Research on the bioactivity of isoquercetin extracted from marestail on bladder cancer EJ cell and the mechanism of its occurrence.

Research studies in recent years have found that isoquercetin has an inhibiting effect on multiple carcinogens, but research studies filed on isoquercetin in bladder cancer are quite few. This paper observed the influence of isoquercetin on biological activity of the EJ cell of bladder cancer through HC dyeing and trypan blue counting, studied the EJ cell cycle by flow cytometry (FCM), and then analyzed the influence of isoquercetin and its effect on the protein expression of STAT3 and STAT3-inhibiting factors (PIAS3) in EJ cells. Research has shown that isoquercetin has an inhibitory effect on the EJ cells of bladder cancer, but it is not obvious.

Astragalus membranaceus up-regulate Cosmc expression and reverse IgA dys-glycosylation in IgA nephropathy.

BACKGROUND: Decreased Core I ß3-Gal-T-specific molecular chaperone (Cosmc) expression induced IgA1 aberrant glycosylation is the main characteristic of IgA nephropathy (IgAN). This study tried to elucidate the effect of Astragalus membranaceus on Cosmc expression and IgA O-glycosylation of peripheral B lymphocytes in IgAN patients. METHODS: Peripheral B lymphocytes of 21 IgAN patients and 10 normal controls were isolated and cultured with or without lipopolysaccharide (LPS) and Astragalus membranaceus injection (AMI). Cosmc mRNA and protein expression levels were measured by real-time RT-PCR and Western blot. IgA1 and glycosylation level were determined by enzyme-linked immunosorbent assay (ELISA) and VV lectin-binding method. RESULTS: Cosmc mRNA expression and IgA1 O-glycosylation level in IgAN patients was significantly lower than normal controls at baseline. Treatment of LPS could obviously inhibit Cosmc expression and increase the IgA1 secretion in peripheral B lymphocytes of IgAN patients, which resulted in a significantly increase in IgA1 aberrant glycosylation level. Addition of AMI could remarkably up regulated Cosmc expression, decrease IgA1 secretion, and reverse glycosylation level in a dose related manner. CONCLUSION: AMI can up-regulate Cosmc expression of peripheral B lymphocytes and reverse IgA1 aberrant O-glycosylation level, which might be the underlying mechanism of AMI therapy in treating IgAN. TRIAL REGISTRATION: TCTR20140515001 (Registration Date: 2014-05-15).

[Antibiotic-dependent selection of the E. coli clones with increased chaperone activity for highly-efficient production of the full-length soluble New Delhi metallo-beta-lactamase].

The spread of the New Delhi metallo-beta-lactamase (NDM-1), a plasmid-borne enzyme conferring bacterial resistance to any known beta-lactam antibiotics, represents the global health threat. There is an urgent need to develop the efficient NDM-1 inhibitors of various mode of action thereby necessitating structural studies of the enzyme as well as analysis of the secretion pathway and localization of the protein. The recombinant full-length NDM-1 is produced in E. coli in the inactive form and is mostly accumulated in the inclusion bodies. The secreted recombinant NDM-1 forms are several N-terminally truncated species. The robust expression system capable of high-level production of the full-length NDM-1 and derivatives thereof is required to obtain NDM-1 in the quantities necessary for drug discovery, diagnostics, and research purposes. Therefore, we developed a new system that utilizes antibiotic pressure to select E. coli producing increased quantity of soluble NDM-1 and showed that an increase in the NDM-1 solubility occurs in the bacterial clones producing increased amounts in the chaperones.

Gene expression analysis of the mechanisms whereby black cohosh inhibits human breast cancer cell growth.

