Proteins In Culture Supernatant Research Articles

Continuous Taurocholic Acid Exposure Promotes Esophageal Squamous Cell Carcinoma Progression Due to Reduced Cell Loss Resulting from Enhanced Vascular Development

BackgroundRefluxogenic effects of smoking and alcohol abuse may be related to the risk of esophageal squamous cell carcinoma (ESCC). The present study attempts to clarify the effects of continuous taurocholic acid (TCA) exposure, which is neither mutagenic nor genotoxic, on ESCC progression.MethodsA squamous carcinoma cell line (ESCC-DR) was established from a tumor induced in a rat model of gastroduodenal reflux. ESCC-DR cells were incubated with 2 mM TCA for ≥2 months. The effects of continuous TCA exposure were evaluated in vitro on cell morphology, growth, and invasion and in vivo on xenograft tumor growth in nude mice. Moreover, the mean level of secreted transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) proteins in cell culture supernatants and mRNA synthesis of TGF-β1 and VEGF-A of ESCC cells were measured. The angiogenic potential was further examined by a migration assay using human umbilical vein endothelial cells (HUVECs).ResultsContinuous TCA exposure induced marked formation of filopodia in vitro. Expression levels of angiogenic factors were significantly higher in the cells treated with TCA than in control cells. Tumor xenografts derived from cells pre-exposed to TCA were larger and more vascularized than those derived from control cells. In addition, TCA exposure increased HUVEC migration.ConclusionContinuous TCA exposure enhanced ESCC progression due to reduced cell loss in vivo. Cell loss was inhibited by TCA-induced vascular endothelial cell migration, which was mediated by TGF-β1 and VEGF-A released from ESCC cells.

A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris

This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut(s) Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25% of the total proteins in the culture supernatant, after 120h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45% of the total culture supernatant proteins) with concomitant reduction in the induction time to 48h. The amount of BbST obtained was 148mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Mut(s) strains of P. pastoris.

Proteome analysis for antifungal effects of Bacillus subtilis KB-1122 on Magnaporthe grisea P131

Rice blast, caused by Magnaporthe grisea threatens rice production worldwide. It is important to develop novel and environment-safe strategies to control the fungus. Here we reported that Bacillus subtilis KB-1122 could strikingly inhibit the growth of M. grisea P131 in agar diffusion assays. To further understand the molecular mechanism on the suppressive role of B. subtilis on M. grisea, the antagonist-pathogen interaction of the two strains was studied by using comparative proteome analysis in this report. The cellular and culture supernatant (CSN) proteins were prepared from co-culture and subjected to two-dimensional polyacrylamide gel electrophoresis. Proteome analysis revealed 33 cellular and 18 CSN proteins showing changes upon co-culture respectively. Importantly, down-regulated cellular proteins came from M. grisea, whereas up-regulated proteins derived from B. subtilis. Results suggested that glyceraldehyde-3-phosphate dehydrogenase and serine protein kinase might contribute to antifungal activity of B. subtilis KB-1122. Of CSN proteins identified, the endo-1,4-beta-glucanase (involved in degradation of polysaccharides) was up-regulated consistently at different times of incubation. This suggests that this enzyme plays an important role in the interaction between B. subtilis KB-1122 with M. grisea P131.

Autocrine/Paracrine Function of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) for Glucose Homeostasis in Pancreatic β-Cells and Adipocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca(2+) from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca(2+) signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN3, a metabolic inhibitor or under Ca(2+)-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.

Secretome of Transmissible Pseudomonas aeruginosa AES-1R Grown in a Cystic Fibrosis Lung-Like Environment

Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%-80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on BCG [corrected] secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.

Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood mononuclear cells in dogs with sino-nasal aspergillosis

The causal agent of sino-nasal aspergillosis is usually Aspergillus fumigatus, which is a saprophytic and ubiquitous fungus that causes a severe rhinosinusitis in apparent healthy dogs. Affected dogs do not have systemic immuno-suppression. It has been shown previously that dogs affected by this disease have local over-expression of interleukin (IL)-10 and Th1 cytokines in nasal mucosal tissue.The aim of the present study was to assess the response of peripheral blood mononuclear cells (PBMC) from affected and unaffected dogs to antigen-specific stimulation with heat-inactivated Aspergillus spp. conidia, by quantifying gene expression for specific Th1, Th2, Th17 and Treg cytokines and their related transcription factors. Quantification of IL-4 and IFN-γ protein in culture supernatant was performed by enzyme-linked immunosorbent assay (ELISA).PBMC from dogs with SNA produced adequate mRNA encoding IFN-γ and IFN-γ protein. The expression of IL-17A mRNA was significantly greater in PBMC of affected compared with unaffected dogs. The amount of IL-10 mRNA in PBMC from affected dogs decreased after antigen-specific challenge.These results suggest that the incapacity of affected dogs to clear these fungal infections is not related to a defect in Th1 immunity or to an overwhelming regulatory reaction, but rather to an uncontrolled pro-inflammatory reaction driven by Th17 cells.

Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis

Lacticin Q is a 53-amino acid Class II bacteriocin produced by Lactococcus lactis QU 5. It shows antibacterial activity comparable to that of nisin A in terms of both spectrum and intensity. Moreover, it remains stable at alkaline pH values, while nisin A was inactivated. It may possibly be employed as an alternative to or in combination with nisin A. The objective of this study was to express lacticin Q extracellularly with Small ubiquitin-related modifier (SUMO) fusion technology in Bacillus subtilis. Secretory SUMO-lacticin Q fusion protein was efficiently produced in B. subtilis WB600 transformed with the recombinant expression plasmid and accounted for 19% of the culture supernatant proteins. Fusion SUMO-lacticin Q was purified by nickel nitrilotriacetate (Ni–NTA) affinity chromatography and digested with SUMO protease to release lacticin Q. Lacticin Q was further purified by Ni–NTA chromatography to yield about 2.5mg of lacticin Q with more than 93% purity from 1L of supernatant of fermentation culture. An activity assay indicated that the recombinant bacteriocin exhibited excellent antimicrobial activity against indicator strains. The results obtained suggest that the secretory lacticin Q was efficiently expressed using SUMO fusion technology in B. subtilis. The expression and purification system could promote the application of lacticin Q in food and medicine.

Effects of ginsenoside Rh2 on growth and migration of pancreatic cancer cells

To investigate the effects of ginsenoside Rh2 on the human pancreatic cancer cell line Bxpc-3. The human pancreatic cancer cell line Bxpc-3 was cultured in vitro and treated with or without ginsenoside Rh2. Growth rates for Bxpc-3 cells were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle changes were analyzed by flow cytometry. Apoptosis was measured by flow cytometry and Hoechst 33258 fluorescence staining. A scratch assay and a Matrigel invasion assay were used to detect cell migration and invasion. Expression of Bax, Bcl-2, survivin, cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, caspase-8, and caspase-9 mRNA were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Bax, Bcl-2, survivin, cyclin D1, cleaved caspase-3, caspase-8 and caspase-9 protein levels were examined by western blotting. Expression of MMP-2 and MMP-9 proteins in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Rh2 significantly inhibited Bxpc-3 cell proliferation in a dose- and time-dependent manner, as evaluated by the MTT (P < 0.05) and colony formation assays (P < 0.05). Compared to the control group, Rh2 significantly increased the percentage of Bxpc-3 cells in the G0/G1 phase from 43.32% ± 2.17% to 71.32% ± 1.16%, which was accompanied by a decrease in S phase (from 50.86% ± 1.29% to 28.48% ± 1.18%) and G2/M phase (from 5.81% ± 1.19% to 0.20% ± 0.05%) in a dose-dependent manner (P < 0.05), suggesting that Rh2 arrested cell cycle progression at the G0/G1 phase, as measured by flow cytometry. Compared to the control group, cells treated with Rh2 showed significantly higher apoptosis ratios in a dose-dependent manner (percentage of early apoptotic cells: from 5.29% ± 2.28% to 38.90% ± 3.42% (F = 56.20, P < 0.05); percentage of late apoptotic cells: from 4.58% ± 1.42% to 36.32% ± 2.73% (F = 86.70, P < 0.05). Rh2 inhibited Bxpc-3 cell migration and invasion, as detected by scratch wound healing assay and Matrigel invasion assay [percentages of scratch wound healing for 12 h, 24 h and 48 h (control vs experimental group): 37.3% ± 4.8% vs 18.30% ± 1.65%, 58.7% ± 3.5% vs 38.00% ± 4.09% and 93.83% ± 4.65% vs 65.50% ± 4.09%, respectively; t = 6.489, t = 6.656 and t = 7.926, respectively, P < 0.05; the number of cells invading at various concentrations (0 μmol/L, 35 μmol/L, 45 μmol/L and 55 μmol/L): 81.10 ± 9.55, 46.40 ± 6.95, 24.70 ± 6.88 and 8.70 ± 3.34, respectively (F = 502.713, P < 0.05)]. RT-PCR, western blotting or ELISA showed that mRNA and protein expression of Bax, cleaved caspase-3 and caspase-9 were upregulated (P < 0.05), while mRNA and protein expression of Bcl-2, survivin, cyclin D1, MMP-2 and MMP-9 were downregulated (P < 0.05). Ginsenoside Rh2 inhibits proliferation, migration and invasion and induces apoptosis of the human pancreatic cancer cell line Bxpc-3.

