Alterations in ß­catenin/E­cadherin complex formation during the mechanotransduction of Saos­2 osteoblastic cells.

Mechanical load application promotes bone formation, while reduced load leads to bone loss. However, the underlying mechanisms that regulate new bone formation are not fully understood. Wnt/ß­catenin signaling has an important role in bone formation, bone growth and remodeling. The aim of the present study was to investigate whether mechanical stimuli regulated bone formation through the Wnt/ß­catenin signaling pathway. Saos­2 osteoblastic cells were subjected to mechanical strain using a Flexcell strain loading system. The results demonstrated that 12% cyclical tensile stress significantly stimulated Saos­2 cell proliferation, increased the activity of alkaline phosphatase and promoted the formation of mineralized nodules, as determined by MTT and p­nitrophenyl phosphate assays and Alizarin Red S staining, respectively. Furthermore, western blot analysis demonstrated that, following mechanical strain, increased phosphorylation of glycogen synthase kinase­3ß and nuclear ß­catenin expression was observed in cells, compared with static control culture cells. Results of reporter gene and reverse transcription­polymerase chain reaction assays also demonstrated that mechanical strain significantly increased T­cell factor reporter gene activity and the mRNA expression of cyclooxygenase (COX)­2, cyclin D1, c­fos and c­Jun in Saos­2 cells. Co­immunoprecipitation analysis revealed that elongation mechanical strain activated Wnt/ß­catenin signaling and reduced ß­catenin and E­cadherin interaction in Saos­2 cells. In conclusion, the results of the current study indicate that mechanical strain may have an important role in the proliferation and differentiation of osteoblasts. The disassociation of the ß­catenin/E­cadherin complex in the osteoblast membrane under stretch loading and the subsequent translocation of ß­catenin into the nucleus may be an intrinsic mechanical signal transduction mechanism.

UCH-L1 Expressed by Podocytes: a Potentially Therapeutic Target for Lupus Nephritis?

Systemic lupus erythematosus (SLE) is a multisystem disease affecting many organs, and the most severe complication is lupus nephritis. Podocyte injury and loss play vital roles in the pathogenesis of lupus nephritis. Studies have shown that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is involved in the pathogenesis and progression of many diseases, such as neurodegenerative disorders, cancers, and diabetes. Recently, numerous studies have indicated that UCH-L1 was upregulated in the podocytes in immune complex-mediated glomerulonephritis. This increase was correlated with disease aggravation. In this review, we discuss the role and mechanism of UCH-L1 in the pathogenesis of many diseases and, particularly, in lupus nephritis. Hence, we highlight the role of UCH-L1 in lupus nephritis.

Proteomics based detection of differentially expressed proteins in human osteoblasts subjected to mechanical stress.

Mechanical stress is essential for bone development. Mechanical stimuli are transduced to biochemical signals that regulate proliferation, differentiation, and cytoskeletal reorganization in osteoblasts. In this study, we used proteomics to evaluate differences in the protein expression profiles of untreated Saos-2 osteoblast cells and Saos-2 cells subjected to mechanical stress loading. Using 2-D electrophoresis, MALDI-TOF mass spectroscopy, and bioinformatics, we identified a total of 26 proteins differentially expressed in stress loaded cells compared with control cells. Stress loaded Saos-2 cells exhibited significant upregulation of 17 proteins and significant downregulation of 9 proteins compared with control cells. Proteins that were most significantly upregulated in mechanically loaded cells included those regulating osteogenesis, energy metabolism, and the stress response, such as eukaryotic initiation factor 2 (12-fold), mitochondrial ATP synthase (8-fold), and peptidylprolyl isomerase A (cyclophilin A)-like 3 (6.5-fold). Among the proteins that were significantly downregulated were those involved in specific signaling pathways and cell proliferation, such as protein phosphatase regulatory (inhibitor) subunit 12B (13.8-fold), l-lactate dehydrogenase B (9.4-fold), Chain B proteasome activator Reg (Alpha) PA28 (7.7-fold), and ubiquitin carboxyl-terminal esterase L1 (6.9-fold). Our results provide a platform to understand the molecular mechanisms underlying mechanotransduction.

