Serine O-acetyltransferase derived NV14 peptide reduces cytotoxicity in H2O2 induced MDCK cells and inhibits MCF-7 cell proliferation through caspase gene expression.

BACKGROUND: Most of the bioactive peptides exhibit antioxidant effect and do elicit inhibitory effect on proliferation of cancer cells. This study investigates the in-vitro antioxidant and anti-cancer properties of NV14 peptide, derived from serine O-acetyltransferase (SAT) of spirulina, Arthrospira platensis. METHODS: The anti-cancer effect of the peptide was evaluated using human adenocarcinoma epithelial cells (MCF-7), while the anti-oxidant potential, as in reduction in ROS concentration, has been established using the H2O2-exposed, Madin-Darby canine kidney (MDCK) cells. The outcome of the in vitro analyses has been evaluated by in silico molecular docking analyses. RESULTS: The peptide, dose-dependently, reduced oxidative stress as well as cell proliferation. Besides, based on the binding scores between NV14 peptide and the important proteins associated with apoptosis and antioxidant defense, it is evident that the peptide has antioxidant and anti-cancer effect, in vitro. CONCLUSIONS: Together, this study demonstrates that NV14 has a potent antioxidant and anti-cancer capability; however, further direction needs to be focused on clinical or pharmacodynamics aspects.

Lysozyme microspheres incorporated with anisotropic gold nanorods for ultrasound activated drug delivery.

We report on the fabrication of lysozyme microspheres (LyMs) incorporated with gold nanorods (NRs) as a distinctive approach for the encapsulation and release of an anticancer drug, 5-Fluorouracil (5-FU). LyMs with an average size of 4.0 ± 1.0 µm were prepared by a sonochemical method and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). The LyMs were examined using hydrophobic (nile red) as well as hydrophilic (trypan blue) dyes under confocal laser scanning microscopy (CLSM) to obtain information about the preferential distribution of fluorescent molecules. Notably, the fluorescent molecules were accumulated in the inner lining of LyMs as the core was occupied with air. The encapsulation efficiency of 5-FU for LyMs-NR was found to be ∼64%. The drug release from control LyMs as well as LyMs incorporated with NRs was investigated under the influence of ultrasound (US) at 200 kHz. The total release for control LyMs and LyMs incorporated with gold NRs was found to be ∼70 and 95% after 1 h, respectively. The density difference caused by NR incorporation on the shell played a key role in rupturing the LyMs-NR under US irradiation. Furthermore, 5-FU loaded LyMs-NR exhibited excellent anti-cancer activity against the THP-1 cell line (∼90% cell death) when irradiated with US of 200 kHz. The enhanced anti-cancer activity of LyMs-NR was caused by the transfer of released 5-FU molecules from bulk to the interior of the cell via temporary pores formed on the surface of cancer cells, i.e., sonoporation. Thus, LyMs-NR demonstrated here has a high potential for use as carriers in the field of drug delivery, bio-imaging and therapy.

Design and characterization of a novel Arthrospira platensis glutathione oxido-reductase-derived antioxidant peptide GM15 and its potent anti-cancer activity via caspase-9 mediated apoptosis in oral cancer cells.

