Formation and nuclear export of pre-ribosomes requires many nucleolar complexes, hNoc4L which contains a conserved Noc doman is a homolog of nucleolar complex associated 4 (S. cerevisiae), but its function is completely unclear. Here, we successfully got the recombinant lentiviral vector p113.7-EF1-hNoc4L-Flag by replacing the U6 promoter in p113.7 with EF1alpha promoter, and then inserted hNoc4L to down-stream of the EF1alpha prompter. We determined the transduction efficiency in different mammalian cell lines based on lentiviral packaging system. Subsequently, we analyzed the immunogenicity of the recombinant lentivirus and stable expression of hNoc4L in RAW264.7 cells. The results showed that the recombinant lentivirus characterized a high transduction efficiency, long-term expression and low immunogenicity. Therefore, we pave the way for further identification of the biological activity of hNoc4L protein during ribosome biogenesis in mammalian.
Animals ; Cell Line ; Genetic Vectors ; genetics ; Lentivirus ; genetics ; metabolism ; Mice ; Nuclear Proteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics

OBJECTIVETo acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.

METHODSThe coding sequence of hNASP gene was amplified from human testis RNA with specific primers, and the PCR product was cloned first into pMD-18T and then into pET-28a ( + ) after restriction digestion with BamH I and Hind III. The fusion protein was expressed in E. coli BL21 (DE3) after induction with IPTG. The recombinant protein NASP was purified from the supernatant with Ni2 -NTA His-bind resin under native conditions.

RESULTSThe results of DNA sequencing and SDS-PAGE analysis showed the protein to be what we had hoped to acquire. ELISA showed that we had acquired rabbit anti-hNASP serum with high titer.

CONCLUSIONHigh purity recombinant hNASP protein could be obtained with the above-mentioned prokaryotic expression method, and so could the rabbit anti-hNASP serum with high titer and high specificity.

Adult ; Animals ; Antibody Formation ; Autoantigens ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Humans ; Male ; Nuclear Proteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2), human mut-l homologue 1 (hMLH1) and human mut-s homologue 2 (hMSH2) proteins in human paired gastric carcinoma (GC) and adjacent normal mucosa, and analyze their relationship with microsatellite instability (MSI).

METHODSThe protein expressions were examined by western blotting. Five MSI loci were assessed by PCR.

RESULTSIn 30 surgically excised GC tissues, the overexpression rate of COX-2, the low expression rate of hMLH1 and hMSH2 were 66.7%, 40% and 33.3%, respectively. Significant differences were found when compared with those of adjacent normal mucosa (P < 0.05). MSI was detected in 13 GC. The number of MSI-H (MSI-High, > or = 2 loci), MSI-L (MSI-Low, only one locus), and MSS (microsatellite stable) were 9, 4 and 17, respectively. The number of low expression rates of COX-2, hMLH1 and hMSH2 in MSI-H were 6, 8 and 5, respectively. There were significant differences compared to that of MSS (P < 0.05).

CONCLUSIONThe results suggest that microsatellite instability pathway is probably involved in the carcinogenesis of gastric carcinoma, which is frequently accompanied by low expression of hMLH1 and hMSH2, and may be also by low expression of COX-2.

Adaptor Proteins, Signal Transducing ; biosynthesis ; genetics ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Humans ; Microsatellite Repeats ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the expression of gene BRG1 in prostatic intraepithelial neoplasia and adenocarcinoma, and the relationship between gene BRG1 expression and the clinicopathological features of prostate carcinoma.

METHODSGene BRG1 expression was evaluated in 37 cases of human prostate carcinoma, 13 human prostatic intraepithelial neoplasia (PIN) and 14 human benign prostatic hyperplasia (BPH) by using immunohistochemistry (EnVision method) and tissue microarray.

RESULTSThe positive rates of BRG1 protein were 81.08% (30/37), 38.46% (5/13) and 14.28% (2/14) in prostate carcinoma, PIN and BPH, respectively, significantly higher in the first group than in the latter two (P < 0.05). There was no statistically significant difference in BRG1 gene expression either between PIN and BPH (P > 0.05) or between the groups of the moderate differentiation (the Gleason histologic grading: 5-7) and the lower one (the Gleason histologic grading: 8-10) (P > 0.05).

CONCLUSIONBRG1 may play an important role in the development of prostate carcinoma. Tissue microarray technology, with the advantages of high throughput, conciseness, rapidity, high efficiency, low cost, and nice reproducibility, has significant practical value and broad application prospects in pathology.

Aged ; Aged, 80 and over ; DNA Helicases ; biosynthesis ; Humans ; Immunohistochemistry ; Male ; Microchip Analytical Procedures ; Middle Aged ; Nuclear Proteins ; biosynthesis ; Prostatic Neoplasms ; metabolism ; pathology ; Reproducibility of Results ; Transcription Factors ; biosynthesis

OBJECTIVETo study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts.

METHODSCalvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction.

RESULTSThe mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3.

CONCLUSION1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals ; Animals, Newborn ; Calcitriol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Glycoproteins ; biosynthesis ; genetics ; physiology ; Ligands ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; NF-kappa B ; biosynthesis ; genetics ; Osteoclasts ; metabolism ; physiology ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; analysis ; biosynthesis ; genetics ; physiology ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo study the expressions of p53 and Gadd45a proteins and their clinicopathological significance in human pancreatic cancer.

METHODSThe expression of p53 and Gadd45a proteins was detected with immunohistochemistry in a series of 59 pancreatic cancers. Their relationships with the clinicopathological parameters including gender, tumor site, TNM stage, histological differentiation, and the prognosis of pancreatic cancer patients were analyzed.

