Search for lectins in seeds of tropical trees of Kerala, India

Tissue specific plant lectins were searched in the seeds of 44 tropical trees of Kerala, India. Seeds of only 12 plant species showed lectin activity. N-acetyl-D-galactosamine was the best inhibitor of lectin activity for the majority of the seeds. Lectin activity in the seeds of 4 species were not inhibited by any of the mono- or polysaccharides used. This revised version was published online in July 2006 with corrections to the Cover Date.     

Homology modeling and atomic level binding study of Leishmania MAPK with inhibitors

The current therapy for leishmaniasis is not sufficient and it has two severe drawbacks, host-toxicity and drug resistance. The substantial knowledge of parasite biology is not yet translating into novel drugs for leishmaniasis. Based on this observation, a 3D structural model of Leishmania mitogen-activated protein kinase (MAPK) homologue has been developed, for the first time, by homology modeling and molecular dynamics simulation techniques. The model provided clear insight in its structure features, i.e. ATP binding pocket, phosphorylation lip, and common docking site. Sequence-structure homology recognition identified Leishmania CRK3 (LCRK3) as a distant member of the MAPK superfamily. Multiple sequence alignment and 3D structure model provided the putative ATP binding pocket of Leishmania with respect to human ERK2 and LCRK3. This analysis was helpful in identifying the binding sites and molecular function of the Leishmania specific MAPK homologue. Molecular docking study was performed on this 3D structural model, using different classes of competitive ATP inhibitors of LCRK3, to check whether they exhibit affinity and could be identified as Leishmania MAPK specific inhibitors. It is well known that MAP kinases are extracellular signal regulated kinases ERK1 and ERK2, which are components of the Ras-MAPK signal transduction pathway which is complexed with HDAC4 protein, and their inhibition is of significant therapeutic interest in cancer biology. In order to understand the mechanism of action, docking of indirubin class of molecules to the active site of histone deacetylase 4 (HDAC4) protein is performed, and the binding affinity of the protein-ligand interaction was computed. The new structural insights obtained from this study are all consistent with the available experimental data, suggesting that the homology model of the Leishmania MAPK and its ligand interaction modes are reasonable. Further the comparative molecular electrostatic potential and cavity depth analysis of Leishmania MAPK and human ERK2 suggested several important differences in its ATP binding pocket. Such differences could be exploited in the future for designing Leishmania specific MAPK inhibitors.     

Manganese and cobalt recovery by surface display of metal binding peptide on various loops of OmpC in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In a cell-surface display (CSD) system, successful display of a protein or peptide is highly dependent on the anchoring motif and the position of the display in that anchoring motif. In this study, a recombinant bacterial CSD system for manganese (Mn) and cobalt (Co) recovery was developed by employing OmpC as an anchoring motif on three different external loops. A portion of Cap43 protein (TRSRSHTSEG)₃ was employed as a manganese and cobalt binding peptide (MCBP), which was fused with OmpC at three different external loops. The fusions were made at the loop 2 [fusion protein-2 (FP2)], loop 6 (FP6), and loop 8 (FP8) of OmpC, respectively. The efficacy of the three recombinant strains in the recovery of Mn and Co was evaluated by varying the concentration of the respective metal. Molecular modeling studies showed that the short trimeric repeats of peptide probably form a secondary structure with OmpC, thereby giving rise to a difference in metal recovery among the three recombinant strains. Among the three recombinant strains, FP6 showed increased metal recovery with both Mn and Co, at 1235.14 (1 mM) and 379.68 (0.2 mM) µmol/g dry cell weight (DCW), respectively.     

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment

1. Download high-res image (200KB)
2. Download full-size image

     

Immunogenic gangliosides in human ovarian carcinoma

Ganglioside signatures of four poorly and three moderately differentiated ovarian epithelial cancer (OEC) cell lines reveal the presence of GM3, GM2, GD2, O-AcGD2, GD1a and GM1b. The expression of GM3, presence of GD1a and GM1b in the ascitic fluid and plasma, together with a positive correlation in the total-gangliosides levels between ascitic fluid and plasma of OEC patients support the earlier contention that the tumor-gangliosides may be released (or shed) into the tumor-microenvironment. The immunogenicity of OEC-gangliosides is determined by comparing anti-ganglioside-IgM titers in ascitic fluid (n = 14) and plasma (n = 23) of OEC-patients and age-matched healthy (n = 14). The titers were measured by ELISA. Strikingly, the level of anti-GD1a-IgM is significantly higher in ascitic fluid and plasma of patients than in the plasma of healthy volunteers. Paired sample analysis of ascitic fluid and plasma from the same patients confirmed the significant expression of anti-GD1a IgM in OEC patients, while no such difference was observed with other anti-ganglioside IgMs among different groups. The significance of the endogenous IgM response to GD1a may be to eliminate this immunosuppressive-ganglioside from the tumor-microenvironment.     

