Synthesis and evaluation of 2,5 and 2,6 pyridine-based CXCR4 inhibitors

Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000 nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10 nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.     

Degradation of cholecystokinin octapeptide,related fragments and analogs by human and rat plasma in vitro

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products.CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min).The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide.CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids.CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol®), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.     

A Conservative Point Mutation in a Dynamic Antigen-binding Loop of Human Immunoglobulin λ6 Light Chain Promotes Pathologic Amyloid Formation

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ∼30 Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.     

The association of a distinct plasma proteomic profile with the cervical high-grade squamous intraepithelial lesion of Uyghur women: a 2D liquid-phase chromatography/mass spectrometry study

Objective: To identify plasma protein biomarkers of cervical high-grade squamous intraepithelial lesion (HSIL) of Uyghur women by proteomics approach.

Methods: Plasma protein samples of Uyghur women with HSIL and chronic cervicitis were analyzed with 2D HPLC followed by detection of target proteins with Linear Trap Quadrupole Mass Spectrometer (LTQ MS/MS).

Results: We detected three upregulated and one downregulated protein peaks representing protein constituents distinguishing HSIL from controls by 2D HPLC, identified 31 target proteins by LTQ MS/MS. Further confirmed analysis with online software IPA^® 8.7 and ELISA assay showed APOA1 and mTOR as potential biomarkers.

Conclusions: A distinct plasma proteomic profile may be associated with HSIL of Uyghur women.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer’s Disease Brain

Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS–based glycoproteomics. We analyzed brain tissue from 10 persons with no cognitive impairment or AD, 10 with asymptomatic AD, and 10 with symptomatic AD, detecting over 300 glycoproteins and 1900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic or complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain N-glycoproteome, occurring at <9% among the glycoforms. We observed AD-associated differences in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among samples from our three groups. Further analysis revealed glycosylation differences in subcellular compartments across disease stage, including glycoproteins in the lysosome frequently modified with paucimannosidic glycans. These results illustrate the N-glycoproteomics landscape across the spectrum of AD clinical and pathologic severity and will facilitate a deeper understanding of progression and treatment development.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.