BACKGROUND: Previous studies indicate that specific extracts and the pure triterpene glycoside actein obtained from black cohosh inhibit growth of human breast cancer cells. Our aim is to identify alterations in gene expression induced by treatment with a methanolic extract (MeOH) of black cohosh. MATERIALS AND METHODS: We treated MDA-MB-453 human breast cancer cells with the MeOH extract at 40 microg/ml and collected RNA at 6 and 24 h; we confirmed the microarray results with real-time RT-PCR for 18 genes. RESULTS: At 6 h after treatment there was significant increase in expression of ER stress (GRP78), apoptotic (GDF15), lipid biosynthetic (INSIG1 and HSD17B7) and Phase 1 (CYP1A1) genes and, at 24 h, decrease in expression of cell cycle (HELLS and PLK4) genes. CONCLUSION: Since the MeOH extract activated genes that enhance apoptosis and repressed cell cycle genes, it may be useful in the prevention and therapy of breast cancer.

The Agrobacterium VirE3 effector protein: a potential plant transcriptional activator.

During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-alpha and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development.

[Effects of yisui shengxue granules on expressions of alpha-hemoglobin stabilizing protein and erythroid transcription factor GATA-1 mRNAs in bone marrow of patients with beta-thalassemia].

OBJECTIVE: To evaluate the effects of Yisui Shengxue Granules on expressions of alpha-hemoglobin stabilizing protein (AHSP) and erythroid transcription factor GATA-1 mRNAs in bone marrow of patients with beta-thalassemia, and to explore its possible molecular mechanism. METHODS: Twelve patients with beta-thalassemia intermedia were treated with Yisui Shengxue Granules for three months. The blood indexes including hemoglobin (Hb), RBC, fetal hemoglobin (HbF) and reticulated corpuscles (Ret) were examined before and after treatment. Total RNA was extracted from bone marrow karyocyte in 8 patients selected from these 12 patients before and after treatment, and the expression levels of the AHSP and GATA-1 mRNAs were measured by real-time PCR. RESULTS: Yisui Shengxue Granules could not only obviously improve the clinical symptoms of patients with beta-thalassemia intermedia, but also obviously increased the contents of Hb, RBC, HbF and Ret (P<0.05, or P<0.01). The expression levels of AHSP and GATA-1 mRNAs also significantly increased after treatment as compared with those before treatment (P<0.05, or P<0.01). CONCLUSION: The results revealed that one of the possible molecular mechanism of the effects caused by Yisui Shengxue Granules is that it can up-regulate the expression levels of AHSP and erythroid transcription factor GATA-1 mRNAs, enhance the protein synthesis of AHSP which can bind the relative excess free alpha-globin, prevent the formation of alpha -globin-cytotoxic precipitates in red blood cells and decrease the hemolysis.

Genistein induces glucose-regulated protein 78 in mammary tumor cells.

Preliminary studies have shown that genistein modulates the expression of some heat shock proteins in mammary tumor cells. In this study, we investigated the effect of genistein pretreatment on the expression of glucose-regulated protein 78 (GRP78) in both estrogen receptor-positive (MCF-7) and -negative (MDA-MB-231) cells. Genistein increased the expression of GRP78 in a dose- and time-dependent manner and suppressed glucose uptake in both cell lines. However, induction of GRP78 by genistein appears not to be directly associated with inhibition of glucose uptake. Genistein treatment also made MDA-MB-231 cells more sensitive to doxorubicin, probably via increased GRP78 expression, but had no effect or even decreased drug sensitivity in MCF-7 cells. These results suggest that genistein may be exploited as an enhancer of chemotherapeutic agents in certain types of breast cancer.

Multiple co-transfection and co-expression of human beta-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells.

Human beta-1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, beta3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25-1500 microg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed beta3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying beta3GnT2. In this study we correlated the activity of beta3GnT2 with the amount of beta3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and beta3GnT2 cDNA were in a rough ratio of 1:1, the beta3GnT2 activity was at a high level. In order to achieve better expression levels of beta3GnT2 with less cost and time, efficient ways have to be devised.

Ge-Jee-Bok-Ryung-Hwan induces apoptosis in human cervical carcinoma HeLa cells--an endoplasmic reticulum stress pathway--.

