High-Level Expression and Purification of a Recombinant Human α-1,3-Fucosyltransferase in Baculovirus-Infected Insect Cells

A human α-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 μg/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG–Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed α-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal α-2,3-linked sialic acid among various acceptors. The apparentK_mvalues of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.     

Effects of Huangqi （Hex） on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells

InterestintraditionalChineseherbalremedieshasboomedin the western countries. It is very important to study theirmolecular mechanisms and purify effective compoundswith new knowledge and new techniques to meet a greatneed for human health. Ephedrine, the f…     

CD4-CD8-αβ and γδ T Cells Display Inflammatory and Regulatory Potentials during Human Tuberculosis

T-cells play an important role controlling immunity against pathogens and therefore influence the outcome of human diseases. Although most T-lymphocytes co-express either CD4 or CD8, a smaller T-cell subset found the in the human peripheral blood that expresses the αβ or γδ T-cell-receptor (TCR) lacks the CD4 and CD8 co-receptors. These double negative (DN) T-cells have been shown to display important immunological functions in human diseases. To better understand the role of DN T-cells in human Mycobacterium tuberculosis, we have characterized their frequency, activation and cytokine profile in a well-defined group of tuberculosis patients, categorized as severe and non-severe based on their clinical status. Our data showed that whereas high frequency of αβ DN T-cells observed in M. tuberculosis-infected patients are associated with disease severity, decreased proportion of γδ DN T-cells are found in patients with severe tuberculosis. Together with activation of CD4⁺ and CD8⁺ T-cells, higher frequencies of both αβ and γδ DN T-cells from tuberculosis patients also express the chronic activation marker HLA-DR. However, the expression of CD69, an early activation marker, is selectively observed in DN T-cells. Interestingly, while αβ and γδ DN T-cells from patients with non-severe tuberculosis display a pro-inflammatory cytokine profile, characterized by enhanced IFN-γ, the γδ DN T-cells from patients with severe disease express a modulatory profile exemplified by enhanced interleukin-10 production. Overall, our findings suggest that αβ and γδ DN T-cell present disparate immunoregulatory potentials and seems to contribute to the development/maintenance of distinct clinical aspects of TB, as part of the complex immunological network triggered by the TB infection.     

Pegylated Interferon-α2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

Purpose

We investigated the effects of pegylated interferon-α2a (PEG-IFN-α2a) on the growth of human liver cancer cells.

Methods

The effect of PEG-IFN-α2a on the proliferation of 13 liver cancer cell lines was investigated in vitro. Cells were cultured with medium containing 0–4,194 ng/mL of PEG-IFN-α2a, and after 1, 2, 3, or 4 days of culture, morphologic observation and growth assay were performed. After hepatocellular carcinoma (HCC) cells (HAK-1B and KIM-1) were transplanted into nude mice, various doses of PEG-IFN-α2a were subcutaneously administered to the mice once a week for 2 weeks, and tumor volume, weight, and histology were examined.

Results

PEG-IFN-α2a inhibited the growth of 8 and 11 cell lines in a time- and dose-dependent manner, respectively, although the 50% growth inhibitory concentrations of 7 measurable cell lines on Day 4 were relatively high and ranged from 253 ng/mL to 4,431 ng/mL. Various levels of apoptosis induction were confirmed in 8 cell lines. PEG-IFN-α2a induced a dose-dependent decrease in tumor volume and weight, and a significant increase of apoptotic cells in the tumor. Subcutaneous administration of clinical dose for chronic hepatitis C (3 μg/kg, 0.06 μg/mouse) was effective and induced about 30-50% reduction in the tumor volume and weight as compared with the control.

Conclusions

Although in vitro anti-proliferative effects of PEG-IFN-α2a were relatively weak, PEG-IFN-α2a induced strong anti-tumor effects on HCC cells in vivo. The data suggest potential clinical application of PEG-IFN-α2a for the prevention and treatment of HCC.     

Effects of Antioxidant and Nitric Oxide on Chemokine Production in TNF-α-stimulated Human Dermal Microvascular Endothelial Cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1?mM) and spermine NONOate (Sper-NO, 1?mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-κB) induced by TNF-α (10?ng/ml). Treatment with TNF-α for 8?h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1?h, but not by 1?mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-κB were induced by TNF-α for 2?h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-α are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-κB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Immunomodulatory Activity of a Novel,Synthetic Beta-glucan (β-glu6) in Murine Macrophages and Human Peripheral Blood Mononuclear Cells

Natural β-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural β-glucans limits their clinical applicability. We have synthesized β-glu6, which is an analog of the lentinan basic unit, β-(1→6)-branched β-(1→3) glucohexaose, that contains an α-(1→3)-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which β-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of β-glu6. We showed that β-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, β-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1α, IL-1β, IL-16, IL-17, IL-23, IFN-γ, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1) and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF) in human PBMCs. In summary, it demonstrates the immunomodulatory activity of β-glu6 in innate immunity.     

