Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8⁺ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR⁺ cells within CD8⁺ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8⁺ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8⁺ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth.     

The Impact of Microbial Biotransformation of Catechin in Enhancing the Allelopathic Effects of Rhododendron formosanum

Rhododendron formosanum is distributed widely in the central mountains in Taiwan and the major allelopathic compound in the leaves has been identified as (-)-catechin, which is also a major allelochemical of an invasive spotted knapweed in North America. Soil microorganisms play key roles in ecosystems and influence various important processes, including allelopathy. However, no microorganism has been identified as an allelochemical mediator. This study focused on the role of microorganisms in the allelopathic effects of R. formosanum. The microorganism population in the rhizosphere of R. formosanum was investigated and genetic analysis revealed that the predominant genera of microorganisms in the rhizosphere of R. formosanum were Pseudomonas, Herbaspirillum, and Burkholderia. The dominant genera Pseudomonas utilized (-)-catechin as the carbon source and catalyzed the conversion of (-)-catechin into protocatechuic acid in vitro. The concentrations of allelochemicals in the soil were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry. The concentration of (-)-catechin in the soil increased significantly during the extreme rainfall in the summer season and suppressed total bacterial populations. Protocatechuic acid accumulation was observed while total bacterial populations increased abundantly in both laboratory and field studies. Allelopathic interactions were tested by evaluating the effects of different allelochemicals on the seed germination, radicle growth, and photosynthesis system II of lettuce. Protocatechuic acid exhibited higher phytotoxicity than (-)-catechin did and the effect of (-)-catechin on the inhibition of seed germination was enhanced by combining it with protocatechuic acid at a low concentration. This study revealed the significance of the allelopathic interactions between R. formosanum and microorganisms in the rhizosphere. These findings demonstrate that knowledge regarding the precise biotransformation process of (-)-catechin by microorganisms in the environment is necessary to increase our understanding of allelopathy.     

Phospholipid membrane-interaction of a peptide from S4 segment of KvAP K(+) channel and the influence of the positive charges and an identified heptad repeat in its interaction with a S3 peptide

In order to examine the ability of S3 and S4 segments of a Kv channel to interact with each other, two wild type short peptides derived from the S3 and S4 segments of KvAP channel were synthesized. Additionally, to evaluate the role of positive charges and an identified heptad repeat in the S4 segment, two S4 mutants of the same size as the S4 peptide, one with substitution of two leucine residues in the heptad repeat sequence by two alanine residues and in the other two arginine residues replaced by two glutamines residues were synthesized. Our results show that only the wild type S4 peptide, but not its mutants, self-assembled and permeabilized negatively charged phospholipid vesicles. The S3 peptide showed lesser affinity toward the same kind of lipid vesicles and localized onto its surface. However, the S3 peptide interacted only with S4 wild type peptide, but not with S4 mutants, and altered its localization onto the phospholipid membrane with increased resistance against the proteolytic enzyme, proteinase-k, in the presence of the S4 peptide. The results demonstrate that the selected, synthetic S3 and S4 segments possess the required amino acid sequences to interact with each other and show that the positive charges and the identified heptad repeat in S4 contribute to its assembly and interaction with S3 segment.     

Studies on the assembly of a leucine zipper antibacterial peptide and its analogs onto mammalian cells and bacteria

Membrane-interaction and assembly of a leucine zipper peptide (LZP), and its single (SASA) and double (DASA) alanine-substituted analog onto mammalian, hRBCs and 3T3 cells and bacteria, Escherichia coli and Staphylococcus aureus were studied as a model system to understand the plausible role of assembly on their contrasting cytotoxic but similar bactericidal activities. Peptides’ ability to depolarize and damage the membrane organization of hRBC and 3T3 cells decreased from LZP to SASA and to DASA which may be related to their decrease in assembly onto these mammalian live cells and oligomerization states in the presence of these cell membranes or zwitterionic PC/Chol lipid vesicles. However, LZP and its analogs showed appreciable similarities in damaging or depolarizing the E. coli or S. aureus cells, which further matched with their comparable assembly and oligomerization either onto these live cells or the cell membranes or in the presence of negatively charged PC/PG lipid vesicles.     

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.     