Glutathione oxido-reductase (GR) is a primary antioxidant enzyme of most living forms which protects the cells from oxidative metabolism by reducing glutathione (GSH) from its oxidized form (GSSG). Although the antioxidant role of the enzyme is well characterized, the specific role of conserved N' peptide sequence in antioxidant mechanism remains unclear. In this study, we have identified an RNA sequence encoding GR enzyme from spirulina, Arthrospira platensis (Ap) and the changes in its gene expression profile was analysed during H2O2 stress. Results showed that H2O2 (10 mM) stimulated the expression of ApGR throughout the timeline of study (0, 5, 10, 15 and 20 days) with highest expression at 5th day post-exposure which confirmed the antioxidant role of ApGR in spirulina during H2O2 induced oxidative stress. A dithiol containing short antioxidant peptide, 39GGTCVIRGCVPKKLM53 (GM15) from ApGR was predicted and its radicals (superoxide and hydroxyl radical) scavenging potential was confirmed by in vitro cell-free assays. GM15 (12.5 µM) reduced the intracellular generalized oxidative stress level, as measured using DCFDA assay in H2O2 exposed leucocytes without affecting any of the cellular population. Further, the biomedical application of the radical scavenging property of GM15 was validated in oral carcinoma (KB) cells where GM15 exhibited significant cytotoxicity. Also, GM15 exhibited heterogenous effects on intracellular oxidative stress level in KB cells: at lower concentration (6.25 µM), the peptide reduced oxidative stress whereas, at higher concentration (25 µM) it increased the intensity of oxidative stress. GM15 (25 µM) induced caspase-9 mediated apoptosis in KB cells along with membrane disruption and DNA degradation which are confirmed by propidium iodide (PI) internalization and comet assays, respectively. Overall, the study shows that GM15 peptide i) scavenges superoxide, hydroxyl radicals, and influences intracellular oxidative stress, and ii) has anti-cancer effect in oral cancer cells.

Role of pterostilbene in attenuating immune mediated devastation of pancreatic beta cells via Nrf2 signaling cascade.

Nrf2 (nuclear factor erythroid 2-related factor-2) is a transcription factor that regulates oxidative/xenobiotic stress response and also suppress inflammation. Nrf2 signaling is associated with an increased susceptibility to various kinds of stress. Nrf2 has been shown as a promising therapeutic target in various human diseases including diabetes. Our earlier studies showed Pterostilbene (PTS) as a potent Nrf2 activator, and it protects the pancreatic ß-cells against oxidative stress. In this study, we investigated PTS confer protection against cytokine-induced ß-cell apoptosis and its role on insulin secretion in streptozotocin (STZ)-induced diabetic mice. The Nrf2 activation potential of PTS was assessed by dissociation of the Nrf2-Keap1 complex and by expression of ARE-driven downstream target genes in MIN6 cells. Further, the nuclear Nrf2 translocation and blockage of apoptotic signaling as demonstrated by the reduction of BAX/Bcl-2 ratio, Annexin-V positive cells and caspase-3 activity conferred the cyto-protection of PTS against cytokine-induced cellular damage. In addition, PTS treatment markedly improved glucose homeostasis and abated inflammatory response evidenced by the reduction of proinflammatory cytokines in diabetic mice. The inhibition of ß-cell apoptosis by PTS as assessed by BAX/Bcl-2 ratio and caspase-3 activity in the pancreas was associated with the activation of Nrf2 and the expression of its downstream target genes. PTS also inhibited the activation of iNOS and decreased nitric oxide (NO) formation in the pancreas of diabetic animals. The results obtained from both in vitro and in vivo experiments showed that PTS improves ß-cell function and survival against cytokine stress and also prevents STZ-induced diabetes.

Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan.

Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.

Hydrogen bonded capsules by layer-by-layer assembly of tannic acid and poly(2-n-propyl-2-oxazoline) for encapsulation and release of macromolecules.

We report hydrogen bonded capsules with the built-in ability to release loaded bioactive molecules at a physiological temperature of 37 °C. The use of neutral and non-toxic building blocks such as tannic acid (TA) and poly(2-n-propyl-2-oxazoline)s (PnPropOx) as hydrogen bonding donor and acceptor results in stable hollow capsules. The temperature induced morphological changes of the shell were investigated using a scanning electron microscope and an optical microscope and revealed pore formation in the shell when the temperature (T) increases beyond the cloud point temperature (TCP) of PnPropOx. Furthermore, confocal laser scanning microscopic investigation of the hollow capsules loaded with different probes of varying hydrodynamic diameters revealed that the open and closed state of the capsules could be effectively manipulated by varying the incubation time and hydrodynamic radius of the probes. Such hydrogen bonded capsules have high potential for use in temperature responsive sustained drug delivery applications.

Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin.

