Saurabh Srivastava

Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2.

Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine-zipper-like sequences in melittin and other naturally occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in the presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. However, Mag-mut bound and localized strongly on to the mammalian cells tested and exhibited significantly higher cytotoxicity than magainin 2. Only Mag-mut, but not magainin 2, permeabilized human red blood cells and zwitterionic lipid vesicles. In contrast with magainin 2, Mag-mut self-assembled in an aqueous environment and bound co-operatively on to zwitterionic lipid vesicles. The peptides formed pores of different sizes on to a selected mammalian cell. The results of the present study indicate an important role of the leucine zipper sequence in the cytotoxicity of Mag-mut and demonstrate that its introduction into a non-toxic peptide, without altering the amino acid composition, can render cytotoxicity.

Consequences of Alteration in Leucine Zipper Sequence of Melittin in Its Neutralization of Lipopolysaccharide-induced Proinflammatory Response in Macrophage Cells and Interaction with Lipopolysaccharide

Background: Bee venom antimicrobial peptide, melittin, neutralizes LPS-induced proinflammatory response in macrophage cells. Results: Alteration in the leucine zipper sequence of melittin impaired its anti-LPS property and interaction with LPS. Conclusion: The leucine zipper sequence of melittin plays a crucial role in maintaining its antiendotoxin properties. Significances: The results suggest an overlap of structural requirements for the cytotoxic and antiendotoxin properties of melittin. The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.

Design of Nontoxic Analogues of Cathelicidin-Derived Bovine Antimicrobial Peptide BMAP-27: The Role of Leucine as Well as Phenylalanine Zipper Sequences in Determining Its Toxicity

BMAP-27 is a cathelicidin-derived bovine antimicrobial peptide, which shows moderate cytotoxicity and potent antibacterial activity against a wide variety of microorganisms. Despite a number of studies, very little is known about the amino acid sequences of this peptide that controls its antibacterial and cytotoxic activities. Small stretches of phenylalanine and leucine zipper sequences were identified at the N- and C-termini of the molecule, respectively. To understand the structural and functional roles of these sequence elements, we synthesized and characterized several analogues of BMAP-27 after substituting leucine or phenylalanine residue(s) at a and/or d positions of the leucine and phenylalanine zipper sequences, respectively, with alanine. BMAP-27 analogues exhibited significantly reduced cytotoxicity against the human red blood (hRBC) and murine 3T3 cells as compared to that of the wild-type peptide. Interestingly, BMAP-27 and its analogues exhibited comparable antibacterial activity against the selected Gram-positive and Gram-negative bacteria. Moreover, BMAP-27 and its analogues exhibited similar localization and assembly onto the selected bacteria and induced comparable permeability in these cells. However, only BMAP-27, not its analogues, assembled and bound strongly onto the hRBCs and permeabilized them. The results indicated that not only a leucine zipper but also a phenylalanine zipper sequence plays an important role in maintaining the assembly of BMAP-27 on the mammalian cells examined here and cytotoxic activity against them. To the best of our knowledge, this is the first report of the evaluation of structural and functional roles of a phenylalanine zipper sequence in a naturally occurring antimicrobial peptide.

Introduction of a Lysine Residue Promotes Aggregation of Temporin L in Lipopolysaccharides and Augmentation of Its Antiendotoxin Property

ABSTRACT Temporin L (TempL) is a 13-residue frog antimicrobial peptide that shows moderate bactericidal activity and antiendotoxin properties in macrophages. We envisioned that, due to its very hydrophobic nature, the peptide might fail to show its desired biological properties. It was predicted by employing the available algorithms that the replacement of a glutamine by lysine at position 3 could appreciably reduce its aggregation propensity in an aqueous environment. In order to investigate the structural, functional, and biological consequences of replacement of glutamine by lysine at its third position, TempL and the corresponding analog, Q3K-TempL, was synthesized and characterized. Introduction of the lysine residue significantly promoted the self-assembly and oligomeric state of TempL in lipopolysaccharide (LPS). Q3K-TempL exhibited augmented binding to LPS and also dissociated LPS aggregates with greater efficacy than TempL. Further, Q3K-TempL inhibited the LPS-induced proinflammatory cytokines in rat primary macrophages in vitro and in vivo in BALB/c mice with greater efficacy than TempL. The results showed that a simple amino acid substitution in a short hydrophobic antimicrobial peptide, TempL, enhanced its antiendotoxin properties and illustrate a plausible correlation between its aggregation properties in LPS and LPS detoxification activity.

Sialylation controls prion fate in vivo.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC. The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc. Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K+ channel with six transmembrane (S1–S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218–239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Molecular Dynamics Simulations of the Host Defense Peptide Temporin L and Its Q3K Derivative: An Atomic Level View from Aggregation in Water to Bilayer Perturbation

Temporin L (TempL) is a 13 residue Host Defense Peptide (HDP) isolated from the skin of frogs. It has a strong affinity for lipopolysaccharides (LPS), which is related to its high activity against Gram-negative bacteria and also to its strong tendency to neutralize the pro-inflammatory response caused by LPS release from inactivated bacteria. A designed analog with the Q3K substitution shows an enhancement in both these activities. In the present paper, Molecular Dynamics (MD) simulations have been used to investigate the origin of these improved properties. To this end, we have studied the behavior of the peptides both in water solution and in the presence of LPS lipid-A bilayers, demonstrating that the main effect through which the Q3K substitution improves the peptide activities is the destabilization of peptide aggregates in water.

Study of Microstructure, Tribological, Thermal and Mechanical Properties of Ultrahigh Molecular Weight Polyethylene (UHMWPE)/Copper Nanocomposite

Nanomaterials possess many special physical and chemical properties based on small size, surface and interface effects. The use of nanomaterials, as additives, will provide a well bonded interface that will enable polymer based nano composites to exhibit higher performance. Higher performance is very much influenced by various techniques of preparation of nanocomposite. Melt mixing technique involving a co‐rotating intermeshing twin screw extruder for preparation of nanocomposite is an effective processing method and can play an important role in preparation of nanocomposite with uniform mircostructure.Currently, there is considerable interest in ultra high molecular weight polyethylene (UHMWPE) because of their superior abrasion resistance, reduced coefficient of friction, enhanced work of fracture and improved moisture barrier. The UHMWPE/HDPE blend has drawn much attention as a promising human joint repair material because such a blend has higher wear resistance than neat UHMWPE.Keeping these advantage...

Gamma response study of radiation sensitive MOSFETs for their use as gamma radiation sensor

Continuous monitoring of gamma dose is important in various fields like radiation therapy, space-related research, nuclear energy programs and high energy physics experiment facilities. The present work is focused on utilization of radiation-sensitive Metal-Oxide-Semiconductor Field Effect Transistors (MOSFETs) to monitor gamma radiation doses. Static characterization of these detectors was performed to check their expected current-voltage relationship. Threshold voltage and transconductance per unit gate to source voltage (K factor) were calculated from the experimental data. The detector was exposed to gamma radiation in both, with and without gate bias voltage conditions, and change in threshold voltage was monitored at different gamma doses. The experimental data was fitted to obtain equation for dependence of threshold voltage on gamma dose. More than ten times increase in sensitivity was observed in biased condition (+3 V) compared to the unbiased case.