Expression of FGF4 mRNA is mediated by mating behaviours in mice.

In mammals, breeding is preceded by species-specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16 h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5 µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c-Myc) in oocytes, apoptosis-related genes (i.e. Bax, Bcl2 and c-Myc) in cumulus cells and pluripotency-related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (p < 0.05). Embryo development was primarily blocked at the eight-cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25 ng/ml FGF4 to the medium at the four-cell stage (p > 0.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression.

[Effects of tetramethylpyrazine on nitric oxide synthase activity and calcium ion concentration of skeletal muscle in hindlimb unloading rats].

OBJECTIVE: To explore the effects of tetramethylpyrazine on the nitric oxide synthase activity and calcium ion concentration in skeletal muscle fiber and decipher the possible mechanisms of anti-muscle atrophy function of tetramethylpyrazine in hindlimb unloading rats. METHODS: Hindlimb unloading (HLU) rats were used as a muscle atrophy model to study the activity of nitric oxide synthase by colorimetry. The concentration of intracellular calcium ion was measured by laser scanning confocal microscope. A total of 18 female rats were randomly divided into 3 groups: control (CON), hindlimb unloading with water (HLU + W) and hindlimb unloading with tetramethylpyrazine (HLU + Tmp) (n = 6 each). RESULTS: (1) Compared with CON, the activity of nitric oxide synthase decreased by 28% in HLU + W (P < 0.05) and decreased by 46% in HLU + Tmp (P < 0.01). The activity of nitric oxide synthase less decreased in HLU + Tmp than that in HLU + W, but it was not statistically significant (P > 0.05). (2) Compared with CON, the concentrations of intracellular calcium ion in HLU + W and HLU + Tmp increased by 330% and 86% respectively (P < 0.01). Compared with HLU + W, the concentration of intracellular calcium ion decreased by 130% in HLU + Tmp (P < 0.01). CONCLUSION: The activity of nitric oxide synthase decreases and the concentration of calcium ion increases in hindlimb unloading rats. And tetramethylpyrazine may suppress the calcium ion overloading but not the activity of NOS associated with disuse muscular atrophy.

Effect of ergosta-4,6,8(14),22-tetraen-3-one (ergone) on adenine-induced chronic renal failure rat: a serum metabonomic study based on ultra performance liquid chromatography/high-sensitivity mass spectrometry coupled with MassLynx i-FIT algorithm.

BACKGROUND: Ergosta-4,6,8(14),22-tetraen-3-one (ergone) has been proven to prevent the progression of renal injury and the subsequent renal fibrosis. We investigated the therapeutic effects and mechanism of ergone on a chronic renal failure model of rats induced by adenine. METHODS: A serum metabonomic method based on the UPLC Q-TOF/MS was undertaken to explore the excretion pattern of low molecular mass metabolites. RESULTS: Coupled with blood biochemistry and kidney histopathology results, the significant difference in metabolic profiling between adenine-induced chronic renal failure group and the ergone treated group by using pattern recognition analysis indicated that changes in global serum metabolites occurred. Some significantly changed metabolites like lysophosphatidylcholines, adenine, dopamine, creatinine, aspartic acid and phenylalanine have been found and identified. These biochemical changes in serum metabolites are related to the perturbations of amino acid metabolism and lecithin metabolism, which may be helpful to further understand the chronic renal failure and therapeutic mechanisms of ergone. CONCLUSION: The work shows that the metabonomic method is a valuable tool for studying the essence of chronic kidney disease and therapeutic effect mechanism of preclinical or clinical drug.

Increased apoptosis and expression of FasL, Bax and caspase-3 in human lupus nephritis class II and IV.

