Homing of radiolabelled recombinant interleukin-2 activated natural killer cells and their efficacy in adoptive immunotherapy against murine fibrosarcoma

Natural killer (NK) cells are spontaneously cytotoxic against tumour target cells. Their number was found to be four times more in the spleen of tumour-bearing Swiss albino mice. After activation with recombinant interleukin-2 (rIL-2), NK cells were tested and found to seek out the tumour site when injected intravenously in tumour-bearing mice. Their potential for fighting tumours in vivo was further seen following adoptive transfer of rIL-2 activated NK (A-NK) cells in tumour-bearing mice. After surgical removal of tumour load, adoptive transfer of A-NK cells inhibited tumour recurrence in 92.3% cases, thereby suggesting the use of this protocol for therapeutic purposes to obtain a better outcome.     

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

Viper and cobra venom neutralization by β-sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae)

We reported previously that the methanolic root extract of the Indian medicinal plant Pluchea indica Less. (Asteraceae) could neutralize viper venom-induced action [Alam, M.I., Auddy, B., Gomes, A., 1996. Viper venom neutralization by Indian medicinal plant (Hemidesmus indicus and P. indica) root extracts. Phytother. Res. 10, 58-61]. The present study reports the neutralization of viper and cobra venom by beta-sitosterol and stigmasterol isolated from the root extract of P. indica Less. (Asteraceae). The active fraction (containing the major compound beta-sitosterol and the minor compound stigmasterol) was isolated and purified by silica gel column chromatography and the structure was determined using spectroscopic analysis (EIMS, (1)H NMR, (13)C NMR). Anti-snake venom activity was studied in experimental animals. The active fraction was found to significantly neutralize viper venom-induced lethal, hemorrhagic, defibrinogenation, edema and PLA(2) activity. Cobra venom-induced lethality, cardiotoxicity, neurotoxicity, respiratory changes and PLA(2) activity were also antagonized by the active component. It potentiated commercial snake venom antiserum action against venom-induced lethality in male albino mice. The active fraction could antagonize venom-induced changes in lipid peroxidation and superoxide dismutase activity. This study suggests that beta-sitosterol and stigmasterol may play an important role, along with antiserum, in neutralizing snake venom-induced actions.     

Novel molecular insights into the anti‐oxidative stress response and structure–function of a salt‐inducible cyanobacterial Mn‐catalase

KatB, a salt‐inducible Mn‐catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological–biochemical function of KatB at the molecular level. Anabaena overexpressing the wild‐type KatB protein (KatBWT) detoxified H₂O₂ efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F‐2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X‐ray crystallography‐based analysis showed that F‐2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F‐2, through its interaction with F‐66 and W‐43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn‐catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.     

Polymorphism of growth hormone gene and its association with seminal and sexual behavioral traits in crossbred cattle

The decline in the male reproductive ability in terms of sexual behavior and seminal traits might lead to nonavailability of required number of bulls in a progeny testing program. The present study was conducted in 493 crossbred cattle (Bos taurus × Bos indicus) bulls to study polymorphisms of growth hormone (GH) gene and its association with seminal and sexual behavioral characteristics. A 428-base pair fragment of GH gene spanning over the fourth exon, fourth intron, and fifth exon was amplified and digested with AluI restriction enzyme. Bulls were found to be polymorphic, with two variants, LL and LV, and higher genotypic frequency for LL being 0.88. Twelve nucleotide changes and a single nonsynonymous substitution of Leucine by Valine were observed from GH1 (L) to GH2 (V). Statistical analysis revealed that the genotype of the GH gene had a significant effect on libido score, reaction time, Flehmen response, requirement of mounting stimulus, sperm mass activity, number of semen doses per collection, individual fresh sperm motility, postthaw sperm motility, acrosome integrity, hypo-osmotic swelling test, live and dead count, total morphological abnormality, and head abnormality of sperm in crossbred bulls. Growth hormone gene might be considered a candidate gene for seminal and sexual behavioral traits in crossbred cattle.     

Oxidative stress modulates theophylline effects on steroid responsiveness

Oxidative stress is a central factor in many chronic inflammatory diseases such as severe asthma and chronic obstructive pulmonary disease (COPD). Oxidative stress reduces the anti-inflammatory corticosteroid action and may therefore contribute to the relative corticosteroid insensitivity seen in these diseases. Low concentrations of theophylline can restore the anti-inflammatory action of corticosteroids in oxidant exposed cells, however the mechanism remains unknown. Here, we demonstrate that a low concentration of theophylline restores corticosteroid repression of pro-inflammatory mediator release and histone acetylation in oxidant exposed cells. Global gene expression analysis shows that theophylline regulates distinct pathways in naïve and oxidant exposed cells and reverses oxidant mediated modulated of pathways. Furthermore, quantitative chemoproteomics revealed that theophylline has few high affinity targets in naive cells but an elevated affinity in oxidant stressed cells. In conclusion, oxidative stress alters theophylline binding profile and gene expression which may result in restoration of corticosteroid function.     

Residue depth: a novel parameter for the analysis of protein structure and stability.

BACKGROUND: Accessible surface area is a parameter that is widely used in analyses of protein structure and stability. Accessible surface area does not, however, distinguish between atoms just below the protein surface and those in the core of the protein. In order to differentiate between such buried residues we describe a computational procedure for calculating the depth of a residue from the protein surface. RESULTS: Residue depth correlates significantly better than accessibility with effects of mutations on protein stability and on protein-protein interactions. The deepest residues in the native state invariably undergo hydrogen exchange by global unfolding of the protein and are often significantly protected in the corresponding molten-globule states. CONCLUSIONS: Depth is often a more useful gage of residue burial than accessibility. This is probably related to the fact that the protein interior and surrounding solvent differ significantly in polarity and packing density. Hence, the strengths of van der Waals and electrostatic interactions between residues in a protein might be expected to depend on the distance of the residue(s) from the protein surface.     

What signals do herbivore-induced plant volatiles provide conspecific herbivores?

Herbivore-induced plant volatiles (HIPVs) have been opined as ‘indirect or direct defenses’ of plants and are extensively studied. In contrast, HIPVs may also indicate that plant defenses have been overcome by herbivores infesting the plant; however, studies on this aspect have so far received little attention. Using the interaction of Capsicum annum (Bell pepper) with its pest Scirtothrips dorsalis (Chilli thrips) as a model system, we studied the role of HIPVs in this selected insect–plant interaction. Multiple-choice olfactometer assays with headspace volatiles collected from different growth stages of un-infested C. annum plants represented by pre-flowering (PF), flowering (FL) and fruiting stages (FR) proved FR volatiles to be highly attractive to S. dorsalis. Further, FR plants were infested with S. dorsalis adults and HIPVs released by infested plants were collected and subjected to multiple-choice olfactometer bioassays. Thrips were significantly attracted to HIPVs than to headspace volatiles of un-infested FR plants or thrips body odour. Coupled GC-EAG with S. dorsalis and HIPVs or FR plant volatile revealed specific compounds that elicited an EAG response. Individual EAG-active compounds were less attractive to thrips, however, synthetic blends of EAG-active compounds at the ratio similar to headspace samples were found to be highly attractive. However, when given a choice between synthetic blends of HIPVs and FR, thrips were significantly attracted to synthetic blend of HIPVs. Our study provides empirical data on signals HIPVs may provide to conspecific herbivores and suggests that the role of HIPVs, mostly generalized as defense, may vary based on the interaction and must be studied closely to understand their ecological functions.