Ultracytochemical observation of the intracellular localization of H+-adenosine triphosphatase / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.</p><p><b>METHODS</b>The localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.</p><p><b>RESULTS</b>H(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.</p><p><b>CONCLUSION</b>This finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.</p>

Establishment of molecular beacon real-time PCR for detecting p53 gene single nucleotide polymorphism / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method based on molecular beacon real-time PCR for detecting single nucleotide polymorphisms (SNP) in codon 72 of scar-related p53 gene.</p><p><b>METHODS</b>Two fluorescence-labeled molecular beacon probes were synthesized targeting CCC/CGC SNP of p53 codon 72. The genomic DNA was extracted from the peripheral blood of 28 patients with keloid, and the CCC/CGC SNP of P53 gene codon 72 were assayed with molecular beacon real-time PCR. The results of SNP typing were compared with the results of reverse dot hybridization and confirmed by direct DNA sequencing.</p><p><b>RESULTS</b>The goodness of fit of this method was 100% in comparison with direct DNA sequencing, higher than that of reverse dot hybridization.</p><p><b>CONCLUSION</b>Molecular beacon real-time PCR is suitable for rapid clinical detection of SNPs in p53 gene.</p>

Effect of metformin on apoptosis of renal cell carcinoma cells in vitro and its mechanisms / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms.</p><p><b>METHODS</b>Fluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9.</p><p><b>RESULTS</b>Metformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin.</p><p><b>CONCLUSION</b>Metformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.</p>

Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on nuclear factor-kappa B and cytokine expression in the intestine of septic mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the expression of nuclear factor-kappa B (NF-κB) and cytokines in the intestinal tissue of septic mice.</p><p><b>METHODS</b>Male BALB/c mice were randomly divided into 4 groups, namely the sham-operated group, model group, TSP-2 treatment group and rabbit IgG treatment group. Sepsis was induced by cecal ligation and puncture (CLP), and at 6, 12 or 24 h after the operation, the ileal tissues were harvested from the mice for HE staining. NF-κB expression was detected with immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expressions were detected with qRT-PCR and their protein expressions by ELISA.</p><p><b>RESULTS</b>The NF-κB expression in the intestinal tissue significantly increased in the model group as compared with that in the sham- operated group, and decreased after TSP-2 treatment. The model group also showed significantly increased expression levels of TNF-α and IL-6 mRNA and protein in the intestinal tissue (P<0.05), which were lowered by TSP-2 (P<0.05) but not by rabbit IgG treatment (P>0.05).</p><p><b>CONCLUSION</b>The TSP-2 antibody can protect the intestine and delay the development of sepsis by inhibiting NF-κB activation and down-regulating TNF-α and IL-6 expressions in mice.</p>

Progesterone promotes the proliferation and migration of cultured breast cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of progesterone on the growth and migration of breast cancer cells.</p><p><b>METHODS</b>MCF-7 and T-47D cells were cultured in DMEM and stimulated with 100 nmol/L progesterone for 48 h, and the cell proliferation was evaluated by MTT assay, cell migration by wound-healing assay and E-catherin expression by Western blotting.</p><p><b>RESULTS</b>Progesterone stimulated the cell proliferation and migration and down-regulated the expression of E-catherin in both MCF-7 and T-47D cells.</p><p><b>CONCLUSIONS</b>Progesterone stimulates the cell proliferation and migration of cultured breast cancer cells, suggesting the clinical significance of anti-progesterone therapy in breast cancer.</p>

Positional cloning of a novel allele of zebrafish cloche mutant / 南方医科大学学报

<p><b>OBJECTIVE</b>To perform the genetic identification of cloche(172) mutant zebrafish.</p><p><b>METHODS</b>The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization (3dpf) stage were observed and the cloche(172) gene was identified by mapping and complementation test.</p><p><b>RESULTS</b>We selected 4 lyC-deficient zebrafish by WISH. cloche(172) mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche(172) gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche(172) mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining.</p><p><b>CONCLUSION</b>cloche(172) gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.</p>

Expression of oxyntomodulin in bifidobacteria and effect of oxyntomodulin-transformed bifidobacteria on the body weight of obese mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of pBBADs-OXM-transformed bifidobacteria on the body weight of obese mice.</p><p><b>METHODS</b>B. longum was transformed with pBBADs-OXM by electroporation, and arabopyranose-induced oxyntomodulin expression by the bacterium was detected by ELISA. pBBADs-OXM-transformed bifidobacteria was administered orally obese mice on a daily basis with pBBADs-GFP-transformed bifidobacteria as the negative control, and the body weight changes of the mice were observed.</p><p><b>RESULTS</b>OXM was detected by ELISA not only in the supernatant but also the precipitant of the transformed bacterial culture. The body weight of the obese mice fed with pBBADs-OXM-transformed bifidobacteria decreased significantly compared with that of the mice in the obese model group (P<0.05).</p><p><b>CONCLUSION</b>Administration of pBBADs-OXM-transformed B.longum can reduce the body weight of obese mice.</p>

Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis.</p><p><b>METHODS</b>In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80.</p><p><b>CONCLUSION</b>TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.</p>

Role of phospholipase C-gamma1 signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells.</p><p><b>METHODS</b>PC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis.</p><p><b>RESULTS</b>After inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells.</p><p><b>CONCLUSION</b>PLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.</p>

Prokaryotic expression and bioactivity of human platelet-derived growth factor B chain mature peptide / 南方医科大学学报

<p><b>OBJECTIVE</b>To express human platelet-derived growth factor (hPDGF) B chain mature peptide gene in a prokaryotic expression system and detect the bioactivity of the expressed protein.</p><p><b>METHODS</b>hPDGF B chain mature peptide gene was amplified and expressed in E. coli, and the recombinant protein, rhPDGF-BB, was purified and renatured in GSSG/GSS system. The bioactivity of rhPDGF-BB in vitro was evaluated with SD rat osteoblasts.</p><p><b>RESULTS</b>The full-length PDGF-B mature peptide gene was obtained and verified, and successfully expressed in E. coli. Bioactivity detection results showed that the expressed rhPDGF-BB obviously promoted the proliferation and DNA replication of SD rat osteoblasts in vitro (P<0.01).</p><p><b>CONCLUSION</b>he PDGF-B chain mature peptide cDNA has been successfully cloned and the PDGF-B precursor highly expressed in E. coli, and renatured rhPDGF-BB displays high bioactivity as shown by MTT assay and flow cytometry. This success provides the basis for production of functional PDGF-BB and facilitates further studies of its role in fracture healing and trauma reconstruction.</p>

Matrine-induced erythroid differentiation of K562 cells is associated with activation of the apoptotic pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe matrine-induced erythroid differentiation of K562 cells in relation to activation of the apoptotic pathway in vitro.</p><p><b>METHODS</b>K562 cells were cultured in the presence or absence of matrine at different concentrations for 4 days, and the morphological and ultramicrostructural changes of the cells were observed using inverted microscopy and transmission electron microscopy, respectively. The expression of apoptosis-related protein p27kip1 was detected by immunocytochemistry.</p><p><b>RESULTS</b>Compared to untreated K562 cells, the cells treated with matrine at 0.10 g/L exhibited apoptostic characteristics in the cellular morphology and ultramicrostructure, with the expression of p27kip1 protein upregulated in a concentration- and time-dependent manner.</p><p><b>CONCLUSION</b>Matrine-induced erythroid differentiation of K562 cells is associated with cell apoptosis, and upregulation of p27kip1 protein expression may play a crucial role in the process of apoptosis.</p>

Construction and expression of different mutants of human p53 and their effects on arsenite-induced cell apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis.</p><p><b>METHODS</b>Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry.</p><p><b>RESULTS</b>The recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells.</p><p><b>CONCLUSION</b>The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.</p>

BRCA1 regulates progesterone receptors A and B protein expressions in breast cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells.</p><p><b>METHODS</b>Breast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting.</p><p><b>RESULTS</b>The protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased.</p><p><b>CONCLUSION</b>In breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.</p>

Adenovirus-mediated overexpression of AMP-activated protein kinase induces LX2 cell apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of adenovirus-mediated overexpression of AMP-activated protein kinase (AMPK) on apoptosis of hepatic stellate cell line LX2.</p><p><b>METHODS</b>After adenovirus infection of the LX2 cells, the exogenous gene expression was detected by Western blotting, and cell apoptosis evaluated by flow cytometry with PI staining. Agarose gel electrophoresis was performed to detect the DNA ladder, and the expressions of caspase-3, Bcl-2 and Bax are detected by Western blotting.</p><p><b>RESULT</b>With AMPK overexpression, apoptotic peak and DNA ladder of the infected cells appeared, and pro-caspase-3 was activated to transform into caspase-3 accompanied by up-regulated Bax expression, whereas Bcl-2 expression was hardly detectable by Western blotting.</p><p><b>CONCLUSION</b>Overexpression of AMPK mediated by the adenovirus can induce LX2 cell apoptosis, possible as a result of up-regulated Bax expression with low Bcl-2 expression.</p>

Antitumour effects on human colorectal carcinomas cells by stable silencing of phospholipase C-gamma 1 with lentivirus-delivered siRNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.</p><p><b>METHODS</b>Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.</p><p><b>RESULTS</b>Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.</p><p><b>CONCLUSIONS</b>PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.</p>