Arsenic Uptake by Lemna minor in Hydroponic System

Arsenic is hazardous and causes several ill effects on human beings. Phytoremediation is the use of aquatic plants for the removal of toxic pollutants from external media. In the present research work, the removal efficiency as well as the arsenic uptake capacity of duckweed Lemna minor has been studied. Arsenic concentration in water samples and plant biomass were determined by AAS. The relative growth factor of Lemna minor was determined. The duckweed had potential to remove as well as uptake arsenic from the aqueous medium. Maximum removal of more than 70% arsenic was achieved at initial concentration of 0.5 mg/l arsenic on 15th day of experimental period of 22 days. Removal percentage was found to decrease with the increase in initial concentration. From BCF value, Lemna minor was found to be a hyperaccumulator of arsenic at initial concentration of 0.5 mg/L, such that accumulation decreased with increase in initial arsenic concentration.     

Theoretical studies on the pyridoxal-5'-phosphate dependent enzyme dopa decarboxylase: effect of thr 246 residue on the co-factor-enzyme binding and reaction mechanism

Decarboxylation of amino acid is a key step for biosynthesis of several important cellular metabolites in the biological systems. This process is catalyzed by amino acid decarboxylases and most of them use pyridoxal-5'-phosphate (PLP) as a co-factor. PLP is bound to the active site of the enzyme by various interactions with the neighboring amino acid residues. In the present investigation, density functional theory (DFT) and real-time dynamics studies on both ligand-free and ligand-bound dopa decarboxylases (DDC) have been carried out in order to elucidate the factors responsible for facile decarboxylation and also for proper binding of PLP in the active site of the enzyme. It has been found that in the crystal structure Asp271 interacts with the pyridine nitrogen atom of PLP through H-bonding in both native and substrate-bound DDC. On the contrary, Thr246 is in close proximity to the oxygen of 3-OH ofPLP pyridine ring only in the substrate-bound DDC. In the ligand-free enzyme, the distance between the oxygen atom of 3-OH group of PLP pyridine ring and oxygen atom of Thr246 hydroxyl group is not favorable for hydrogen bonding. Thus, present study reveals that hydrogen bonding with 03 of PLP with a hydrogen bond donor residue provided by the enzyme plays an important role in the decarboxylation process.     

Synergistic effect of salt mixture on the gelation temperature and morphology of methylcellulose hydrogel

Gelation temperature of methylcellulose (MC) can be altered by adding different additives. Pure MC showed sol-gel transition at 60°C. Sodium citrate and sodium tartrate were used alone and in combination to see the effect of individual salt and combination of salts on the gelation temperature of MC. The gelation temperature of all the binary and ternary combinations of MC and salts were measured with different methods such as test tube tilting method (TTM), UV-vis spectroscopy, viscometry, and by rheometer and also the morphology of gels were characterized with the help of environmental scanning electron microscopy (ESEM). It was observed that when 0.1M sodium citrate (NaC) and 0.1M sodium tartrate (NaT) were used separately, the gelation temperature of MC was reduced up to 44°C and 47°C respectively but when mixture of NaC and NaT (0.1(M) NaC and 0.1(M) (NaT)) were used the gelation temperature was further reduced to 36°C. It was clear from ESEM images that when NaC and NaT were used separately the formation of network was not distinguishable. But, well-connected network structure was observed when a mixture 0.1M NaC and 0.1M NaT was used.     

Effect of PVA on the gel temperature of MC and release kinetics of KT from MC based ophthalmic formulations

The effect of molecular weight of poly(vinyl alcohol) (PVA) and sodium chloride on the gelation temperature of methylcellulose (MC) was studied with the objective to develop a MC based formulation for sustained delivery of ketorolac tromethamine a model ophthalmic drug. Pure MC showed sol-gel transition at 61.2 °C. In order to reduce the gelation temperature of MC and to increase the drug release time, PVA was used. Different techniques such as test tube tilting method, UV-vis spectroscopy, viscometry and rheometry were used to measure gelation temperature of all the binary combinations of MC and PVA. It was observed that the gelation temperature of MC was reduced with the addition of 4% PVA and also the extent of reduction of the gelation temperature of MC was dependent on the molecular weight of PVA. The strong interactions between MC and PVA molecules were established using Fourier transform infrared spectroscopy. To study the in vitro drug release properties of the MC-PVA binary combinations, 6% sodium chloride was used to reduce the gelation temperature further up to physiological temperature. It was observed that the drug release time increased from 5 to 8h with the increase of molecular weight of PVA from 9×10(3) to 1.3×10(5) and this was due to the higher viscosity, better gel strength and greater interactions between the drug and PVA molecules in case of PVA (1.3×10(5)) compared to PVA (9×10(3)). In order to have an idea about the nature of interactions between the functional moieties of the drug and the polymer unit of PVA, a theoretical study was carried out.