Ge-Jee-Bok-Ryung-Hwan (GJBRH), a commonly used herb formulation in Korea, Japan and China, caused a decrease of viability in HeLa human cervical carcinoma cells. The treatment of GJBRH resulted in genomic DNA fragmentation as well as the increase of Sub-G1 portion in cell cycle analysis. In this study, GFP-Bax over-expression system showed that Bax, pro-apoptotic Bcl-2 family protein, was translocated to mitochondria by the presence of GJBRH. The treatment of BAPTA-AM, permeable endogenous calcium chelator, inhibited GJBRH-induced caspase-3 and -9 activations, the release of cytochrome c and Smac/DIABLO into cytoplasm and the resultant cell death in HeLa human cervical carcinoma cells. The treatment of BAPTA-AM increased the expression of XIAP, which mediates binding to and inhibiting caspases and showed protective effect, in GJBRH-treated cells. GJBRH induced the expression of Glucose Response Protein 78 (GRP 78), a positive ER stress marker protein. However, BAPTA-AM did not interfere with the ER-stress response pathway that triggers the expression of GRP 78. This study showed that GJBRH induces cell death, which occurs downstream of or parallel to this point in the ER-stress pathway linked to apoptosis. In conclusion, GJBRH induces apoptosis in HeLa cells via ER stress-pathway associated mitochondria-dependent apoptosis mechansim.

Clusterin as a biomarker in murine and human intestinal neoplasia.

Early detection of colorectal cancer is critical for the management of this disease. Biomarkers for early detection of several cancers have been developed and applied clinically in recent years. We have sought to discover candidate biomarkers without the restricted choice of markers placed on microarrays, and without the biological complications of genetic and environmental heterogeneity. We have compared by cDNA subtraction two genetically matched sets of mice, one developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then subjected to a series of validation steps. In situ hybridization and immunohistochemistry were used to analyze clusterin expression at a cellular level on a series of murine intestinal and human colonic neoplasms. Elevated clusterin expression was characterized within certain regions of murine and human tumors regardless of tumor stage, location, or mode of initiation. The cells showing high clusterin levels generally lacked differentiation markers and adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed low levels of clusterin. Its specific expression patterns and correlation with cellular events during tumorigenesis make it a useful diagnostic tool in the mouse and a potential contributor to the set of biomarkers for early detection of human colon cancer.

Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens.

Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action.

The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex.

When mammalian cells are subjected to calcium depletion stress or protein glycosylation block, the transcription of a family of glucose-regulated protein (GRP) genes encoding endoplasmic reticulum (ER) chaperones is induced to high levels. The consensus mammalian ER stress response element (ERSE) conserved among grp promoters consists of a tripartite structure CCAAT(N9)CCACG, with N being a strikingly GC-rich region of 9 bp. The ERSE, in duplicate copies, can confer full stress inducibility to a heterologous promoter in a sequence-specific but orientation-independent manner. In addition to CBF/NF-Y and YY1 binding to the CCAAT and CCACG motifs, respectively, we further discovered that an ER stress-inducible complex (ERSF) from HeLa nuclear extract binds specifically to the ERSE. Strikingly, the interaction of the ERSF with the ERSE requires a conserved GGC motif within the 9 bp region. Since mutation of the GGC triplet sequence also results in loss of stress inducibility, specific sequence within the 9 bp region is an integral part of the tripartite structure. Finally, correlation of factor binding with stress inducibility reveals that ERSF binding to the ERSE alone is not sufficient; full stress inducibility requires integrity of the CCAAT, GGC and CCACG sequence motifs, as well as precise spacing among these sites.

Overproduction of DnaJ in Escherichia coli improves in vivo solubility of the recombinant fish-derived transglutaminase.

