Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery

Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.     

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47^phox-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3–Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H₂O₂ (10 μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2 μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47^phox, and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Three MTP Transporters Sequestrate Zn in Sedum alfredii Hance

Russian Journal of Plant Physiology - Hyperaccumulating ecotype (HE) of Sedum alfredii Hance is a Zn/Cd hyperaccumulator, which can accumulate Zn in shoot up to 2% of dry weight, understanding the...     

BRD4 inhibition by JQ1 protects against LPS-induced cardiac dysfunction by inhibiting activation of NLRP3 inflammasomes

Molecular Biology Reports - JQ1, a BRD4 inhibitor, first identified its therapeutic role in cancer, has gradually demonstrated a protective effect on the heart in recent years; however, it is...     

Ocular Hypertension after Pediatric Cataract Surgery: Baseline Characteristics and First-Year Report

Monitoring intraocular pressure (IOP) is essential for pediatric cataract treatment but always difficult due to lack of cooperation in young children. We present the baseline characteristics and the first-year results of a long-term prospective cohort study, which are aimed to determine the relationship of the incidence of ocular hypertension (OH) in children after cataract surgery during the first-year period and the risk of developing late-onset glaucoma. Children were included with the following criteria: they were≤10 years old and scheduled to undergo cataract surgery with/without intraocular lens implantation; they were compliant with our follow-up protocol, which included monitoring IOP using a Tono-Pen under sedation or anesthesia. Incidence of OH, peak OH value, OH onset time and OH duration within a 12-month period following surgery were measured. In brief, 206 patients (379 eyes) were included and OH developed in 66 of 379 (17.4%) eyes. The mean follow-up period was 14.0±3.2 months (median, 12 months; range, 10–16 months). Moreover, 33 of 196 (16.8%) aphakic eyes and 33 of 183 (18.0%) IOL eyes were diagnosed with OH. The peak OH onset times were at 1-week (34/66, 51.5%) and 1-month (14/66, 21.2%) appointments postsurgery. The peak IOP value in the OH eyes was 29.9±7.5 mmHg (median, 29 mmHg; range, 21–48 mmHg). The duration of OH was 30.9±31.2 days (median, 30 days; range, 3–150 days). OH recurred in 13 eyes with a history of OH diagnosed within 1 month postsurgery (13/54, 24.1%), which needed temporary or long term use of antiglaucoma medications. In conclusion, the incidence of OH in children after cataract surgery was 17.4% during the first-year period. Children who have suffered elevated IOP in the first year after cataract surgery should be followed closely to determine if there is an increased risk of developing late-onset glaucoma.