Dual antimicrobial and anticancer activity of a novel synthetic α-helical antimicrobial peptide.

Antimicrobial peptides (AMPs) are increasingly sought-after and researched antimicrobial agents due to its desired pharmacological properties and the continuous diminishing efficacy of antibiotics. In addition to this line of research, the aim of the present study is to determine the antimicrobial and anticancer activity of a de novo designed α-helical peptide. Circular dichroism showed 100% helical nature of the peptide in 10 mM SDS. Notably, the peptide exerted significant antimicrobial activity against the reference and antibiotic-resistant clinical isolates belonging to Pseudomonas sp. at a MIC and MBC of 2 and 8 µM, respectively. The progressive disruption and disturbance of cell membrane in the overall topography was observed in the scanning electron microscopy (SEM) micrographs of Pseudomonas aeruginosa ATCC 27853 treated with the peptide as compared to untreated control. The results of time-kill kinetics showed complete lysis at 3x MIC after 50 min of incubation of the microbe with the peptide. Moreover, the peptide did not lyse human RBCs even at the highest concentration of the peptide (10 mM) and retained its activity upon treatment at 0.5 mg/ml trypsin. Cancer cell lines, viz. A549 and MCF-7 were also found to be sensitive to peptide activity showing 50% reduction in survivability at 4 and 2 µM, respectively; however, L929 cells were unaffected. Drastic membrane permeability and necrotic mode of lysis of peptide-treated-A549 cells were affirmed by propidium iodide and live/dead cell staining. The results showed that the designed peptide could be an efficient drug molecule for clinical studies subjected to successful experiments on animal models.

Multidimensional significance of crystallin protein-protein interactions and their implications in various human diseases.

BACKGROUND: Crystallins are the important structural and functional proteins in the eye lens responsible for refractive index. Post-translational modifications (PTMs) and mutations are major causative factors that affect crystallin structural conformation and functional characteristics thus playing a vital role in the etiology of cataractogenesis. SCOPE OF REVIEW: The significance of crystallin protein-protein interactions (PPIs) in the lens and non-lenticular tissues is summarized. MAJOR CONCLUSIONS: Aberrancy of PPIs between crystallin, its associated protein and metal ions has been accomplished in various human diseases including cataract. A detailed account on multidimensional structural and functional significance of crystallin PPI in humans must be brought into limelight, in order to understand the biochemical and molecular basis augmenting the aberrancies of such interaction. In this scenario, the present review is focused to shed light on studies which will aid to expand our present understanding on disease pathogenesis related to loss of PPI thereby paving the way for putative future therapeutic targets to curb such diseases. GENERAL SIGNIFICANCE: The interactions with α-crystallins always aid to protect their structural and functional characteristics. The up-regulation of αB-crystallin in the non-lenticular tissues always decodes as biomarker for various stress related disorders. For better understanding and treatment of various diseases, PPI studies provide overall outline about the structural and functional characteristics of the proteins. This information not only helps to find out the route of cataractogenesis but also aid to identify potential molecules to inhibit/prevent the further development of such complicated phenomenon. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

C-phycocyanin modulates selenite-induced cataractogenesis in rats.

The present investigation is aimed to evaluate the anticataractogenic potential of C-phycocyanin (C-PC), extracted and purified from Spirulina platensis. Enucleated rat lenses were maintained in vitro in Dulbecco's modified Eagle medium (DMEM). Group I contained DMEM, Group II and Group III contained 100 µM of sodium selenite, Group III was subdivided into three viz IIIa, IIIb, IIIc supplemented with 100, 150, 200 µg of C-PC respectively. In the in vivo study, on tenth day post partum: Group I rat pups received an intraperitoneal injection of saline, Group II, IIIa, IIIb, and IIIc rat pups received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight) Group IIIa, IIIb, IIIc also received an intraperitoneal injection of 100, 150, 200 mg/kg body weight of C-PC, respectively, from postpartum days 9-14. On termination of the experiment, the lenses from both in vitro and in vivo studies were subjected to morphological examination and subsequently processed to estimate the activities of antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, levels of reduced glutathione and lipid peroxidation products. Sodium selenite-exposed, C-PC-treated rat lenses (Group IIIc), showed significant restoration of antioxidant enzyme activity (p < 0.05) when compared to their counterpart Group II. Group IIIc conserved the levels of GSH and lipid peroxidation products at near to normal levels as compared with Group II. Results conclude the possible role of C-PC in modulating the antioxidant enzyme status, thereby retarding sodium selenite-induced cataract incidence both in vitro and in vivo.

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate.

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVß-gal, resulted in enhanced ß-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ß-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.