Induction of CXCL1 in Osteoblasts by Myeloma Cells Promotes Migration of Osteoclast Precursors

AbstractAbstract 441 IntroductionMultiple myeloma (MM) causes a dysbalance in the bone microenvironment between bone building osteoblasts and bone resorbing osteoclasts (OCs), with an increase in OC recruitment, differentiation and activation, leading to myeloma bone disease (MBD). Presence of MBD has a major impact on the quality of life of MM patients and novel treatment approaches for MBD are urgently needed. Several factors have been identified that play a role in this process, e.g. receptor activator of NF-kB ligand (RANKL). However, the pathomechanism of increased osteoclast recruitment and activation is not completely understood. Here, we investigated the role of the chemokine CXCL1 and its receptor CXCR2 in the bone microenvironment in MM. Material and MethodsSerum samples from 52 patients with newly diagnosed MM and from 22 healthy volunteers were assayed using a CXCL1 ELISA. Primary human mesenchymal stem cells (hMSCs) were cultured from bone marrow aspirates and primary human differentiated osteoblasts (hOBs) were cultured from trabecular bone fragments, both from healthy volunteers. Osteoclast precursors (pre-OCs) were generated by immunomagnetic sorting of CD14-positive cells from the peripheral blood of healthy volunteers. Human myeloma cell lines (HMCLs) U-266, RPMI-8226 and LP-1 and primary bone marrow myeloma cells (pMMCs) selected using CD138 immunomagnetic sorting were used for the experiments. Co-cultures of HMCLs and pMMCs with hMSCs or hOBs were performed using 0.45 μm transwell inserts, allowing for the exchange of soluble mediators. Migration assays were performed using 8 μm transwell inserts and human recombinant CXCL1. Immunohistochemistry was performed on paraffin-embedded bone marrow biopsies from MM patients using an anti-CXCR2 monoclonal antibody. All experimental procedures involving patient material were approved by the local ethics committee and conducted after informed consent was obtained. ResultsCXCL1 serum levels were found to be significantly higher in MM patients than in healthy individuals (193.4 pg/mL vs. 137 pg/mL, respectively, p<0.05), indicating a role for CXCL1 in MM pathophysiology. We went on to investigate the role of CXCL1 in MBD and performed co-cultures of HMCLs and pMMCs with hMSCs or hOBs. Baseline CXCL1 expression was absent in HMCLs and low or absent in hMSCs or hOBs at baseline. RNA expression as well as protein excretion by hMSCs and hOBs were induced after co-culture with myeloma cells. For example, pMMCs from different individuals led to a mean 154-fold upregulation of CXCL1 mRNA levels in hMSCs and to a mean upregulation of CXCL1 protein in cell culture supernatants from <31.5 pg/mL at baseline to 2140 pg/mL after co-cultures. In order to investigate the potential function of elevated CXCL1 levels in the bone marrow microenvironment, the expression of CXCR2, the receptor for CXCL1, was analyzed. Pre-OCs as well as a majority of pMMCs expressed CXCR2 mRNA. CXCR2 protein expression in pMMCs was verified using immunohistochemistry on MM bone marrow biopsies. Human recombinant CXCL1 significantly increased pre-OC cell migration in a dose-dependent manner. For example, 50 ng/mL or 100 ng/mL of CXCL1 increased mean pre-OC migration along a CXCL1 gradient 2.5-fold and 5.6-fold over baseline, respectively. In addition, mean pMMC migration was increased 3.8-fold compared to baseline along a 100 ng/mL gradient of recombinant CXCL1. The osteoclastogenic capacity of the migrated pre-OCs was confirmed by TRAP expression after stimulation with RANKL and M-CSF. ConclusionWe describe here a novel role for the chemokine CXCL1 in myeloma bone disease. We demonstrate that CXCL1 is induced in hMSCs and hOBs by co-culture with MM cells. CXCL1 leads to chemoattraction of both pre-OCs and pMMCs. These effects could lead to co-localization of OCs and MM cells in the bone marrow microenvironment and contribute to the tumor-promoting interaction between these cell types. Our data indicate the CXCL1-CXCR2 axis as a therapeutic target in myeloma bone disease. Disclosures:No relevant conflicts of interest to declare.

Physiologic Erythropoiesis with Enhanced Globin Switching and Endogenous Cytokine Production in a Long-Term Serum-free in Vitro human Bone Marrow Biomimicry

AbstractAbstract 276Cord blood mononuclear cells (CBMNCs) are an ideal cell source for therapeutic applications although maintenance of fetal globin in expanded erythroid cells is problematic. We have previously developed a 3D bone marrow (BM) biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which expanded CBMNCs in a cytokine-free environment for at least 28 days, with or without addition of serum. Addition of near physiological concentrations (0.2U/mL) recombinant human erythropoietin (rhuEPO) to the 3D CBMNCs serum- and cytokine-free cultures at day 7 enhanced normal erythropoiesis to enucleation (from CD45+/CD71+/CD235− to CD45−/CD71+/CD235+ stages) and promoted erythroid clonogenic capacity (CFU-E and BFU-E). However, mechanisms of erythroid expansion and globin-maturity of the cells are ill-defined. We have now extended our investigation to evaluate in situ hemoglobin and cytokine gene expression and detect cellular hemoglobin protein and cytokine production in culture supernatants. CBMNCs were separated by Ficoll-Paque density gradient centrifugation and seeded onto collagen-coated PU 3D scaffolds at 2.5×106cells/scaffold (5×5×5mm3). Cultures were established with full-medium exchange every other day in 4 conditions: 2 controls (serum-free and serum-containing cultures without rhuEPO) and serum-free cultures with addition of rhuEPO at 1.845U/mL or 0.2U/mL from day 7 onwards. Culture output was evaluated by in situ analysis and by physically extracting cells from the scaffolds. β-globin (HBB) and γ-globin (HBG) genes were detected by in situ PCR from day 0 and were consistently expressed throughout the culture period in all conditions (HBB expression was consistently higher). A decline in expression of both globins was noted at days 21 and 28 only in serum-free cultures without addition of rhuEPO. Western blot analysis of extracted cells confirmed both HBB and HBG expression in the serum-containing rhuEPO-free culture. In serum-free conditions, both HBB and HBG proteins were noted only at day 7; once rhuEPO was added to these cultures, a hemoglobin switch occurred with persistence of only HBB at days 21 and 28. Even in serum-free and rhuEPO-free cultures, HBB was present, although in lower amounts than in the rhuEPO-exposed cells, which suggested that fetal globin switching and maturation towards adult erythropoiesis occurred primarily in the serum-free cultures exposed to rhuEPO. Increased expression of GM-CSF, IL-1β and TNF was observed to day 7 and declined thereafter while IL-6 expression was observed only at day 7. There was constant high expression of TGF-β throughout the culture in all conditions, whereas TPO was not detected in any condition. The erythropoietin-receptor (EPO-R) gene was detected in all conditions, yet EPO-R was shed into the supernatant mostly in the first 7 days (maximum 42pg/ml) of serum-free cultures, declining by day 14, which corresponded to the timing of rhuEPO addition. Flt-3 was consistently detected in supernatant of the serum-containing cultures, yet declined after day 14 in serum-free cultures with and without rhuEPO. Stem cell factor (SCF), critical in early stages of erythropoiesis, was not present in culture supernatants at any time-point. Interestingly, endogenous production of EPO (maximum 0.8 mU/ml) was detected by ELISA in the first 2 weeks of both serum-containing and serum-free cultures, prior to addition of rhuEPO. High EPO concentration (maximum 2600mU/ml) was still observed in the serum-free, rhuEPO-containing cultures over the entire 28 days, suggesting that EPO was not completely utilized by the maturing cells during culture and that even lower concentrations of EPO could be beneficial. EPO detection was maximal at day 21 for serum-free cultures exposed to rhuEPO; this day 21 peak, in conjunction with the known erythroid maturation kinetics within the 3D culture, the detection of endogenous EPO production at day 14, the shedding profile of EPO-R and the hemoglobin switch, suggested that the 14–21 day time-points of culture will be important for the future study of erythroid physiology within this system. In conclusion, the 3D BM biomimicry is a good model to study erythropoiesis ex vivo, using physiological concentrations of rhuEPO and serum-free conditions rendering it suitable for future clinical applications. Disclosures:No relevant conflicts of interest to declare.

Anti-inflammatory effects of 1,25-dihydroxyvitaminD3in monocytes cultured in serum from patients with type2 diabetes mellitus and diabetic nephropathy with uremia via Toll-like receptor4 and nuclear factor-κB p65.

Type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) seriously affect the quality of human life. 1,25-dihydroxyvitaminD3 (VD3) is known to exert anti‑inflammatory and immunomodulatory effects. The present study investigated the effects of VD3 on Toll‑like receptor4 (TLR4) and nuclear factor (NF)‑κB p65 expression in monocytes from patients with T2DM and investigated the underlying mechanisms. Serum from subjects of the control, T2DM and DN with uremia groups was isolated, and THP‑1 monocytes were cultured in these sera with or without VD3. After treatment with lipopolysaccharide (LPS) and interleukin (IL)‑15, monocytes and culture supernatants were collected. The expression of TLR4, TLR9 and IL‑15 mRNA was detected using reverse‑transcription polymerase chain reaction analysis. Furthermore, the protein expression of TLR4, NF‑κB p65 and inhibitor of NF‑κB (IκB) was determined using western blot analysis. The levels of IL‑6 and monocyte chemotactic protein in culture supernatants were detected using ELISAs. The impact of LPS, IL‑15 and VD3 on the TLR/NF‑κB signaling pathway in the T2DM and DN uremia groups was also investigated. In the T2DM and DN uremia groups, LPS and IL‑15 downregulated the expression of IκB and upregulated levels of proteins, including TLR4, NF‑κB p65, IL‑6 and monocyte chemoattractant protein1, as well as TLR4 and IL‑15 mRNA. There was no significant difference in TLR9 mRNA protein expression among the three groups. VD3 partially blocked the above effects; However, pre‑treatment with VD3 had no significant effect on TLR4 mRNA expression, protein expression of NF-κB p65 and IκB, or the levels of associated inflammatory cytokines. In conclusion, the present study indicated that anti‑inflammatory effects of VD3 in inflammatory immune responses in T2DM and DN uremia are associated with the TLR4/NF-κB p65 signaling pathway.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

The Type III Secreted Protein BspR Regulates the Virulence Genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.

Rigid polyurethane foams obtained from tall oil and filled with natural fibers: Application as a support for immobilization of lignin-degrading microorganisms

The forest biomass represents an abundant, renewable, non-food competition, and low-cost resource that can play an alternative role to petro-resources. The first topic of the research activity is focused on the use of wood and a pulp mill by-product—tall oil —as raw materials for the production of rigid polyurethane foams. The maximum content of the renewable resource in ready foams is 26%. By using biopolymers as a matrix, a natural way is to reinforce them with natural fibers. Further advantages are significant weight and cost savings and, at the same time, replacement of petrochemical raw materials. Three different natural fibers (cellulose, wood, and modified cellulose) were tested as a filler of foams. Rigid polyurethane foams was used as biomass support particles for immobilization of microorganisms. Suspension cultures of the organism with biomass support particles can promote the adhesion of cells to the porous matrix surface, and subsequently the cells become immobilized during the cultivation. This method for cell immobilization has a great potential for enhancing the production of proteins or chemicals in culture supernatants. The presence of natural fibers in the matrix promotes the enzyme production because the material not only functions as an attachment place but also supplies some nutrients to the microorganism and induces the production of ligninolytic enzymes. This paper discusses the studies into the use of tall oil as a renewable source in rigid polyurethane foam production

Additive Effects of Ulinastatin and Docetaxel on Growth of Breast Cancer Xenograft in Nude Mice and Expression of PGE2, IL-10, and IL-2 in Primary Breast Cancer Cells

Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

Abstract 501: The carboxypeptidase inhibitor TIG1 interacts with cathepsin L2 and inhibits cathepsin L2-stimulated cellular invasion

Abstract The tazarotene-induced gene 1 (TIG1), also known as retinoic acid receptor responder 1 (RARRES1), is a putative tumor suppressor gene. The gene encodes a carboxypeptidase inhibitor for ATP/GTP binding protein-like 2 (AGBL2). This study identified TIG1 binding proteins and characterized the activities of TIG1 on cellular invasion of HCT116 colon cancer cells stimulated by the cathepsin L2 (CTSL2). The cytoplasmic portion of the TIG1A isoform was used for identification of TIG1 binding proteins using the yeast two-hybrid screening, and six genes, CTSL2, DNAJC8, SPATA22, SPINK2, VAC14, and C6orf225, were selected. CTSL2, a cysteine protease with an increased expression shown in breast and colon carcinomas, was selected for analysis. Immunoprecipitation analysis using transiently transfected cytosols confirmed the binding between both TIG1 isoforms and CTSL2. Both TIG1A and CTSL2 were secreted proteins; however, the interaction of both proteins in culture supernatants was not detected. Further immunoprecipitation analysis using purified recombinant proteins demonstrated direct binding between both TIG1 isoforms and CTSL2. Cellular invasion of HCT116 cells was increased by 52 to 71% in CTSL2 transiently transfected cells. Both TIG1 isoforms had no effects on cellular invasion of HCT116 cells. However, TIG1A and TIG1B significantly inhibited CTSL2-stimulated cellular invasion by 65.1 and 62.9%, respectively. In conclusion, both TIG1 isoforms interact with CTSL2 and inhibited CTSL2-stimulated cellular invasion of HCT116 cells. The results suggest that TIG1 may suppress CTSL2-mediated cellular invasion through inhibiting the activation of CTSL2 protease, and merit for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 501. doi:1538-7445.AM2012-501

New IMAC media enabling purification of histidine‐tagged proteins directly from eukaryotic cell culture supernatants

Immobilized metal affinity chromatography (IMAC) purification of secreted histidine‐tagged proteins in eukaryotic cell culture supernatants is often problematic. Incompatibility with the cell culture media appears as stripping of the immobilized metal ions required for binding of target proteins. Due to low target protein concentration in cell culture supernatants, large sample volumes are often needed, aggravating the stripping effect.In this study, purifications of histidine‐tagged proteins were performed using two novel Sepharose™ based IMAC media; Ni Sepharose excel and magnetic His Mag Sepharose excel. Both media have a new type of chelating ligand with exceptionally strong binding of nickel ions. Data showing successful purification of histidine‐tagged proteins from CHO cell culture supernatants will be presented.Furthermore, the characteristics of new media enabled purification of target protein from insect cell culture supernatants. The purification was easily scaled up from 20 μl His Mag Sepharose excel beads to 1 mL pre‐packed columns.

Identification of the autotransporter Pet toxin in &amp;lt;i&amp;gt;Proteus mirabilis&amp;lt;/i&amp;gt; strain isolated from patients with urinary tract infections

Proteus mirabilis, a motile Gram-negative bacterium, represents a common cause of complicated urinary tract infections. Autotransporters are a family of secreted proteins from Gram-negative bacteria that direct their own secretion across the outer membrane (type V autotransporter secretion mechanism). Serine protease autotransporters of Enterobacteriaceae (SPATEs) include adhesins, toxins, and proteases that can contribute to the virulence. Plasmid-encoded toxin (Pet) is the predominant protein in culture supernatants of enteroaggregative E. coli prototype strain 042 and has been extensively studied. Pet toxin is encoded on the 65-MDa adherence-related plasmid of EAEC 042 strain. In this work, Pet protein was found in the supernatant obtained from Proteus mirabilis RTX339 strain isolated from a psychiatric patient suffering complicated urinary tract infections (UTIs). The nucleotide sequence of pet gene was obtained using primers designed from E. coli 042 pet gene reported. The alignment of the sequence showed 100% identity with the pet gene reported. Is important to note that Proteus mirabilis RTX339 pet gene has chromosomal location. The chromosomal location of the gene was established since no plasmids were harbored by this strain.

Expression and characterization of recombinant human serum albumin fusion protein with C-peptide

C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host Pichia pastoris GS115. After 72 h of growth on methanol, the recombinant HSA-CP concentration reached a level of 145 mg/L, representing 70% of total proteins in culture supernatant. The recombinant fusion protein was purified by ultra filtration, Q-sepharose fast flow column and Superdex 75 size-exclusion column. It was specifically recognized by the anti-human HSA antibody and CP antibody in Western blotting assay. In vitro, the recombinant HSA-CP can stimulate HEK293 cell proliferation. In Zucker diabetic fatty (ZDF) rats, 12 weeks recombinant HSA-CP treatment could prevent the accumulation of glomerular extracellular matrix, which leads to mesangial expansion and glomerular hypertrophy. The terminal biological half-time of the protein was 3.38±1.87 h after single administration of 500 nmol/kg of HSA-CP in Wister rats. The pharmacokinetic analysis of the protein indicate its promising application in clinical medicine. Key words : C-peptide, human serum albumin, recombinant fusion protein, Pichia pastoris, bioactivity,biological half-time.