Exploration of the alternative splicing variants of rat phospholipase C-gamma 1 pre-mRNA / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of phospholipase C-gamma 1 (PLC-gamma1) alternative splicing variants in rats.</p><p><b>METHODS</b>According to the sequence of human PLCG1 splicing variant, specific primers for rat PLC-gamma1 were designed and synthesized. The rat RNA was reverse transcribed into cDNA, which was amplified using the specific primers, and the PCR products were sequenced and analyzed using BLAST and bioinformatics methods. Totally 21 rat tissue samples were examined, including the heart, liver, lung, kidney, eyeball, and brain obtained in 3 different embryonic stages, 7 different early postnatal stages, and in adulthood.</p><p><b>RESULTS</b>The result did not show that rat PLC-gamma1 had the same splicing variant (PLC-gamma1a, NM_002660) as human does.</p><p><b>CONCLUSIONS</b>The same splicing variant of PLC-gamma1 detectable in human may not exist in rats, and the pre-mRNA may undergo splicing resulting predominantly in PLC-gamma1b mRNA. Very likely, the alternative splicing site of rat PLC-gamma1 is not identical to that of human.</p>

Construction of lentivirus producing PLC-gamma1 siRNA and its effect on apoptosis of human colorectal carcinomas cell lines / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct recombinant lentivirus that stably suppresses phospholipase C (PLC) gamma1 expression in human colorectal carcinoma LoVo cells to obtain LoVo cell lines deficient in PLC gamma1 for investigation of the role of PLC gamma1 gene.</p><p><b>METHODS</b>Recombinant lentivirus producing PLC gamma1 siRNA were constructed to infect LoVo cells, and the stably transduced cells were selected with blasticidin. The protein and mRNA expression of PLC gamma1 was examined by Western blotting and RT-PCR, and the effect of the lentivirus on cell apoptosis was analyzed by flow cytometry.</p><p><b>RESULTS AND CONCLUSION</b>PLC gamma1 siRNA significantly suppressed PLC gamma1 expression in LoVo cells, suggesting high efficiency of gene silencing induced by the siRNA produced by the recombinant lentivirus. Concomitantly, cell apoptosis induced by 5-FU was significantly increased.</p>

Sensitization of human glioma SWO cell line to tumor necrosis factor-induced apoptosis by blocking phospholipase C-gamma1 signaling pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of phospholipase C-gamma1 (PLC-gamma1) in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis of human glioma SWO cells.</p><p><b>METHODS</b>The PLC-gamma1 pathway was blocked by U73122 in SWO cells, and the inhibitory effect of TNF-alpha on SWO glioma cell proliferation with or without U73122 treatment was investigated by MTT assay. The cell apoptosis induced by TNF-alpha along or in combination with U73122 was detected by flow cytometry with PI staining. The expression of caspase-3 and Bcl-2 was detected by Western blotting.</p><p><b>RESULTS AND CONCLUSION</b>U73122 can sensitize SWO glioma cells to TNF-alpha-induced apoptosis. Blocking the PLC-gamma1 pathway may not induce apoptosis of SWO glioma cells, but can sensitize SWO glioma cells to small-dose TNF-alpha-induced apoptosis, the mechanism of which may involve down-regulation of bcl-2.</p>

Immunocytochemical study of phospholipase C-gamma1 expression in mouse embryonic tissue / 南方医科大学学报

To investigate the expression of phospholipase C-gamma1 (PLC-gamma1) in mouse embryonic tissues, serial tissue sections were prepared routinely for immunocytochemistry for PLC-gamma1. The results showed that PLC-gamma1 was expressed in the cartilage, skeletal muscles, myocardium, the collecting tubule of the kidney, connective tissues and the brain, suggesting the important role PLC-gamma1 and the related signal pathway may play in the development of mouse embryonic tissues.

Phospholipase Cγ1 and NF-κB are required for cell-matrix adhesion of colorectal cancer cells / 第二军医大学学报

Objective: To investigate the function and mechanism of phospholipase Cγ1 (PLCγ1) in cell-matrix adhesion in colorectal cancer. Methods: Highly metastatic colorectal cancer cell line LoVo and lowly metastatic colorectal cancer cell line SW480 were subjected to cell-matrix adhesion assay. U73122 (a specific inhibitor of PLC) and pyrrolidine dithiocarbamate (PDTC) (an inhibitor of NF-κB) were used to study the effect of PLCγ1 and NF-κB on cell-matrix adhesion. Furthermore, Western blot and gel electrophoresis mobility shift assay (EMSA) were performed to detect the mechanism of PLCγ1 in colorectal cancer cell adhesion to matrix. Results: Inhibition of PLCγ1 or NF-κB resulted in reduction of cell-matrix adhesion in a dose-dependent manner in LoVo cells(P<0.05), but had no marked effect on SW480 cells. Western blot analysis showed that epidermal growth factor (EGF) stimulated the phosphorylation of PLCγ1 in LoVo. The results of EMSA indicated that inhibition of PLCγ1 signaling pathway also down-regulated the activity of NF-κB while EGF reversed the function. Conclusion:These data suggest that PLCγ1 plays a pivotal role in the EGF-induced cell-matrix adhesion of highly metastatic colorectal cancer cells and that NF-κB is also functional in this signaling pathway.