BACKGROUND: Apoptosis is involved in glomerular injuries leading to glomerulonephritis. However, the role of renal cellular apoptosis in the pathogenesis and progression of human lupus nephritis (LN) is controversial, and studies on the expression of apoptosis-related proteins, such as FasL, Bax and caspase-3, in different classifications of human LN renal tissues are limited. METHODS: Thirty-two samples of LN tissues, including 10 cases of class II and 22 cases of class IV LN, and 5 cases of human normal renal tissues were obtained. Expression of FasL, Bax and caspase-3 proteins in LN tissues was examined by immunohistochemical staining. Apoptotic cells were evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: Expression of FasL, Bax and caspase-3 and TUNEL-positive cells in glomerular parenchymal cells, renal tubular epithelial cells and interstitial inflammatory cells were higher in LN tissues compared with controls. Expression of Bax and caspase-3, but not FasL, was significantly higher in glomeruli of class IV LN than those of class II LN. The apoptotic cell count per glomerulus was significantly higher in class IV LN than class II LN (p<0.05). CONCLUSIONS: Increased apoptosis and the expression of FasL, Bax and caspase-3 in human LN suggest that apoptosis might be induced through pathways of these proteins in the pathogenesis process and play an important role in LN progression through Bax and caspase-3, but not FasL.

Abnormal expression of NRF-2α in hepatocellular carcinoma identified with a newly prepared monoclonal antibody against human NRF-2α protein.

Human nuclear respiratory factor 2 alpha subunit (NRF-2α) is fundamentally important to cell function and the development. We aimed to establish the monoclonal antibody (MAb) against the human NRF-2α protein and to investigate its distribution in human hepatocellular carcinoma (HCC) and tumor-adjacent tissues. The 6× His-NRF-2α fusion protein was successfully induced and purified. One monoclonal antibody (MAb) against human NRF-2α, 1-D10-E1-B11-G3 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. Using immunohistochemical analysis, the expression of NRF-2α was investigated in 38 cases of HCC specimens and 14 cases of tumor-adjacent specimens. Staining was found positive in 9 cases of HCC tissues (23.7%) and 8 cases of normal hepatic tissues (57.1%). The higher-grade frequency of expression of NRF-2α in tumor-adjacent tissues was significantly higher (P < 0.01) than that in tumor tissues, suggesting that NRF-2α may play important roles in carcinogenesis of HCC.

[Significant increase of glucose transport activity in breast cancer].

OBJECTIVE: To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. METHODS: A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). CONCLUSIONS: Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

Heat shock protein 70 fused to or complexed with hantavirus nucleocapsid protein significantly enhances specific humoral and cellular immune responses in C57BL/6 mice.

Heat shock proteins (HSPs) are known to act as an effective molecular adjuvant to enhance the induction of antigen peptide-specific cellular immunity, when coupled with the antigen or peptide. Hantaan virus (HTNV) nucleocapsid protein (NP) is relatively conserved among hantaviruses and highly immunogenic in both animals and humans. To analyze the influence of HSP70 on NP vaccine potency, and evaluate the possibility of developing a novel effective vaccine against hantaviruses, we constructed prokaryotic expression plasmids, and expressed three recombinant proteins, namely, HTNV NP, HSP70 and HSP70-NP fusion protein. As an alternative to fusion protein, we also generated HSP70 and HTNV NP complexes (HSP70+NP) in vitro. C57BL/6 mice were immunized with those recombinant proteins, the humoral and cellular responses elicited against NP were measured by ELISA, fluorescence flow cytometry, cytotoxicity assays, and IFN-gamma ELISPOT assay. We found that immunization of mice with HSP70-NP fusion protein, or HSP70+NP complexes elicited significantly higher NP-specific antibody titers, frequencies of IFN-gamma-producing cells and cytotoxic T lymphocyte (CTL) activities in vivo than conventional HTNV NP vaccination. Antibody isotype analysis showed that the antibody response was characterized by a higher HTNV NP-specific titer of IgG2a than IgG1 antibodies, resulting in a significant higher IgG2a/IgG1 ratio. By comparison, HSP70-NP fusion protein is significantly superior to HSP70+NP complexes in enhancement of NP antigenicity. These results indicated that HSP70, when fused to or complexed with HTNV NP, greatly enhance NP vaccine potency by preferential induction of a predominant Th1 immune response in a NP-specific, HSP70-dependent manner.

[Expression of Hsp70 Grp94 and IgG in human lung carcinoma].

