Phytochemical and Biological Activities of Pseudocalymma elegans: A False Garlic

Evaluation of phytochemical constituents and antioxidant and antimicrobial activities of hexane (PELH), dichloromethane (PELDCM), ethyl acetate (PELEA), and MeOH (PELM) extracts of young leaves of Pseudocalymma elegans have been carried out. Moreover, extracts have also been explored for the presence of sulphur containing compounds, 1,2‐dithiolane ( 33 ), diallyl disulfide ( 35 ), 3‐vinyl‐1,2‐dithiacyclohex‐5‐ene ( 37 ), and diallyl trisulfide ( 38 ) responsible for the garlic like smell of P. elegans. All the extracts were found to be antioxidant and showed potent inhibition with IC₅₀ values of 0.168 ± 0.001, 0.128 ± 0.002, 0.221 ± 0.011, and 0.054 ± 0.001, respectively, as compared to standard drugs ascorbic acid (AA) and butylated hydroxytoluene (BHT). The ethyl acetate extract (PELE) showed excellent activities against few Gram‐positive and Gram‐negative bacteria and some fungi as compared with standard drug ceftriaxone (3rd generation cephalosporin) and nystatin, respectively. Chemical constituents of hexane, dichloromethane, and ethyl acetate extracts were identified by gas chromatography‐mass spectrometry and mass spectral library search. Over all 55 chemical constituents were first time identified from the leaves which included branched and n‐hydrocarbons, fatty acids, fatty acid methyl esters, fatty alcohols, terpenes, alkaloid, vitamins, glycosides, aromatic compounds, and sulfur containing compounds. Two known chemical constituents, ursolic acid ( 1 ) and β‐amyrin ( 2 ), were also purified for the first time from the MeOH extract. To elucidate the structures of these compounds, UV, IR, EI‐MS, ¹H‐ and ¹³C‐NMR spectroscopy were used.     

Immunogenicity of mitochondrial DNA modified by hydroxyl radical

Mitochondria consume about 90 percent of oxygen used by the body, and are a particularly rich source of reactive oxygen species (ROS). In this research communication mitochondrial DNA (mtDNA) was isolated from fresh goat liver and modified in vitro by hydroxyl radical generated from UV irradiation (254 nm) of hydrogen peroxide. As a consequence of hydroxyl radical modification, mtDNA showed hyperchromicity and sensitivity to nuclease S1 digestion as compared to control mtDNA. Animals immunized with mtDNA and ROS-modified mtDNA induced antibodies as detected by direct binding and competition ELISA. The data suggest that immunogenicity of mtDNA got augmented after treatment with hydroxyl radical. IgG isolated from immune sera showed specificity for respective immunogen and cross-reaction with other nucleic acids. Binding of induced antibodies with array of antigens clearly indicates their polyspecific nature. Moreover, the polyspecificity exhibited by induced antibodies is unique in view of similar multiple antigen binding properties of naturally occurring anti-DNA antibodies derived from SLE patients.     

Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Heterogeneity of Equilibrium Molten Globule State of Cytochrome c Induced by Weak Salt Denaturants under Physiological Condition

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO₄) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε ₄₀₀) and CD ([θ]₄₀₉), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO₄ and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO₄ and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.     

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin,jacalin

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1β, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.     

Isolation and evaluation of probiotic potential of lactic acid bacteria isolated from poultry intestine

The gut is a source of lactic acid bacteria with remarkable functional and technologies properties as well as a potential source of probiotics. In the present study, 13 strains of Lactobacillus were isolated from poultry intestine and identified according to their 16S rDNA sequences, as well as the evaluation of their probiotic potential. The probiotic properties were tested in aspects of antibiotic susceptibility, antimicrobial activity, exopolysaccharide production, lysozyme tolerance, gut condition tolerance (low pH, bile salt tolerance) and adhesion to human colorectal adenocarcinoma cell line (Caco-2). Most isolates were resistance to streptomycin (10 μg/mL), gentamicin (10 μg/mL), kanamycin (30 μg/mL), penicillin (10 μg/mL) and chloramphenicol (30 μg/mL). Isolates shows strong abilities to adhere to Caco-2 cell in the range of (76 to 85%). Isolates SHA101 to SHA113 showed high survival rate under gastrointestinal tract condition (>80%), indicating their potential in application of probiotics. The results of these tests indicate that the lactic acid bacteria isolated from poultry intestine have potential use as probiotic in various products.     

