Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.     

Monitoring of petroleum hydrocarbon degradative potential of indigenous microorganisms in ozonated soil

This study was performed to investigate the petroleum hydrocarbon (PH) degradative potential of indigenous microorganisms in ozonated soil to better develop combined pre-ozonation/bioremediation technology. Diesel-contaminated soils were ozonated for 0–900min. PH and microbial concentrations in the soils decreased with increased ozonation time. The greatest reduction of total PH (TPH, 47.6%) and aromatics (11.3%) was observed in 900-min ozonated soil. The number of total viable heterotrophic bacteria decreased by three orders of magnitude in the soil. Ozonated soils were incubated for 9weeks for bioremediation. The number of microorganisms in the soils increased during the incubation period, as monitored by culture- and nonculture-based methods. The soils showed additional PH-removal during incubation, supporting the presence of PH-degraders in the soils. The highest removal (25.4%) of TPH was observed during the incubation of 180-min ozonated soil during the incubation while a negligible removal was shown in 900-min ozonated soil. This negligible removal could be explained by the existence of relatively few or undetected PH-degraders in 900-min ozonated soil. After a 9-week incubation of the ozonated soils, 180-min ozonated soil showed the lowest TPH concentration, suggesting that appropriate ozonation and indigenous microorganisms survived ozonation could enhance remediation of PH-contaminated soil. Microbial community composition in 9-week incubated soils revealed a slight difference between 900-min ozonated and unozonated soils, as analyzed by whole cell hybridization. Taken together, this study provided insight into indigenous microbial potential to degrade PH in ozonated soils.     

Molecular characterization of spontaneous and growth-factor-augmented chondrogenesis in periosteum–bone tissue transferred into a joint

Multilineage potential of progenitor cells from periosteum is well established, but conditions for differentiation within their native niche are unclear. We evaluated at cellular and molecular levels whether chondrogenesis of periosteal progenitor cells is promoted spontaneously or by growth-factor mixture (GFM) application when transferring periosteum–bone cylinders into cartilage defects. Osteochondral defects in the patellar groove of minipigs were filled with periosteum–bone cylinders and randomly supplemented with GFM. Neochondrogenesis was characterized by histology, immunohistology, and quantitative gene expression analysis. According to morphology and glycosaminoglycan accumulation, spontaneous neocartilage formation occurred in the cambium layer already at 6 weeks, increased after 12 weeks, but declined until 52 weeks, independent of GFM. Multiple cartilage differentiation markers were induced after transfer. Expression of aggrecan, COMP, decorin, and Col10a1 increased significantly within 52 weeks. Sox 9 and Col2a1 mRNA levels were elevated at 6 versus 52 weeks in the GFM group and resulted in higher collagen type II protein accumulation. Neochondrogenesis was promoted in lower periosteum layers by transfer of periosteum–bone plugs into a joint, and collagen type II protein deposition was enhanced by GFM. The final tissue subsumed typical features of periosteum and fibrocartilage but lacked an intact tide mark and features of hyaline cartilage desired for cartilage repair.     

Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.     

Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells

The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.     

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

Characterization of the human GARP (Golgi associated retrograde protein) complex

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.     

Hydrogen-bonded supramolecular manganese(II) complexes with network and sheet structures bridged by terephthalate unit

Two 1D complexes [Mn(4- methylpyrazole)₃(H₂O)(tp)]_n (2) and [Mn(4-methylpyrazole)₄(tp)]_n (3) (tp = terephthalate) were synthesized and characterized by means of X-ray analysis and magnetic studies. The molecular structure of 2 reveals that Mn(II) centers with asymmetric coordination surroundings are bridged by crystallographically different tp ligands, forming a 1D chain. The 1D coordination chains are interconnected by hydrogen bonds between free carboxylate oxygen atoms in a chain and hydrogens of pyrazole nitrogen atoms in neighboring chains, leading to a 3D framework. Compound 3 also exhibits a 1D coordination chain which is hydrogen-bonded to adjacent chains, providing a 2D sheet structure. Interestingly, the structures include intra- and interchain hydrogen bonds contributed from N-H groups of the capping 4-methylpyrazole ligands. Magnetic measurements show weak antiferromagnetic interactions with exchange coupling parameters of J = −0.018 cm⁻¹ for 2 and J = −0.062 cm⁻¹ for 3 through the extended tp ligand on the basis of an infinite chain model (H = −J∑S_i · S_i + 1).     

Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.