Distinct Roles of Glycogen Synthase Kinase (GSK)-3�� and GSK-3�� in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and α-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3β and down-regulation of β-catenin, whereas it stimulated GSK-3α expression only modestly. The promoter region of GSK-3β was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3β potently induced CM differentiation, that of GSK-3α induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3β abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3α facilitated CM differentiation in MSCs. Although GSK-3β is found in both the cytosol and nucleus in MSCs, GSK-3α is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3β failed to stimulate CM differentiation. Down-regulation of β-catenin mediates GSK-3β-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3α knockdown. These results suggest that GSK-3α and GSK-3β have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3β in the cytosol induces CM differentiation of MSCs through down-regulation of β-catenin. In contrast, GSK-3α in the nucleus inhibits CM differentiation through down-regulation of c-Jun.     

Effects of Carbon Source and Vitreoscilla Hemoglobin (VHb) on the Production of β-Galactosidase in Enterobacter aerogenes

At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on β-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of β-galactosidase formation by carbon sources. In parallel, the bacterial O₂ consumption was increased correspondingly to the vgb induction of β-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42°C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37°C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in β-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.     

Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase of Bacillus sp. K40T

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5–7.0 and was stable at pH 6.0–9.0. The enzyme hydrolyzed the α(1 → 2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 〈O-α-L-fucose-(1 → 2)-O-β-D-galactose-(1 → 3)-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α(1 → 3)-, α-(1 → 4)- and α-(1 → 6)-L-fucosidic linkages in LNF-III 〈O-β-D-galactose-(1 → 4)[O-α-L-fucose-(1 → 3)-]-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, LNF-II 〈O-β-D-galactose-(1 → 3)[O-α-L-fucose-(1 → 4)-]-N-acetyl-O-β-D-galactose-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉 or 6-O-α-L-fucopyranosyl-N-acetylglucosamine.     

Feedback Regulation of SREBP and Aromatase in A β(25-35)-Supplemented Human Neuroblastoma Cells

1. The analogies between the processing of amyloid precursor protein (APP) and other transmembrane sterol regulatory element binding proteins (SREBPs) inspired us to conduct further studies on whether β-amyloid (Aβ) affects aromatase by interacting with APP and SREBP.2. In this study, cultured human neuroblastoma cells (SHSY-5Y) were incubated in experimental media (media without FBS, the main cholesterol source) in the presence or absence of Aβ (1 μM) for 24 h.3. Cellular extracts were subjected to immunoblot analysis using anti-APP, anti-aromatase and anti-SREBP-1. In these cell lines, we detected aromatase (55 kDa), SREBP cleavage product (68 kDa) and APP precursor (100–95 kDa) and cleavage product (60 kDa) by immunoblotting. Aromatase and SREBP levels were elevated in the cells incubated 24 h in experimental media and were attenuated in Aβ-supplemented experimental media.4. The disturbance of cholesterol homeostasis appears to be an important factor in the pathogenesis of Alzheimer's disease. These findings may have important implications for understanding the mechanisms of the aromatase enzyme gene in disease states such as Alzheimer’s.     

Leishmania infantum Amastigotes Enhance HIV-1 Production in Cocultures of Human Dendritic Cells and CD4+ T Cells by Inducing Secretion of IL-6 and TNF-α

Background

Visceral leishmaniasis has emerged as an important opportunistic disease among patients infected with HIV-1. Both HIV-1 and the protozoan parasite Leishmania can productively infect cells of the macrophage-dendritic cell lineage.

Methodology/Principal Findings

Here we demonstrate that Leishmania infantum amastigotes increase HIV-1 production when human primary dendritic cells (DCs) are cocultured together with autologous CD4⁺ T cells. Interestingly, the promastigote form of the parasite does not modulate virus replication. Moreover, we report that amastigotes promote virus replication in both cell types. Our results indicate that this process is due to secretion of parasite-induced soluble factors by DCs. Luminex micro-beads array system analyses indicate that Leishmania infantum amastigotes induce a higher secretion of several cytokines (i.e. IL-1α, IL-2, IL-6, IL-10 and TNF-α) and chemokines (i.e. MIP-1α, MIP-1β and RANTES) in these cells. Studies conducted with pentoxifylline and neutralizing antibodies revealed that the Leishmania-dependent augmentation in HIV-1 replication is due to a higher secretion of IL-6 and TNF-α.

Conclusions/Significance

Altogether these findings suggest that the presence of Leishmania within DC/T-cell conjugates leads to an enhancement of virus production and demonstrate that HIV-1 and Leishmania can establish complex interactions in such a cellular microenvironment.     

