Polymeric biocompatible iron oxide nanoparticles labeled with peptides for imaging in ovarian cancer

Compared with other nanomaterials, surface-modified iron oxide nanoparticles (IONPs) have gained attraction for cancer therapy applications due to its low toxicity, and long retention time. An innocuous targeting strategy was developed by generation of fluorescein isothiocyanate (FITC)-labeled peptide (growth factor domain (GFD) and somatomedin B domain (SMB)) functionalized, chitosan-coated IONPs (IONPs/C). It can be used to target urokinase plasminogen activator receptor (uPAR), which is a surface biomarker, in ovarian cancer. Binding affinity between uPAR and peptides (GFD and SMB) were revealed by in-silico docking studies. The biophysical characterizations of IONPs, IONPs/C, and IONPs/C/GFD-FITC or SMB-FITC nanoprobes were assessed via Vibrating Sample Magnetometer (VSM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). Prussian Blue staining, fluorescence spectroscopy, and fluorescence imaging were performed to confirm the targeting of nanoprobes with the surface receptor uPAR. The combination of IONPs/C/GFD+SMB showed efficient targeting of uPAR in the tumor microenvironment, and thus can be implemented as a molecular magnetic nanoprobe for cancer cell imaging and targeting.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC–MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.     

Measurement of drug-protein binding by immobilized human serum albumin-HPLC and comparison with ultrafiltration

An HPLC method employing CHIRAL-I (150 mm x 3 mm), 5 microm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to alpha1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.     

Solubility enhancement of cox-2 inhibitors using various solvent systems

This study examined the solubility enhancement of 4 cox-2 inhibitors, celecoxib, rofecoxib, meloxicam, and nimesulide, using a series of pure solvents and solvent mixtures. Water, alcohols, glycols, glycerol, and polyethylene glycol 400 (PEG 400) were used as solvents and water-ethanol, glycerol-ethanol, and polyethylene glycol-ethanol were used as mixed-solvent systems. A pH-solubility profile of drugs was obtained in the pH range 7.0 to 10.9 using 0.05M glycine-sodium hydroxide buffer solutions. Lower alcohols, higher glycols, and PEG 400 were found to be good solvents for these drugs. The aqueous solubility of celecoxib, rofecoxib, and nimesulide could be enhanced significantly by using ethanol as the second solvent. Among the mixed-solvent systems, PEG 400-ethanol system had highest solubilization potential. In the case of meloxicam and nimesulide, solubility increased significantly with increase in pH value. Physico-chemical properties of the solvent such as polarity, intermolecular interactions, and the ability of the solvent to form a hydrogen bond with the drug molecules were found to be the major factors involved in the dissolution of drugs by pure solvents. The greater the difference in the polarity of the 2 solvents in a given mixed solvent, the greater was the solubilization power. However, in a given mixed-solvent system, the solubilization power could not be related to the polarity of the drugs. Significance of the solubility data in relation to the development of formulations has also been discussed in this study.     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events

CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.     

Functional and Structural Diversity of Insect Glutathione S-transferases in Xenobiotic Adaptation

As a superfamily of multifunctional enzymes that is mainly associated with xenobiotic adaptation, glutathione S-transferases (GSTs) facilitate insects'' survival under chemical stresses in their environment. GSTs confer xenobiotic adaptation through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. In this article, a comprehensive overview of current understanding on the versatile functions of insect GSTs in detoxifying chemical compounds is presented. The diverse structures of different classes of insect GSTs, specifically the spatial localization and composition of their amino acid residues constituted in their active sites are also summarized. Recent availability of whole genome sequences of numerous insect species, accompanied by RNA interference, X-ray crystallography, enzyme kinetics and site-directed mutagenesis techniques have significantly enhanced our understanding of functional and structural diversity of insect GSTs.     

Conformational Plasticity in the HIV-1 Fusion Peptide Facilitates Recognition by Broadly Neutralizing Antibodies

1. Download : Download high-res image (201KB)
2. Download : Download full-size image