In silico discrimination of nsSNPs in hTERT gene by means of local DNA sequence context and regularity

Understanding and predicting the significance of novel genetic variants revealed by DNA sequencing is a major challenge to integrate and interpret in medical genetics with medical practice. Recent studies have afforded significant advances in characterization and predicting the association of single nucleotide polymorphisms in human TERT with various disorders, but the results remain inconclusive. In this context, a comparative study between disease causing and novel mutations in hTERT gene was performed computationally. Out of 59 missense mutations, five variants were predicted to be less stable with the most deleterious effect on hTERT gene by in silico tools, in which two mutations (L584W and M970T) were not previously reported to be involved in any of the human disorders. To get insight into the structural and functional impact due to the mutation, docking study and interaction analysis was performed followed by 6 ns molecular dynamics simulation. These results may provide new perspectives for the targeted drug discovery in the coming future.     

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

Cover Image,Volume 88, Issue 6

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (¹⁴²STN¹⁴⁴) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (¹⁴¹RSTN¹⁴⁴). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.     

Signaling role of nitric oxide in the induction of plant defense by exogenous application of abiotic inducers

The objective of this work was to search out the probable molecule behind the activation of broad spectrum resistance during abiotic elicitors such as arachidonic acid, cupric chloride, chitosan, isonicotinic acid and salicylic acid mediated induced systemic resistance (ISR) in Raphanus sativus L. The elicitor compounds were sprayed on the radish leaves of healthy plant and after 24 h incubation a significant increase of β-1,3 glucanase, peroxidase, polyphenol oxidase and phenolics as well as a remarkable increase of nitric oxide (NO), a probable potent defense-signaling molecule in plant, was observed. Furthermore, treatment of the host with NO donor, sodium nitroprusside, also induced the same defense molecules. The results suggests that NO might be the signaling molecule during abiotic elicitor mediated ISR induction in the host system.     

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.     

Magnetopriming circumvents the effect of salinity stress on germination in chickpea seeds

Chickpea seeds of Pusa 1053 (Mediterranean) and Pusa 256 (native) were magnetoprimed with 100 mT static magnetic field for 1 h to evaluate the effect of magnetopriming on germination of seeds under saline conditions. Enhanced rate of germination and seedling growth parameters (root and shoot length, and vigour indices) under different salinity levels indicated that magnetopriming was more effective in alleviating salinity stress at early seedling stage in Pusa 1053 as compared to Pusa 256. Dynamics of seed water absorption in magnetoprimed seeds showed increased water uptake in Pusa 1053 under non-saline as compared to saline conditions. This could have resulted in faster hydration of enzymes in primed seeds leading to higher rate of germination. Total amylase, protease and dehydrogenase activities were higher in primed seeds as compared to unprimed seeds under both non-saline and saline conditions. Production of superoxide radicals was enhanced in germinating seeds of both the genotypes under salinity irrespective of priming. Increased levels of hydrogen peroxide in germinating magnetoprimed seeds, under both the growing conditions, suggested its role in promotion of germination. Our results showed that magnetopriming of dry seeds of chickpea can be effectively used as a pre-sowing treatment for mitigating adverse effects of salinity at seed germination and early seedling growth.     

An ecological perspective on marine reserves in prey–predator dynamics

This paper describes a prey–predator type fishery model with prey dispersal in a two-patch environment, one of which is a free fishing zone and other is a protected zone. The existence of possible steady states, along with their local stability, is discussed. A geometric approach is used to derive the sufficient conditions for global stability of the system at the positive equilibrium. Relative size of the reserve is considered as control in order to study optimal sustainable yield policy. Subsequently, the optimal system is derived and then solved numerically using an iterative method with Runge–Kutta fourth-order scheme. Numerical simulations are carried out to illustrate the importance of marine reserve in fisheries management. It is noted that the marine protected area enables us to protect and restore multi-species ecosystem. The results illustrate that dynamics of the system is extremely interesting if simultaneous effects of a regulatory mechanism like marine reserve is coupled with harvesting effort. It is observed that the migration of the resource, from protected area to unprotected area and vice versa, is playing an important role towards the standing stock assessment in both the areas which ultimately control the harvesting efficiency and enhance the fishing stock up to some extent.     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of α_vβ₃ expression in malignant tumors. The integrin α_vβ₃ binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a ^99mTc labeled dimeric RGD derivative using a RGD monomer and [^99mTcN]⁺² intermediate. The dithiocarbamate derivative of cyclic RGD peptide G₃-c(RGDfK) (G₃ = Gly-Gly-Gly, f = Phe, K = Lys) was synthesized and radiolabeled with [^99mTcN]⁺² intermediate to form the ^99mTcN-[G₃-c(RGDfK)]₂ complex in high yield (～98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61 ± 0.04% IA/g at 30 min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.