 upregulation by ERK and JNK mediates cadmium-induced necrosis in neuronal cells.

Cadmium (Cd) is a toxic heavy metal that may cause neurological disorders. We studied the mechanism underlying Cd-mediated cell death in neuronal cells. In Cd-induced neurotoxicity, caspase-3 was only modestly activated, and accordingly, zVAD-fmk, a pan-caspase inhibitor, partially attenuated cell death. However, pretreatment with Necrox-2 or Necrox-5, two novel necrosis inhibitors, suppressed cell death more markedly compared with pretreatment with zVAD-fmk. Moreover, the necrosis inhibitors did not prevent cleavage of caspase-3. These results indicate that caspase-independent necrosis is more prevalent in Cd-induced neurotoxicity. Bcl-2 and adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) has been reported to be related to caspase-independent cell death. Cd treatment caused a dramatic upregulation of BNIP3 mRNA and protein levels in vitro and in vivo. Furthermore, knockdown of BNIP3 greatly inhibited Cd-induced cell death. Importantly, BNIP3 RNAi decreased lactate dehydrogenase release and the percentage of propidium iodide-positive cells, two markers of necrotic cell death due to rupture of the cell membrane, whereas it had no effect on activation of caspase-3 in Cd-treated cells. These data suggest that BNIP3 mediates caspase-independent necrosis, but not apoptosis. Moreover, our results indicate that induction of BNIP3 by Cd may not be related to HIF-1 which is generally regarded as a mediator responsible for BNIP3 expression. Finally, we show that mitogen-activated protein kinases (MAPKs) are activated by Cd in vitro and in vivo; ERK and JNK promote BNIP3 upregulation and subsequent necrosis. Taken together, our results suggest BNIP3, upregulated by activation of ERK and JNK, mediates Cd-induced necrosis in neuronal cells.

[Dextran sedimentation for study of neutrophil polarization].

OBJECTIVE: To determine the optimal method for separating neutrophils for studying neutrophil polarization. METHODS: Human neutrophil was separated from healthy human peripheral blood by Percoll density gradient centrifugation and Dextran sedimentation. The cell polarization, purity and activity of the neutrophils were determined, and F-actin polymerization and [Ca2+]i were analyzed. RESULTS: No significant difference was found in cell polarization, purity and activity of the human neutrophils separated by Dextran sedimentation and Percoll density gradient centrifugation (P>0.05), but F-actin polymerization was inhibited in PMNs separated by Dextran sedimentation, and the peak value of [Ca2+]i was decreased by 25% in PMNs separated by Dextran sedimentation compared to the cells separated by Percoll density gradient centrifugation. CONCLUSIONS: Both Percoll density gradient centrifugation and Dextran sedimentation can be used for isolating human neutrophils to study cell polarization, but the former method allows better isolation. Dextran sedimentation can be considered when a large number of neutrophils need to be separated.

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

[Preparation and preliminary characterization of soluble HLA-A2-peptide complex].

AIM: To express HLA-A2 heavy chain (HC) and light chain beta(2m) in bacteria and to prepare soluble HLA-A2-peptide complex. METHODS: HC and beta(2m) expressing engineering bacteria was induced for 5 h with 0.5 mmol/L IPTG. After sonification of the bacteria,crude inclusion body was obtained which was then refined, renaturated, ultrafiltrated, and purified by DEAE Sepherose Fast Flow anion exchange chromatography. Then HC and beta(2m) were connected with two specific peptides, purified through Superdex 75 gel filtration, and identified with mAb W6/32 which can recognize native HLA-A2. RESULTS: The expression rates of HC and beta(2m) in engineering bacteria was both about 50%. The purity of both expression products reached to 95%.Moreover, the two HLA-A2-peptide complexes could be recognized by mAb W6/32, even after being stored at 4 degrees Celsius for 2 months. CONCLUSION: The two soluble HLA-A2-peptide complexes prepared by us are stable and lay the foundation for further research of CTL recognition and response.