Protein modeling,molecular network and molecular dynamics study of newly sequenced interleukin-18 (IL-18) gene in Mus musculus

Interleukin-18 (IL-18) belongs to the superfamily of IL-1 protein and exerts a pleiotropic pro-inflammatory effect on the body. Generally, this protein is significantly involved in immune defense during infection in cells, but sometimes its anomalous activities produce some inflammatory diseases like rheumatoid arthritis and Crohn’s disease. In the present study, the IL-18 gene was isolated from mice and was subsequently cloned and sequenced. Further, the network analysis was carried out to explore the functional role of IL-18 protein in animals. The 3D protein structure of the IL-18 protein was generated and docked with appropriate 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CPS) ligand. Later the complex structure of the protein was subjected to molecular dynamics simulation (MDS) for 50 ns to determine the effect of ligand on protein. The network analysis explored the correlation of IL-18 protein with others proteins and their involvement in the different significant pathway to defend the cell from various diseases. As confirmed by MDS, the CPS:IL-18 complex was found to be highly stable. Our results further indicated that CPS ligand has the potential to act as a drug molecule, in future, for counteracting IL-18 activity. To date, no structural details were available for animal IL-18. Hence, the finding of this study will be useful in broadening the horizon towards a better understanding of the functional and structural aspects of IL-18 in animals.     

Eukaryotic initiation factor 4E (eIF4E): A recap of the cap-binding protein

Eukaryotic initiation factor 4E (eIF4E), a fundamental effector and rate limiting element of protein synthesis, binds the 7-methylguanosine cap at the 5′ end of eukaryotic messenger RNA (mRNA) specifically as a constituent of eIF4F translation initiation complex thus facilitating the recruitment of mRNA to the ribosomes. This review focusses on the engagement of signals contributing to growth factor originated maxim and their role in the activation of eIF4E to achieve a collective influence on cellular growth, with a key focus on conjuring vital processes like protein synthesis. The review invites considerable interest in elevating the appeal of eIF4E beyond its role in regulating translation viz a viz cancer genesis, attributed to its phosphorylation state that improves the prospect for the growth of the cancerous cell. This review highlights the latest studies that have envisioned to target these pathways and ultimately the translational machinery for therapeutic intervention. The review also brings forward the prospect of eIF4E to act as a converging juncture for signaling pathways like mTOR/PI3K and Mnk/MAPK to promote tumorigenesis.     

Molecular Characterization and Phylogenetic Relationship of Wild Type 1 Poliovirus Strains Circulating across Pakistan and Afghanistan Bordering Areas during 2010–2012

Pakistan and Afghanistan share a long uncontrolled border with extensive population movement on both sides. Wild poliovirus transmission has never been interrupted in this block due to war against terrorism, poor public health infrastructure, misconceptions about polio vaccines and inadequate immunization activities. All these issues complicate the eradication operations and reinforce the complexity of wiping out poliomyelitis from this region. This study illustrates the origins and routes of cross-border wild poliovirus type 1 (WPV1) transmission during 2010–2012 between Pakistan and Afghanistan. Sequence analyses were conducted based on complete VP1 capsid protein sequences for WPV1 study strains to determine the origin of poliovirus genetic lineages and their evolutionary relationships. Phylogenetic tree was constructed from VP1 gene sequences applying Maximum Likelihood method using Kimura 2- parameter model in MEGA program v 5.0. A total of 72 (14.3%) out of 502 wild-type 1 polioviruses were found circulating in border areas of both countries during 2010–2012. Molecular phylogenetic analysis classified these strains in to two sub-genotypes with four clusters and 18 lineages. Genetic data confirmed that the most of WPV1 lineages (12; 66.6%) were transmitted from Pakistan to Afghanistan. However, the genetic diversity was significantly reduced during 2012 as most of the lineages were completely eliminated. In conclusion, Pakistan-Afghanistan block has emerged as a single poliovirus reservoir sharing the multiple poliovirus lineages due to uncontrolled movement of people across the borders between two countries. If it is neglected, it can jeopardize the extensive global efforts done so-far to eradicate the poliovirus infection. Our data will be helpful to devise the preventive strategies for effective control of wild poliovirus transmission in this region.