Antimicrobial peptides (AMPs) are innate molecules that are found in a wide variety of species ranging from bacteria to humans. In recent years, excessive usage of antibiotics resulted in development of multi-drug resistant pathogens which made researchers to focus on AMPs as potential substitute for antibiotics. Lily type mannose-binding lectin is an extended super-family of structurally and evolutionarily related sugar binding proteins. These lectins are well-known AMPs which play important roles in fish defense mechanism. Here, we report a full-length lily type lectin-2 (LTL-2) identified from the cDNA library of striped murrel, Channa striatus (Cs). CsLTL-2 protein contained B-lectin domain along with three carbohydrate binding sites which is a prominent characteristic functional feature of LTL. The mRNA transcripts of CsLTL-2 were predominantly expressed in gills and considerably up-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila). To evaluate the antimicrobial activity of the carbohydrate binding region of CsLTL-2, the region was synthesized (QP13) and its bactericidal activity was analyzed. In addition, QP13 was labeled with fluorescein isothiocyanate (FITC) and its binding affinity with the bacterial cell membranes was analyzed. Minimum inhibitory concentration assay revealed that QP13 inhibited the growth of Escherichia coli at a concentration of 80 µM/ml. Confocal microscopic observation showed that FITC tagged QP13 specifically bound to the bacterial membrane. Fluorescence assisted cell sorter (FACS) assay showed that QP13 reduced the bacterial cell count drastically. Therefore, the mechanism of action of QP13 on E. coli cells was determined by propidium iodide internalization assay which confirmed that QP13 induced bacterial membrane disruption. Moreover, the peptide did not show any cytotoxicity towards fish peripheral blood leucocytes. Taken together, these results support the potentiality of QP13 that can be used as an antimicrobial agent against the tested pathogens.

Pellino-1 derived cationic antimicrobial prawn peptide: Bactericidal activity, toxicity and mode of action.

The antimicrobial peptides (AMPs) are multifunctional molecules which represent significant roles in the innate immune system. These molecules have been well known for decades because of their role as natural antibiotics in both invertebrates and vertebrates. The development of multiple drug resistance against conventional antibiotics brought a greater focus on AMPs in recent years. The cationic peptides, in particular, proven as host defense peptides and are considered as effectors of innate immunity. Among the various innate immune molecules, functions of pellino-1 (Peli-1) have been recently studied for its remarkable role in specific immune functions. In our study, we have identified Peli-1 from the cDNA library of freshwater prawn Macrobrachium rosenbergii (Mr) and analyzed its features using various in-silico methods. Real time PCR analysis showed an induced expression of MrPeli-1 during white spot syndrome virus (WSSV), bacteria (Vibrio harveyi) and lipopolysaccharide (LPS) from Escherichia coli challenge. Also, a cationic AMP named MrDN was derived from MrPeli-1 protein sequence and its activity was confirmed against various pathogenic bacteria. The mode of action of MrDN was determined to be its membrane permeabilization ability against Bacillus cereus ATCC 2106 as well as its DNA binding ability. Further, scanning electron microscopic (SEM) images showed the membrane disruption and leakage of cellular components of B. cereus cells induced by MrDN. The toxicity of MrDN against normal cells (HEK293 cells) was demonstrated by MTT and hemolysis assays. Overall, the results demonstrated the innate immune function of MrPeli-1 with a potential cationic AMP in prawn.

A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

Selective deacetylation of zaluzanin D using transformed Escherichia coli cultures.

The use of conventional and unconventional reaction methodology for the hydrolysis of the acetate group in zaluzanin D resulted in hydration of the 11,13 exocyclic bond along with deacetylation. But the microorganism E. coli selectively cleaved the acetate group to yield zaluzanin C.

Complete reduction of 2H-pyran-2-one moiety of coumarin and 6-methyl coumarin by Colletotrichum capsici.

The microbial transformation of coumarin (1) and 6-methyl coumarin (2) using Colletotrichum capsici gave 2-(3'-hydroxypropyl) phenol (3) and 2-(3'-hydroxypropyl)-4-methyl phenol (4). The phytopathogenic fungi effectively reduced the 2H-pyran-2-one moiety of both parent coumarins to respective alcohols.