Functional conservation and divergence of duplicated the suppressor of cytokine signaling 1 in blunt snout bream (Megalobrama amblycephala).

The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensively involved in many different cytokine signaling pathways, such as regulation of the immune system and growth of organism. However, the molecular and functional information on socs1 genes in freshwater fish is unclear. In the present paper, we identified and characterized the full-length closely related but distinct socs1 genes (socs 1a and -1b) in blunt snout bream (Megalobrama amblycephala). The bioinformatic analysis results showed that duplicated socs1s shared majority conserved motifs with other vertebrates. Both socs1a and -1b mRNAs were detected throughout embryogenesis, and gradually increase and then constantly expressed after 16 hpf. Whole-mount in situ hybridization demonstrated that socs1a and socs1b mRNAs were detected in the brain at 12hpf and 24hpf, and in the notochord and brain at 36hpf. In adult fish, the socs1a mRNA were strongly expressed in the heart, eye, kidney, spleen and gonad, but were found to be relatively low in the intestine and liver. On the other hand, the expression of socs1b mRNA was significantly high in the muscle, eye and spleen, and relatively low in the intestine, liver, skin and heart. The results of hGH treatment experiment showed that socs1a and 1b mRNAs were upregulated markedly in the kidney, muscle and liver. Overexpression of socs1s significantly inhibit the GH and JAK/STAT factor stat3 and the inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. These results indicate that SOCS1 plays an important role in regulating growth and development.

EphA3 contributes to tumor growth and angiogenesis in human gastric cancer cells.

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus­mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC­27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC­27 cells in vitro and in vivo. Furthermore, knockdown of the expression of EphA3 in HGC­27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis in vivo was also inhibited upon EphA3 knockdown in HGC­27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.

[Effect of hyperbaric oxygen on the expression of nitric oxide synthase mRNA in cortex after acute traumatic cerebral injury].

OBJECTIVE: To investigate the effect of hyperbaric oxygen (HBO) treatment on the expression of nitric oxide synthase (NOS) mRNA in cortex after acute traumatic cerebral injury, and to study the mechanism of HBO on brain injury. METHODS: Acute traumatic brain injury model was established with rest received free fall injury method in SD rats. 0.25 MPa HBO treatment was used 1 h or 12 h after brain injury and the cortex was isolated 6 h or 24 h after brain injury respectively. The expression of mRNA coding for nNOS, eNOS or iNOS were assayed using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The expression of nNOS, eNOS and iNOS mRNA were significantly decreased in 0.25 MPa HBO treatment groups than those in acute cerebral injury groups (P < 0.01). The amount of nNOS, eNOS and iNOS mRNA was significantly lower in HBOT 24 h group than those in HBOT 6 h group (P < 0.05, P < 0.01). There was no significantly difference among nNOS, eNOS and iNOS mRNA in 0.25 MPa normoxic hyperbaric nitrogen groups and acute cerebral injury groups (P > 0.05). CONCLUSION: HBO may exert significant effects on the expression of nNOS mRNA/iNOS mRNA and protect cortical neuronal from traumatic cerebral injury.

[Expression of high mobility group box chromosomal protein 1 in mice with lupus nephritis].

OBJECTIVE: To determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis. METHODS: Serum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR. RESULT: MRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA. CONCLUSION: The results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.

Analysis of differentially expressed genes in LNCaP prostate cancer progression model.

The LNCaP/C4-2 human prostate cancer progression model was established to mimic phenotypic and genotypic changes during prostate cancer development from androgen dependence to androgen independence, from nonmetastasis to metastasis. In this study, cDNA microarrays were performed using a microarray chip from Affymetrix to characterize and compare gene expression profiles in LNCaP and C4-2, which may provide novel insight into the molecular mechanism mediating prostate cancer progression. Three hundred eighteen genes consistently exhibited differential expression in LNCaP and C4-2 in 2-time microarray data. Based on their function, the differentially expressed genes can be grouped into several subcategories, including growth factors and signal transducers, oncogenes and tumor suppressors, tumor-specific antigens, transcriptional factors, transporters, and factors involved in invasion, metastasis, and metabolism. Some genes are novel and unexplored in prostate cancer progression and are of potential interest for follow-up investigation. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to corroborate the microarray results, and 76 differentially expressed genes were validated out of 104 candidates. Expression pattern analyses were performed in these 76 differentially expressed genes, and a series of genes was found to be positively or negatively correlated to prostate cancer progression in the LNCaP prostate cancer progression model and to possess predominant prostate cell specificity. ELF5/ESE-2b and long-chain acyl coenzyme A dehydrogenase (ACADL) expressions were found to be positively associated with malignant progression in LNCaP, C4-2, and C4-2B, and predominantly expressed in prostate cancer cells. Functional evaluation revealed that ELF5/ESE-2b and ACADL expressions contributed to the malignant phenotypes of prostate cancer cells. Accordingly, our microarray data may provide clues for finding novel genes involved in prostate cancer progression to androgen independent and metastasis, and shed light on finding new targets for diagnosis and therapy of prostate cancer.

Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells.

BACKGROUND: Although the introduction of serum prostate-specific antigen (PSA) measurements into clinical practice has revolutionized the care of patients with prostate cancer, there are well-recognized limitations of PSA, and there is a critical need to identify additional prostate cancer biomarkers to assist in early detection and prognosis. In this regard, high resolution proteomic technology has the unexceptionable superiority to find those high abundance biomarkers. The purpose of this study was to search new tumor markers by proteomic technology. METHODS: The proteins in conditioned medium (CM) of BPH-1 and LNCaP cells were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The corresponding mRNA levels of some identified proteins were analyzed by RT-PCR. RESULTS: Totally 11 differentially expressed proteins (6 up-regulated including creatine kinase, brain (CKB), triosephosphate isomerase 1 (TPI1), isocitrate dehydrogenase 2 (IDH2) and 5 down-regulated including glutathione S-transferase pi (GST-pi)) in the CM were identified using MALDI-TOF-MS and database search. The expression pattern between mRNA and CM protein levels of CKB, IDH2, TPI1 and GST-pi in BPH-1 and LNCaP was similar. CONCLUSION: We proved a feasible and effective way to search new tumor markers by a proteomics-based strategy and identified 11 potentially useful proteins in CM of BPH-1 and LNCaP cells to distinguish prostate cancer from benign prostatic hypertrophy.

[PC-1 enhances c-myc gene expression in prostate cancer cells].

PC-1(Prostate and colon gene 1) gene belongs to TPD52 (Tumor Protein D52) gene family. The expression of PC-1 is found to promote androgen-independent progression. This study was conducted to assess the mechnism of promotion of androgen-independent progression in PC-1 gene. The c-myc gene expression was tested by RT-PCR and Western blotting analyses in the LNCaP-pc-1 and LNCaP-zero cell line. After separation of cytoplasm and nulear proteins of the LNCaP-pc-1 and LNCaP-zero cell line, the beta-catenin protein was detected by Western blotting. C4-2 cell line was used to examine the effects of 10058-F4 on the PC-1 gene expression. The results of RT-PCR and Western blotting indicated that PC-1 enhanced c-myc gene expression in prostate cancer cells, PC-1 was also found to enhance beta-catenin expression in nuclear. Furthermore, a small-molecule c-Myc inhibitor, 10058-F4 represses PC-1 gene expression in C4-2 cell line. Our findings suggest that PC-1 enhances c-myc gene expression in prostate cancer cells through the Wnt/beta-catenin pathway. Meanwhile, c-myc plays a feed-forward role in enhancing PC-1 driven c-myc gene expression, and promotes prostate an-drogen-independent progression.

[Identification and expression of two new secretory proteins associated with prostate cancer].

Our research intends to obtain extra-cellular proteinogram of cell lines representing different advancement stages of prostate cancer and to test whether screened differential expression proteins can be secreted and used as serum biomarkers for prostate cancer. By examining differential expression spots in two extra-cellular protein 2D-PAGE gels and mass spectrum, candidate molecules were obtained. The expressions of these candidate molecules in eight cell lines and response to androgen stimulus in LNCaP were analyzed by RT-PCR. By constructing eukaryotic expression vectors and western-blotting with anti tags antibodies, the candidate molecules were tested to understand whether they can be expressed in transfected 293T cell culture fluid. Two overexpressed molecules-triosephosphate isomerase 1 (TPI1) and syndecan bind-ing protein, syntenin (ST1)-in extra-cellular proteinogram of C4-2 were screened out; both of them are secretary proteins. On transcriptional level, both proteins were up-regulated with the malignancy of prostate cancer cell lines and ST1 was dose-dependently inhibited by androgen. Considering cellular level results, both TPI1 and ST1 have their potential as serum biomarkers for indicating the developmental stage of prostate cancer.

The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP.

Radiation therapy is a relatively effective therapeutic method for localized prostate cancer (PCa) patients. However, radioresistance occurs in nearly 30% of patients treated with potentially curative doses. Therapeutic synergy between radiotherapy and androgen ablation treatment provides a promising strategy for improving the clinical outcome. Accordingly, the androgen deprivation-induced signaling pathway may also mediate radiosensitivity in PCa cells. The C4-2 cell line was derived from the androgen-sensitive LNCaP parent line under androgen-depleted condition and had acquired androgen-refractory characteristics. In our study, the response to radiation was evaluated in both LNCaP and C4-2. Results showed that C4-2 cells were more likely to survive from irradiation and appeared more aggressive in their resistance to radiation treatment compared with LNCaP, as measured by clonogenic assays and cell viability and cell cycle analyses. Gene expression analyses revealed that a set of genes involved in cell cycle arrest and DNA repair were differentially regulated in LNCaP and C4-2 in response to radiation, which was also consistent with the radiation-resistant property observed in C4-2 cells. These results strongly suggested that the radiation-resistant property may develop with progression of PCa to androgen-independent status. Not only can the LNCaP and C4-2 PCa progression model be applied for investigating androgen-refractory progression, but it can also be used to explore the development of radiation resistance in PCa.