Substituting dietary linoleic acid with alpha-linolenic acid improves insulin sensitivity in sucrose fed rats

This study describes the effect of substituting dietary linoleic acid (18:2 n-6) with alpha-linolenic acid (18:3 n-3) on sucrose-induced insulin resistance (IR). Wistar NIN male weanling rats were fed casein based diet containing 22 energy percent (en%) fat with approximately 6, 9 and 7 en% saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) respectively for 3 months. IR was induced by replacing starch (ST) with sucrose (SU). Blends of groundnut, palmolein, and linseed oil in different proportions furnished the following levels of 18:3 n-3 (g/100 g diet) and 18:2 n-6/18:3 n-3 ratios respectively: ST-220 (0.014, 220), SU-220 (0.014, 220), SU-50 (0.06, 50), SU-10 (0.27, 10) and SU-2 (1.1, 2). The results showed IR in the sucrose fed group (SU-220) as evidenced by increase in fasting plasma insulin and area under the curve (AUC) of insulin in response to oral glucose load. In SU-220, the increase in adipocyte plasma membrane cholesterol/phospholipid ratio was associated with a decrease in fluidity, insulin stimulated glucose transport, antilipolytic effect of insulin and increase in basal and norepinephrine stimulated lipolysis in adipocytes. In SU-50, sucrose induced alterations in adipocyte lipolysis and antilipolysis were normalized. However, in SU-2, partial corrections in plasma insulin, AUC of insulin and adipocyte insulin stimulated glucose transport were observed. Further, plasma triglycerides and cholesterol decreased in SU-2. In diaphragm phospholipids, the observed dose dependent increase in long chain (LC) n-3 PUFA was associated with a decrease in LC-n-6 PUFA but insulin stimulated glucose transport increased only in SU-2. Thus, this study shows that the substitution of one-third of dietary 18:2 n-6 with 18:3 n-3 (SU-2) results in lowered blood lipid levels and increases peripheral insulin sensitivity, possibly due to the resulting high LCn-3 PUFA levels in target tissues of insulin action. These findings suggest a role for 18:3 n-3 in the prevention of insulin resistant states. The current recommendation to increase 18:3 n-3 intake for reducing cardiovascular risk may also be beneficial for preventing IR in humans.     

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.     

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.     

Modulatory effect of sesamol on DOCA-salt-induced oxidative stress in uninephrectomized hypertensive rats

The present study was undertaken to investigate the antihypertensive and antioxidant effects of sesamol on uninephrectomized deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats. Hypertension was induced in surgically single-kidney-removed (left) adult male albino Wistar rats, weighing 180–200 g, by injecting DOCA (25 mg/kg BW) subcutaneously twice a week for 6 weeks, with saline instead of tap water for drinking. Rats were treated with three different doses of sesamol (50, 100 and 200 mg/kg BW) post-orally by gavage daily for 6 weeks. Hypertension was revealed by increased systolic and diastolic blood pressure and the toxicity of DOCA-salt was determined using hepatic marker enzymes, aspartate aminotransferase, alanine aminotransferase, alkaline phospatase and gamma-glutamyl transpeptidase; and, lipid peroxidative markers, thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes were assayed. The activities of enzymatic antioxidants, superoxide dismutase, catalase and glutathione peroxidase and the levels of non-enzymatic antioxidants (vitamin C, vitamin E and reduced glutathione) were evaluated in erythrocytes, plasma and tissues. Post-oral administration of sesamol at the dosage of 50 mg/kg BW remarkably decreased systolic and diastolic blood pressure, hepatic marker enzyme activities and lipid peroxidation products and also enhanced the antioxidant activity. The biochemical observations were also supported by histopathological examinations of the rat liver, kidney and heart sections. These results suggest that sesamol possesses antihypertensive and antioxidant effects.