Bala Ramesh

A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia

The ischemic stroke is the third leading cause of death in developed countries. The C‐terminal peptide of mechano‐growth factor (MGF), an alternatively spliced variant of insulin‐like growth factor 1 (IGF‐1), was found to function independently from the rest of the molecule and showed a neuroprotective effect in vivo and in vitro. In vivo, in a gerbil model of transient brain ischemia, treatment with the synthetic MGF C‐terminal peptide provided very significant protection to the vulnerable neurons. In the same model, ischemia evoked increased expression of endogenous MGF in the ischemia‐resistant hippocampal neurons, suggesting that the endogenous MGF might have an important neuroprotective function. In an in vitro organotypic hippocampal culture model of neurodegeneration, the synthetic peptide was as potent as the full‐length IGF‐1 while its effect lasted significantly longer than that of recombinant IGF‐1. While two peptides showed an additive effect, the neuroprotective action of the C‐terminal MGF was independent from the IGF‐1 receptor, indicating a new mode of action for this molecule. Although MGF is known for its regenerative capability in skeletal muscle, our findings demonstrate for the first time a neuroprotective role against ischemia for this specific IGF‐1 isoform. Therefore, the C‐terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage.

Fluorescence nanoparticles “quantum dots” as drug delivery system and their toxicity: a review

Fluorescence nanocrystals or quantum dots (QDs) are engineered nanoparticles (NP) that have shown great promise with potential for many biological and biomedical applications, especially in drug delivery/activation and cellular imaging. The use of nanotechnology in medicine directed to drug delivery is set to expand in the coming years. However, it is unclear whether QDs, which are defined as NPs rather than small molecules, can specifically and effectively deliver drugs to molecular targets at subcellular levels. When QDs are linked to suitable ligands that are site specific, it has been shown to be brighter and photostable when compared with organic dyes. Interestingly, pharmaceutical sciences are exploiting NPs to minimize toxicity and undesirable side effects of drugs. The unforeseen hazardous properties of the carrier NPs themselves have given rise to some concern in a clinical setting. The kind of hazards encountered with this new nanotechnology materials are complex compared with conventional limitations created by traditional delivery systems. The development of cadmium-derived QDs shows great potential for treatment and diagnosis of cancer and site-directed delivery by virtue of their size-tunable fluorescence and with highly customizable surface for directing their bioactivity and targeting. However, data regarding the pharmacokinetic and toxicology studies require further investigation and development, and it poses great difficulties to ascertain the risks associated with this new technology. Additionally, nanotechnology also displays yet another inherent risk for toxic cadmium, which will enter as a new form of hazard in the biomedical field. This review will look at cadmium-derived QDs and discuss their future and their possible toxicities in a disease situation.

The anti-calcification potential of a silsesquioxane nanocomposite polymer under in vitro conditions: Potential material for synthetic leaflet heart valve

Calcification currently represents a major cause of failure of biological tissue heart valves. It is a complex phenomenon influenced by a number of biochemical and mechanical factors. Recent advances in material science offer new polymers with improved properties, potentially suitable for synthetic leaflets heart valves manufacturing. In this study, the calcification-resistance efficacy and mechanical and surface properties of a new nanocomposite polymeric material (polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane; POSS-PCU) which has been developed by our group are assessed by means of in vitro testing. In particular, thin sheets of nanocomposite, glutaraldehyde-fixed bovine pericardium (BP) and polyurethane (PU) were exposed to a calcium solution into a specially designed in vitro accelerated physiological pulsatile pressure system for a period of 31days and a total of 4×10(7) cycles. The samples were investigated for signs of calcification after exposure to calcium solution by means of X-ray, microscopic and chemical inspections. Mechanical and surface properties were also studied using stress-strain behaviour and surface morphology and hydrophobicity. Comparison shows that, in the experimental conditions, the level of calcification for the nanocomposite is considerably lower than for the fixed BP (p=0.008) and PU samples (p=0.015). Also, mechanical properties were unchanged in POSS-PCU, while there was a significant deterioration in PU samples (p<0.05). Hydrophobicity was significantly reduced in both the POSS-PCU and PU samples (p<0.0001). However, the POSS-PCU nanocomposite remained more hydrophobic than the PU sample (p<0.0001). Less platelet adhered to the POSS-PCU compared to the PU (p<0.0001). These results indicate that the use of this nanocomposite in synthetic leaflets heart valves may lead to potential advantages in terms of long-term performances and durability.

Divalent metal‐dependent regulation of hepcidin expression by MTF‐1

Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflammation and infection, but is repressed by anaemia and hypoxia. Here we further reveal that hepcidin transcription also involves interactions between functional metal response elements (MREs) in its promoter, and the MRE‐binding transcription factor‐1. Analysis of hepcidin mRNA and protein levels in hepatoma cells suggests that its expression may be regulated by divalent metal ions, with zinc inducing maximal effects on hepcidin levels. These data suggest that this peptide may be a pleiotropic sensor of divalent metals, some of which are xenobiotic environmental toxins.