Ubiquitous mitochondrial creatine kinase is overexpressed in the conditioned medium and the extract of LNCaP lineaged androgen independent cell lines and facilitates prostate cancer progression.

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.

The application of CdTe@SiO2 particles in immunoassay.

CdTe@SiO(2) fluorescent particles were synthesized via hydrolysis and condensation of tetraethyl orthosilicate (TEOS) in water-in-oil (W/O) emulsion. Uniform luminophore-doped silica nanoparticles with 100nm in diameter were obtained using microemulsion method and characterized by SEM. Antibody proteins were successfully conjugated to the fluorescent particles by the reaction of avidin and biotin, which were confirmed by fluorescence spectra. CdTe@SiO(2) fluorescent particles were potentially useful for the applications in biolabeling and imaging.

PC-1/PrLZ contributes to malignant progression in prostate cancer.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

[Gene fusion of egfp & kan and recombinant plasmid construction by red mediated in vivo homologous recombination].

Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.

[Role of HMGB1 in rheumatic diseases].

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB1) is originally identified as a DNA-binding protein that functions as a structural co-factor. HMGB1 is actively secreted by macrophage/monocytes via inflammatory stimuli. The extracellular HMGB-1 acts as a mediator of acute and chronic systematic inflammation. In this article we briefly review its role in rheumatic diseases: arthritis, polymyositis and dermatomyositis, lupus erythematosus and Sjogren's syndrome. Increased cytoplasmic expression and extracellular deposition of HMGB1 are found in the affected tissues of those diseases, especially stronger in/around focal infiltrates of mononuclear cells. TNFalpha and IL-1beta are co-expressed in areas of extracellular HMGB1. HMGB1 together with TNF alpha and IL-1beta may form a proinflammatory loop promoting the chronic inflammations. The new findings of HMGB1 as a cytokine provide a better understanding of rheumatiod diseases, and could become a clinically relevant therapeutic target that might be more efficient than other known cytokines.

Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter.

Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.

Anticancer effects of Chinese herbal medicine, science or myth?

Currently there is considerable interest among oncologists to find anticancer drugs in Chinese herbal medicine (CHM). In the past, clinical data showed that some herbs possessed anticancer properties, but western scientists have doubted the scientific validity of CHM due to the lack of scientific evidence from their perspective. Recently there have been encouraging results, from a western perspective, in the cancer research field regarding the anticancer effects of CHM. Experiments showed that CHM played its anticancer role by inducing apoptosis and differentiation, enhancing the immune system, inhibiting angiogenesis, reversing multidrug resistance (MDR), etc. Clinical trials demonstrated that CHM could improve survival, increase tumor response, improve quality of life, or reduce chemotherapy toxicity, although much remained to be determined regarding the objective effects of CHM in human in the context of clinical trials. Interestingly, both laboratory experiments and clinical trials have demonstrated that when combined with chemotherapy, CHM could raise the efficacy level and lower toxic reactions. These facts raised the feasibility of the combination of herbal medicines and chemotherapy, although much remained to be investigated in this area.

[Effect of polymorphism of human intestinal fatty acid binding protein gene on the therapeutic efficacy of fenofibrate].

OBJECTIVE: To explore the effect of polymorphism in codon Ala54Thr of human intestinal fatty acid-binding protein gene (IFABP) on the therapeutic efficacy of fenofibrate. METHODS: Totally 147 patients with hyperlipidemia [72 men mean age: (56.2 +/- 8.63) years; 75 women mean age: (58.4 +/- 9.12) years] were enrolled. IFABP genotypes were detected by polymerase chain reaction, Hha I digestion, and sequencing. Four weeks before and after treatment, the levels of fasting serum total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A I (apoA I) and apolipoprotein B (apoB) were detected with biochemical techniques. RESULTS: The frequency of IFABP genotype was 0.47 for A/A, 0.37 for A/T, and 0.16 for T/T, and the allelic frequency was 0.65 for A and 0.35 for T. No significant different was found in lipid levels in every genotype before treatment (P > 0.05). After 4 weeks of treatment, the levels of TC, TG, LDL-C, and apoB significantly decreased (P < 00.01), and the levels of HDH-C and apoA I significantly increased (P < 0.01). The total therapeutic efficacy on A54A and A54T were 97% and 95%, respectively. In the patients with T54T genotype after treatment, no significant difference in lipids levels was found except TG (P < 0.05), and the total efficacy was only 38%. The total therapeutic efficacies of fenofibrate on A54A and A54T were higher than those of T54T, and there was significant different between A54A and T54T (P < 0.01). CONCLUSION: The polymorphism of human IFABP gene in hyperlipidemia is related with the therapeutic efficacy of fenofibrate, and the T54T IFABP genotype may have strong negative effect on such efficacy.

[pBR322-Red mediated gene knockin, sites and expression in E. coli chromosome].

Genes lacZ, lacY and lacA in the lac opron of E. coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides. Then, a series of new strains, CWL2, CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes.