Identification of low-abundance proteins via fractionation of the urine proteome with weak anion exchange chromatography

<p>Abstract</p> <p>Background</p> <p>Low-abundance proteins are difficultly observed on the two-dimensional gel electrophoresis (2-DE) maps of urine proteome, because they are usually obscured by high-abundance proteins such as albumin and immunoglobulin. In this study, a novel fractionation method was developed for enriching low-abundance proteins by removing high-abundance proteins and progressive elution with salts of various concentrations.</p> <p>Results</p> <p>Stepwise weak anion exchange (WAX) chromatography, which applied DEAE-Sephacel resin with non-fixed volume elution, was used to fractionate urine proteome prior to performing 2-DE. Urine proteome was separated into four fractions by progressively eluting the column with 0 M, 50 mM, 100 mM, and 1 M NaCl solutions. Most of the heavy and light immunoglobulin chains appeared in the eluent. After the high-abundance proteins were removed, various low-abundance proteins were enriched and could be easily identified. The potential of this method for obtaining diversified fractionations was demonstrated by eluting the column separately with Na₂SO₄and MgCl₂solutions. The 2-DE maps of the fractions eluted with these different salt solutions of identical ionic strength revealed markedly different stain patterns.</p> <p>Conclusion</p> <p>The present study demonstrated that this fractionation method could be applied for purposes of enriching low-abundance proteins and obtaining diversified fractionations of urine, and potentially other proteomes.</p

A panel of tumor markers, calreticulin, annexin A2, and annexin A3 in upper tract urothelial carcinoma identified by proteomic and immunological analysis

BACKGROUND: Upper tract urothelial carcinoma (UTUC) is a tumor with sizable metastases and local recurrence. It has a worse prognosis than bladder cancer. This study was designed to investigate the urinary potential tumor markers of UTUC. METHODS: Between January 2008 and January 2009, urine was sampled from 13 patients with UTUC and 20 healthy adults. The current study identified biomarkers for UTUC using non-fixed volume stepwise weak anion exchange chromatography for fractionation of urine protein prior to two-dimensional gel electrophoresis. RESULTS: Fifty five differential proteins have been determined by comparing with the 2-DE maps of the urine of UTUC patients and those of healthy people. Western blotting analysis and immunohistochemistry of tumor tissues and normal tissues from patients with UTUC were carried out to further verify five possible UTUC biomarkers, including zinc-alpha-2-glycoprotein, calreticulin, annexin A2, annexin A3 and haptoglobin. The data of western blot and immunohistochemical analysis are consistent with the 2-DE data. Combined the experimental data in the urine and in tumor tissues collected from patients with UTUC, the crucial over-expressed proteins are calreticulin, annexin A2, and annexin A3. CONCLUSIONS: Calreticulin, annexin A2, and annexin A3 are very likely a panel of biomarkers with potential value for UTUC diagnosis

Proteomic Analysis of Anti-Tumor Effects of 11-Dehydrosinulariolide on CAL-27 Cells

The anti-tumor effects of 11-dehydrosinulariolide, an active ingredient isolated from soft coral Sinularia leptoclados, on CAL-27 cells were investigated in this study. In the MTT assay for cell proliferation, increasing concentrations of 11-dehydrosinulariolide decreased CAL-27 cell viability. When a concentration of 1.5 μg/mL of 11-dehydrosinulariolide was applied, the CAL-27 cells viability was reduced to a level of 70% of the control sample. The wound healing function decreased as the concentration of 11-dehydrosinulariolide increased. The results in this study indicated that treatment with 11-dehydrosinulariolide for 6 h significantly induced both early and late apoptosis of CAL-27 cells, observed by flow cytometric measurement and microscopic fluorescent observation. A comparative proteomic analysis was conducted to investigate the effects of 11-dehydrosinulariolide on CAL-27 cells at the molecular level by comparison between the protein profiling (revealed on a 2-DE map) of CAL-27 cells treated with 11-dehydrosinulariolide and that of CAL-27 cells without the treatment. A total of 28 differential proteins (12 up-regulated and 16 down-regulated) in CAL-27 cells treated with 11-dehydrosinulariolide have been identified by LC-MS/MS analysis. Some of the differential proteins are associated with cell proliferation, apoptosis, protein synthesis, protein folding, and energy metabolism. The results of this study provided clues for the investigation of biochemical mechanisms of the anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells and could be valuable information for drug development and progression monitoring of oral squamous cell carcinoma (OSCC)

Primary Small Cell Carcinoma of the Stomach Successfully Treated With Cisplatin and Etoposide

We report a 44-year-old man with primary gastric small cell carcinoma who showed a remarkable response to chemotherapy specific for pulmonary small cell carcinoma. The patient had been admitted to another local hospital because of intermittent epigastralgia. An upper gastrointestinal examination there revealed an ulcerative tumor, 5 cm in diameter, on the lesser curvature side of the cardia, and endoscopic biopsy reported adenocarcinoma. Computed tomography revealed a mass over the lesser curvature of the stomach and some enlarged regional lymph nodes. Radical total gastrectomy, lymph node dissection, Roux-en-Y esophagojejunostomy and splenectomy were performed at our hospital. Pathology revealed gastric mucosa infiltrated by small-sized tumor cells with scanty cytoplasm and hyperchromatic nuclei. Immunohisto- chemically, the tumor cells were positive for synaptophysin, chromogranin A, and CD56. Primary gastric small cell carcinoma was diagnosed. The postoperative course, complicated by shock due to bleeding, wound infection and intra-abdominal abscess, took more than 2 months to resolve. Follow-up computed tomography showed tumor recurrence with multiple enlarged lymph nodes in the aortocaval region and hepatic hilum. The patient received palliative chemotherapy consisting of cisplatin 80 mg/m2 on day 1 and etoposide 80 mg/m2 on days 1–3 every 28 days, and had partial response to the chemotherapy, with a progression-free survival of 10 months. Chemotherapy with cisplatin and etoposide used for small cell carcinoma of the lung is a good treatment for gastric small cell carcinoma

Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction

Natural compounds from soft corals have been increasingly used for their antitumor therapeutic properties. This study examined 11-epi-sinulariolide acetate (11-epi-SA), an active compound isolated from the cultured soft coral Sinularia flexibilis, to determine its potential antitumor effect on four hepatocellular carcinoma cell lines. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the results demonstrated that 11-epi-SA treatment showed more cytotoxic effect toward HA22T cells. Protein profiling of the 11-epi-SA-treated HA22T cells revealed substantial protein alterations associated with stress response and protein synthesis and folding, suggesting that the mitochondria and endoplasmic reticulum (ER) play roles in 11-epi-SA-initiated apoptosis. Moreover, 11-epi-SA activated caspase-dependent apoptotic cell death, suggesting that mitochondria-related apoptosis genes were involved in programmed cell death. The unfolded protein response signaling pathway-related proteins were also activated on 11-epi-SA treatment, and these changes were accompanied by the upregulated expression of growth arrest and DNA damage-inducible protein (GADD153) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), the genes encoding transcription factors associated with growth arrest and apoptosis under prolonged ER stress. Two inhibitors, namely salubrinal (Sal) and SP600125, partially abrogated 11-epi-SA-related cell death, implying that the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)–activating transcription factor (ATF) 6–CHOP or the inositol-requiring enzyme 1 alpha (IRE1α)–c-Jun N-terminal kinase (JNK)–cJun signal pathway was activated after 11-epi-SA treatment. In general, these results suggest that 11-epi-SA exerts cytotoxic effects on HA22T cells through mitochondrial dysfunction and ER stress cell death pathways

Endoscopic papillary balloon dilatation less than three minutes for biliary stone removal increases the risk of post-ERCP pancreatitis.

OBJECTIVES:The adequate duration for EPBD was unclear. Therefore, we aimed to investigate the effect of balloon dilatation duration of EPBD on the occurrence of PEP. METHODS:One hundred and ninety-eight patients with common bile duct (CBD) stone treated by EPBD were retrospectively recruited. The dilatation duration was determined according to adequate opening of the biliary orifice without bleeding. The clinical outcomes and complications of EPBD were recorded. RESULTS:We stratified the patients according to dilatation duration (Group A, <3 minutes; Group B, 3-5 minutes; Group C, ≥5 minutes). The group C patients had a higher proportion of large CBD stones (stones ≥10 mm) (33.3% vs. 26.8% vs. 53.5%, p = 0.01). Patients in group A had a significantly higher PEP rate than patients in group B (13.3 vs. 3.1, p = 0.032). There were no significant differences in perforation and bleeding rate among the three groups. Univariate and multivariate analyses showed that a dilatation duration of <3 minutes, CBD diameter < 10 mm and age ≤ 75 years were independent risk factors of PEP in post-EPBD patients. CONCLUSIONS:In patients receiving EPBD, dilatation duration <3 minutes, lower CBD diameter, and younger age were independent risk factors of PEP

Function-Oriented Development of CXCR4 Antagonists as Selective Human Immunodeficiency Virus (HIV)‑1 Entry Inhibitors

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound <b>24</b>, with an EC₅₀ of 0.5 nM against HIV-1 entry into host cells and an IC₅₀ of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound <b>24</b> complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound <b>24</b> showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4⁺/CD34⁺ stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function