Expression and Purification of Human Membrane Progestin Receptor α (mPRα)

Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3–1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.     

α1,6-Fucosyltransferase (Fut8) is implicated in vulnerability to elastase-induced emphysema in mice and a possible non-invasive predictive marker for disease progression and exacerbations in chronic obstructive pulmonary disease (COPD)

Fut8 (α1,6-Fucosyltransferase) heterozygous knock-out (Fut8(+/-)) mice had an increased influx of inflammatory cells into the lungs, and this was associated with an up-regulation of matrix metalloproteinases, MMP-2 and MMP-9, after treatment with porcine pancreatic elastase (PPE), exhibiting an emphysema-prone phenotype as compared with wild type mice (Fut8(+/+)). The present data as well as our previous data on cigarette-smoke-induced emphysema [8] led us to hypothesize that reduced Fut8 levels leads to COPD with increased inflammatory response in humans and is associated with disease progression. To test this hypothesis, symptomatic current or ex-smokers with stable COPD or at risk outpatients were recruited. We investigated the association between serum Fut8 activity and disease severity, including the extent of emphysema (percentage of low-attenuation area; LAA%), airflow limitation, and the annual rate of decline in forced expiratory volume in 1 s (FEV(1)). Association with the exacerbation of COPD was also evaluated over a 3-year period. Serum Fut8 and MMP-9 activity were measured. Fut8 activity significantly increased with age among the at risk patients. In the case of COPD patients, however, the association was not clearly observed. A faster annual decline of FEV(1) was significantly associated with lower Fut8 activity. Patients with lower Fut8 activity experienced exacerbations more frequently. These data suggest that reduced Fut8 activity is associated with the progression of COPD and serum Fut8 activity is a non-invasive predictive biomarker candidate for progression and exacerbation of COPD.     

Effect of Recombinant Interferon-α2b (Laferon) on Transport of Sodium Ions in Human Neuroblastoma Cells

To elucidate molecular mechanisms of neurotropic action of a recombinant interferon, IFN-2b (laferon), its effect on transport of ²²Na⁺ through the membrane of cultured human neuroblastoma cells (line IMR 32) was investigated. Within the first minutes after treatment with IFN-2b, the influx of ²²Na⁺ ions was reduced by 20%, as compared with the control. Depolarization of the plasma membrane by a mixture of veratrine and scorpion (Leiurus quinquestriatus) toxin (200 and 10 g/ml, respectively) increased this flux by 50% in the control and by 70% in the IFN-2b-treated cells. A blocker of voltage-operated sodium channels, tetrodotoxin (TTX, 4 · 10^-7 M), suppressed the inward flux of ²²Na⁺ ions (completely in the control cells and by 75% in the IFN-2b-treated cells). The influx of ²²Na⁺ ions into neuroblastoma cells depended on the concentration of IFN-2b in the incubation medium, reaching a maximum at concentrations of 600-1000 IU/ml. This allows us to suggest that entry of Na⁺ ions into neuroblastoma cells caused by IFN-2b is basically performed through voltage-operated TTX-sensitive sodium channels.     

Crystalline Features of Streptococcal (l→3)-α-D-Glucans of Human Saliva

Crystalline polymorphs of the backbone (l→3)-α-D-glucans of two streptococcal α-glucans were studied by X-ray diffraction measurements in comparison with that of a fungal (l→3)-α-D-glucan. The glucan produced by S. salivarius changed its polymorph from the hydrated form at 100% relative humidity to the dehydrated form under vacuum, that produced by cariogenic S. mutans took the dehydrated form only, and the fungal glucan always showed the hydrated form. The difference of polymorphic behavior was ascribed to the molecular weight of the glucan since the fungal glucan showed the highest viscosity, the saliverius glucan, middle, and the mutans glucan, the lowest.     

Production of d(—)-α-Aminobenzylpenicillin by Kluyvera citrophila KY 3641

Intact cells of Kluyvera citrophila KY 3641 produced enzymaticaily d(—)-α-aminobenzyl-penicillin from 6-arainopenicillanic acid and phenylglycine derivatives. The optimum pH of the acylase was 6.5. Among various phenylglycine derivatives examined as substrates, d-phenylglycine methylester HC1 was the best compound giving the yields of about 10.7 mg/ml of d(—)-α-aminobenzy]penicillin in the enzymic reaction mixture. The product was isolated in a crystalline form and identified as d(—)-α-aminobenzylpenicillin.     

Preferential Energy- and Potential-Dependent Accumulation of Cisplatin–Gutathione Complexes in Human Cancer Cell Lines (GLC4 and K562): A Likely Role of Mitochondria

cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20–30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.