RESULTSThe positive expression rate of p53 protein was 67.8% (40/59) and that of Gadd45a protein was 42.4% (25/59). The positive expression rate of p53 protein is significantly higher in patients < 65 years than in patients > or = 65 years (chi squared = 4.711, P = 0.030). Gadd45a expression was not correlated to the age of the patients. No significant difference was found between the expression of p53 proteins and histological differentiation and TNM stage of the tumors. Gadd45a expression was correlated with histological differentiation of pancreatic cancer (chi squared = 10.052, P = 0.007), but not with TNM stage of the tumors. No significant differences in the prognosis were found between the groups with and without p53 expression (chi squared = 0.09, P = 0.764) and the groups with and without Gadd45a expression (chi squared = 0.14, P = 0.704).

CONCLUSIONSBoth p53 and Gadd45a are highly expressed in human pancreatic cancer and may be associated with biological features of pancreatic cancer. Their expression alone or co-expression may be not helpful to evaluate the prognosis of patients with pancreatic cancer.

Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; Carcinoma, Pancreatic Ductal ; metabolism ; pathology ; Cell Cycle Proteins ; biosynthesis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Staging ; Nuclear Proteins ; biosynthesis ; Pancreatic Neoplasms ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; biosynthesis

OBJECTIVEResearch was done on the possible roles of beta-catenin, p53 and proliferating cell nuclear antigen (PCNA) in the carcinogenesis of colorectal adenoma (CRA).

METHODSbeta-catenin and p53 and PCNA expressions were studied with immunohistochemical stain in 77 specimens of CRA together with mild epithelial dysplasia (CRA-MD), CRA with moderate/severe epithelial dysplasia (CRA-D/SD) and CRA with cancerous changes (CRA-C).

RESULTSThe percentage of abnormal expression of beta-catenin increased during the transition from CRA-MD to CRA-D/SD to CRA-C (P < 0.01). The nuclear expressions of beta-catenin in CRA-D/SD and CRA-C were all significantly higher than that in CRA-MD. Expression of p53 and PCNA were increased from CRA-MD to CRA-D/SD to CRA-C, with the positive rates in these three groups of 10.3%, 43.8%, 75.0% for p53 and 17.2%, 62.5%, 87.5% for PCNA, respectively. 69.7% of cases with positive nuclear beta-catenin expression showed strong PCNA positivity which was much higher than the 36.4% of cases without nuclear beta-catenin expression (P < 0.05). The percentage of strong PCNA expression in the p53 positive cases was significant higher than that in cases with negative p53 expression (72.4% vs 37.5%, P < 0.05). Nuclear beta-catenin and p53 co-expression rates in CRA-C reached 50%.

CONCLUSIONbeta-catenin, p53 and PCNA may play important roles in the carcinogenesis of colorectal adenoma.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; Colorectal Neoplasms ; diagnosis ; metabolism ; Cytoskeletal Proteins ; biosynthesis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prognosis ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Trans-Activators ; biosynthesis ; Tumor Suppressor Protein p53 ; biosynthesis ; beta Catenin

OBJECTIVE: To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection. METHODS: Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine. RESULTS: There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum. CONCLUSION There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.
Animals ; Calcium Channels, N-Type ; biosynthesis ; Corpus Callosum ; cytology ; metabolism ; DNA-Binding Proteins ; Male ; Nerve Tissue Proteins ; biosynthesis ; Neural Pathways ; physiology ; Neurons ; cytology ; Nuclear Proteins ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To determine the expressions of survivin and proliferating cell nuclear antigen (PCNA)in non-small cell lung cancer (NSCLC) and to explore its clinical pathological significance. METHODS: Immunohistochemical SP method was used to detect the expressions of survivin and PCNA in 43 patients with NSCLC and 15 normal epithelial tissues of the lung. PCNA labeling proliferative index was assessed. Forty-three patients with NSCLC were followed up for more than 5 years. RESULTS: The positive expression of survivin in NSCLC (79.1%) was significantly higher than that in normal epithelial tissues of the lung (P < 0.01). The survivin expression in Stage I + II was lower than in Stage III (P < 0.05). The overall survival time was significantly shorter in patients with high survivin expression than that in patients with absent or low survivin expression. The survivin expression was not related to sex, age, tumor size and site, histological type, grade, and lymphoid node metastasis (P > 0.05). The mean proliferative index of PCNA in NSCLC was much higher than that in normal epithelial tissues of the lung (P < 0.01). A positive correlation was present between the proliferative index and the tumor size, lymph node metastase, and clinical stage (P <0.01), while a negative correlation between the proliferative index and survival time (P <0.01). There was no correlation between proliferative index and age, sex, site, histological type and grade. The proliferative index was larger in patients with moderate or strong positive survivin expression than that in patients with negative or weak survivin expression (P < 0.05). CONCLUSION Over expression of survivin and PCNA is closely correlated to the progression and prognosis of patients with NSCLC, which is helpful to evaluate the progression of cancer and to predict the prognosis of NSCLC. The up-regulation of survivin expression and its close relationship with the cell proliferation in NSCLC suggest that survivin may play an important role in the carcinogenesis and development of lung cancer.
Adult ; Aged ; Biomarkers, Tumor ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Lung Neoplasms ; metabolism ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; genetics ; Neoplasm Staging ; Prognosis ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Survivin

Cell Line ; Humans ; Liver ; metabolism ; Membrane Potential, Mitochondrial ; Mitochondria, Liver ; metabolism ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Proteins ; pharmacology