The overexpression of red sea bream (Pagrus major) transglutaminase (TGase, E.C. 2.3.2.13) in Escherichia coli mostly leads to the accumulation of biologically inactive enzyme. Although the solubility of the gene products could be improved by cultivation at a lower temperature (26-28 degrees C), most of the synthesized TGase was still in the form of insoluble aggregates. The effects of overproduction of molecular chaperones on the intracellular solubility of newly produced recombinant TGase were examined. The overexpression of dnaK or groES/EL did not improve solubility. However, DnaJ greatly increased the solubility of the recombinant TGase, resulting in active enzyme in the presence of calcium ions. Co-expression of dnaK along with dnaJ further increased the content of soluble TGase. Under our experimental conditions, supplementation with both DnaJ and DnaK elevated the TGase activity in the producer cells by roughly 4-fold, compared with the control strain cultured at 30 degrees C. Thus, we found that DnaJ is important in controlling the solubility of protein overproduced in E. coli.

Molecular chaperones in the etiology and therapy of cancer.

Molecular chaperones are ubiquitous, well-conserved proteins that account for 2-5 % of all cellular proteins in most cells. The present review summarizes our current knowledge about their involvement in the etiology and therapy of cancer with special emphasis on the expression of chaperones in malignant cells, their role in folding of (proto)oncogene products, cell cycle regulation, cell differentiation and apoptosis, development of metastasis, and their participation in the recognition of malignant cells. We also overview the importance of chaperones in hyperthermia, drug resistance, and recent approaches in chaperone-immunotherapy.

The expression of the molecular chaperone calnexin is decreased in cancer cells grown as colonies compared to monolayer.

Differential display was used to identify genes which were differentially expressed when HT-29 human colon adenocarcinoma cells were grown as monolayers attached to plastic or as colonies in soft agarose. One of the gel bands differentially displayed corresponded to a 171 bp fragment that showed 99% identity with a sequence of mRNA for human calnexin. The decrease in calnexin gene expression by HT-29 cells growing as colonies in soft agarose was confirmed by reverse transcriptase-PCR (RT-PCR) using calnexin-specific primers. We also used RT-PCR to show that the expression of calnexin was decreased in HT-29 cells and MCF-7 human breast adenocarcinoma cells growing in suspension in poly(hydroxyethyl methacrylate)-coated wells compared to cells growing as monolayers. The results suggest that there is a signal for the up-regulation of calnexin expression when cells contact a substrate which allows cell adhesion.

Selective induction of the glucose-regulated protein grp78 in human monocytes by bacterial extracts (OM-85): a role for calcium as second messenger.

Heat shock/stress proteins (HSP) act as molecular chaperones, protect cells from injury, and are involved in the immune response. We investigated the effects of the immunomodulating bacterial extracts OM-85 on the stress response in normal human peripheral blood monocytes. While OM-85 did not induce the classical HSP, we show here, using 2D gel electrophoresis combined with immunoblotting, the induction of the glucose regulated protein grp78 (the immunoglobulin heavy chain binding protein BiP) along with the described accumulation of pro-interleukin-1 beta. The increased Ca2+ mobilization observed with OM-85 is the likely second messenger for grp78 induction. Recent studies are in favor of a protective role of grp78 against cytokine-mediated cytotoxicity and apoptosis. We suggest that grp78 induction following exposure to OM-85 explains, at least in part, the immunodulatory and protective effects of the bacterial extracts.

Molecular characterization of seizure-related genes isolated by differential screening.

To isolate seizure-related genes, we applied differential screening technique to the cDNA library which constructed from primary cultured cerebral cortical cells treated with pentylenetetrazol (PTZ). Northern blotting analysis of mRNA levels in the cerebra after systemic administration of PTZ confirmed the results of differential screening procedure: 6 clones showed increased mRNA level and 3 clones showed decreased expression with PTZ. Interestingly, 4 genes which were isolated by this technique were related to intracellular calcium action. They were cytosolic phospholipase A2, 78 kDa glucose regulated protein, SEZ-15 which has an EF hand motif and PTZ-17 that causes calcium current in Xenopus oocyte with PTZ application. These data and our previous results suggest that intracellular calcium may play an important role for seizure-related pathophysiological changes in neuronal cells.