AIM: To explore the expression and significance of heat shock protein 70 (Hsp70), glucose regulated protein 94 (Grp94) and immunoglobulin G (IgG) in human lung carcinoma. METHODS: The expression of Hsp70, Grp94 and IgG in 40 human lung carcinomas was studied using immunohistochemical technique and image analysis. The localization among Hsp70, Grp94 and IgG was analyzed by double labeling immunofluorescent staining and laser scanning confocal microscopy. RESULTS: Hsp70, Grp94 and IgG in Human lung carcinomas showed high expression. The positive rate of Hsp70, Grp94 and IgG was 65% (26/40), 45% (18/40), and 82.5% (33/40), respectively. The average value of optical density was 5.10 +/- 0.32, 3.52 +/- 0.35, and 8.12 +/- 0.31, respectively. Hsp70 was localized in nucleus and cellular cytoplasm while Grp94 and IgG were mainly localized in cellular cytoplasm. Ten cases showed Hsp70 was co-localized with IgG and Eighteen cases showed Grp94 was co-localized with IgG. CONCLUSION: High expression of Hsp70, Grp94 and IgG in Human lung carcinomas suggested that Hsp70, Grp94 and IgG might play an important role in the development of human lung carcinoma. IgG is co-localized with Hsp70 or Grp94. The study will lay a theorical basis for further research on anti-tumor immunotherapy.

Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver.

BACKGROUND: The development of new ultrasound contrast agents (UCAs) has become one of the most promising fields in ultrasound medicine. This paper evaluates a new self-made contrast agent enhancement effect developed to study the fibrotic stages of the liver in perfusion models in vivo. METHODS: We constructed experimental models of hepatic fibrosis involving five stages from F0 to F4 via administration of CCL4 (0.01 ml/kg BW) every 3 days for 3 months. The intrahepatic circulatory time of the contrast agent was analyzed via an image and Cine-loop display. Calculations of the perfusion-related parameters including the peak signal intensity (PSI) and peak signal intensity time (PIT) of the portal vein and parenchyma were obtained from an analysis of the time-acoustic intensity curve. RESULTS: Hepatic artery to vein transmit time (HA-HVTT) was significantly shorter at F4 stage (mean 5.1 seconds) compared with those in other stages (mean 8.3 s, 7.5 s, 6.9 s, 6.6 s, P < 0.01). The average PSI difference of PV-parenchyma was 13.62 dB in F4 stage, demonstrating significant differences between F4 stage and other early stages (P < 0.001). CONCLUSION: These results indicate that the new self-made contrast agent is capable of indicating intrahepatic hemodynamic changes. HA-HVTT and the PSI difference of the microbubble perfusion in liver parenchyma and PV were considered to differentiate the degree of hepatic fibrosis between F4 and other early stages.

HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo.

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, fluorescence activated cell sorter (FACS) analysis, cytotoxicity assay, and IFN-gamma ELISPOT assay. We found that HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG2a/IgG1 ratio, IFN-gamma producing CD8+ T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection.

[Expression of NF-kappaB/IkappaB signal pathway in renal tissues of hepatitis B virus-associated nephritis].

AIM: To investigate the expression of nuclear transcription factor-kappa B (NF-kappaB)/IkappaB in renal tissues of hepatitis B virus-associated nephritis (HBVGN) and to study its role in the pathogenesis of HBVGN. METHODS: The expression of NF-kappaB p65 and IkappaB-alpha in renal tissues was examined in biopsy specimens from 42 HBVGN patients, 20 patients with primary membranous nephropathy and 5 normal subjects by immunohistochemical staining. RESULTS: The NF-kappaB p65 expression in glomerular and tubular of HBVGN tissues was notably higher than that in normal control tissues (P<0.05) and in primary membranous nephropathy tissues (P<0.01). The IkappaB-alpha expression in glomerular and tubular of HBVGN tissues was lower than that in primary membranous nephropathy tissues (P<0.05). CONCLUSION: There might be protein degradation of IkappaB-alpha and nuclear translocation of NF-kappaB in renal tissues of HBVGN, which suggests that NF-kappaB/IkappaB signal pathway may play an important role in the pathogenesis of HBVGN.