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

The complement inhibitors C4b‐binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier‐transform infrared technique at temperatures ranging from 30°C–90°C. We found that predominantly β‐sheet structure of both proteins was stable up to 70°C, and that a reversible conformational change toward α‐helix was apparent at temperatures ranging from 70°C to 90°C. The ability of both proteins to inhibit complement was not impaired after incubation at 95°C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP‐containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in β‐sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP‐containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.

A novel POSS-coated quantum dot for biological application

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Efficacy of the Specific Endothelin A Receptor Antagonist Zibotentan (ZD4054) in Colorectal Cancer: A Preclinical Study

Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ETA receptor. Here, for the first time, we evaluate zibotentan, a specific ETA receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n = 4) and fibroblast strains (n = 6), isolated from uninvolved areas of colorectal cancer specimens, were exposed to ET-1 and/or ETA/B receptor antagonists. Proliferation (methylene blue), migration (scratch wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (Kd, Bmax) were determined using autoradiography on tissue sections and homogenates and cytospun cells, supported by immunohistochemistry. Proliferation was inhibited by ETA (zibotentan > BQ123; P < 0.05), migration by ETB > ETA, and contraction by combined ETA and ETB antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density and affinity ET-1 binding (Bmax = 2.435 fmol/1 × 106 cells, Kd = 367.7 pmol/L; Bmax = 3.03 fmol/1 × 106 cells, Kd = 213.6 pmol/L). In cancer tissues, ETA receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more effectively (IC50: 0.1–10 μmol/L) than ETB receptor antagonist BQ788 (∼IC50, 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibroblasts, implicates these cells in tumor progression. ETA receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptors importance in therapeutic targeting. Zibotentan, the most specific ETA receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety profile, we provide evidence for zibotentans potential role as adjuvant therapy in colorectal cancer. Mol Cancer Ther; 12(8); 1556–67. ©2013 AACR.

Synthesis and evaluation of amphiphilic RGD derivatives: Uses for solvent casting in polymers and tissue engineering applications

Derivatives containing arginine-glycine-aspartic acid (RGD) inhibit fibrinogen binding to activated platelets and promote endothelial and smooth muscle cell attachment. An amphiphilic derivative of RGD that can be dissolved in an organic solvent has potential in the development of non-thrombogenic biomaterials. Such a derivative, LA-GRGD, was synthesised by coupling glycine-arginine-glycine-aspartic acid (GRGD) with lauric acid (LA). Its solubility and antithrombotic, cytotoxic and cell-binding effects were then evaluated in comparison with heparin (which is used clinically) and a fibronectin-engineered protein polymer (FEPP). Thromboelastography (TEG) was used to measure blood clotting time using fresh whole blood from healthy volunteers. Tissue factor (TF) activity was measured using plasma with a standard prothrombin time assay (PT). Cytotoxicity was assessed on human umbilical cord endothelial cells (HUVECs) using an Alamar blue assay. Solubility of the conjugate was assessed in a co-solvent. These techniques were used to study LA-GRGD, using heparin and FEPP as controls. The amphiphilic property of LA-GRGD, using heparin and FEPP as controls. The amphiphilic GRGD was soluble in acetone:water and water. LA-GRGD inhibited TF by >90% and prolonged TEG-r by 8.2±3.3 min (200 μg ml−1). Heparin inhibited TF by >90%, but prolonged TEG-r by 97.4±1.6 min (1 U ml−1) FEPP inhibited TF by >90% (100μg ml−1) and prolonged TEG-r by 73.7±8.4 min (10μg ml−1). Heparin had no cytotoxic effect on EC metabolism and viability at the concentrations studied (0.1–100 U ml−1). No significant cytotoxic effect was produced by LA-GRGD or FEPP at concentrations ranging from 0.1 μg ml−1, to 50 μg ml−1, but, at higher concentrations (100 μg ml−1 and 200 μg ml−1), a detrimental effect was observed. Cell binding studies showed that LA-GRGD bound 29% of ECs compared with FEPP (60%) and heparin (22%). This new approach for synthesising amphiphilic RGD and its analogues has potential as a drug delivery system for the manufacture of new polymer formulations for use in bypass grafts and other tissue-engineered devices.

Biofunctionalized quantum dots for live monitoring of stem cells: applications in regenerative medicine

AIM This study aimed to live monitor the degree of endothelial progenitor cell (EPC) integration onto tissue-engineering scaffolds by conjugating relevant antibodies to quantum dots (QDs). MATERIALS & METHODS Biocompatible mercaptosuccinic acid-coated QDs were functionalized with two different antibodies to EPC (CD133 with QDs of 640 nm wavelength [λ] and later-stage mature EPCs; and von Willebrand factor with QDs of λ595 and λ555 nm) using conventional carbomide and N-hydroxysuccinimide chemistry. Biofunctionalization was characterized with Fourier-transform infrared spectroscopy. Cell viability assays and gross morphology observations confirmed cytocompatibility and normal patterns of celluar growth. The antigens corresponding to each state of cell maturation were determined using a single excitation at λ488 nm. RESULTS The optimal concentrations of antibody-QD conjugates were biocompatible, hemocompatible and determined the state of EPC transformation to endothelial cells. CONCLUSION Antibody-functionalized QDs suggest new applications in tissue engineering of polymer-based implants where cell integration can potentially be monitored without requiring the sacrifice of implants.

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.