Site-directed mutagenesis study of yeast peptide:N-glycanase. Insight into the reaction mechanism of deglycosylation.

Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases.     

Deciphering molecular aspects of interaction between anticancer drug mitoxantrone and tRNA

Mitoxantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione) is a synthetically designed antineoplastic agent and structurally similar to classical anthracyclines. It is widely used as a potent chemotherapeutic component against various kinds of cancer and possesses lesser cardio-toxic effects with respect to naturally occurring anthracyclines. In the present study, we have investigated the binding features of mitoxantrone–tRNA complexation at physiological pH using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and UV–visible absorption spectroscopic techniques. FTIR analysis reveals that mitoxantrone interacts mainly with heterocyclic base residues of tRNA along with slight external binding with phosphate–sugar backbone. In particular, mitoxantrone binds at uracil (C=O) and adenine (C=N) sites of biomolecule (tRNA). CD spectroscopic results suggest that there is no major conformational transition in native A-form of tRNA upon mitoxantrone–tRNA adductation except an intensification in the secondary structure of tRNA is evident. The association constant calculated for mitoxantrone–tRNA association is found to be 1.27?×?10⁵ M^?1 indicating moderate to strong binding affinity of drug with tRNA. Thermodynamically, mitoxantrone–tRNA interaction is an enthalpy-driven exothermic reaction. Investigation into drug–tRNA interaction can play an essential role in the rational development of RNA targeting chemotherapeutic agents, which also delineate the structural–functional relationship between drug and its target at molecular level.     

Spermidine and Melatonin Attenuate Fluoride Toxicity by Regulating Gene Expression of Antioxidants in <Emphasis Type="Italic">Cajanus cajan</Emphasis> L.

Being regulators of growth, both spermidine (Spd) and melatonin (Mel) are involved actively in the modulation of abiotic stress responses of plants. Hence, the present study was aimed to scrutinize the possible involvements of Spd and Mel in alleviation of fluoride ion (F^?)-induced injuries in Cajanus cajan L. Seeds of C. cajan L. were exposed to 1) control, 2) F^?, 3) Spd, 4) Spd?+?F^?, 5) Mel and 6) Mel?+?F^? for five days. The results unveiled that F^? treatment caused inhibited growth (radicle length and dry mass accumulation), protein content, genomic template stability, membrane stability index, and free radical scavenging capacity, but enhanced the levels of cell death, active oxygen species (AOS), malondialdehyde, lipase, protein carbonylation, and DNA polymorphism. Moreover, F^? toxicity elevated the concentrations of endogenous proline, ascorbic acid, and glutathione, and altered the isoenzyme profiles and gene expressions of stress responsive enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione-S-transferase). In contrast, exogenous supplementation of Spd and Mel alleviated the deleterious effects of F^?, consequently improved growth, free radical scavenging capacity, and accumulations of protein, proline, ascorbic acid, and glutathione in C. cajan L. Additionally, application of Spd or Mel also improved the isoenzyme profiles and gene expressions of stress responsive enzymes, and genomic template stability, thereby reduced cell death, AOS, lipid peroxidation, lipase activity, and DNA polymorphism in stressed tissues. The present study concludes that Spd and Mel, particularly Mel, alleviated the adverse impacts of F^? by improving antioxidant machinery and genomic template stability.     

Essential Role for the Mnk Pathway in the Inhibitory Effects of Type I Interferons on Myeloproliferative Neoplasm (MPN) Precursors

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.     

Photobacterium marinum sp. nov., a marine bacterium isolated from a sediment sample from Palk Bay,India

The novel, cream colored, Gram-staining-negative, rod-shaped, motile bacteria, designated strains AK15^T and AK18, were isolated from sediment samples collected from Palk Bay, India. Both strains were positive for arginine dihydrolase, lysine decarboxylase, oxidase, nitrate reduction and methyl red test. The major fatty acids were C_16:0, C_{18:1 ω7c}, C_{16:1 ω7c} and/or C_{16:1 ω6c} and/or iso-C_15:0 2-OH (summed feature 3). Polar lipids content of strains AK15^T and AK18 were found to bephosphatidylethanolamine (PE), two unidentified phospholipids (PL1 and PL2) and three unidentified lipids (L1-L3). The 16S rRNA gene sequence analysis indicated strains AK15^T and AK18 as the members of the genus Photobacterium and closely related to the type strain Photobacterium jeanii with pair-wise sequence similarity of 96.7%. DNA–DNA hybridization between strain AK15^T and AK18 showed a relatedness of 87%. Based on data from the current polyphasic study, strains AK15^T and AK18 are proposed as novel species of the genus Photobacterium, for which the name Photobacterium marinum sp. nov. is proposed. The type strain of Photobacterium marinum is AK15^T (=MTCC 11066^T = DSM 25368^T).     

Lunatimonas lonarensis gen. nov., sp. nov., a haloalkaline bacterium of the family Cyclobacteriaceae with nitrate reducing activity

Novel pinkish-orange pigmented, Gram-negative staining, half-moon shaped, non-motile, strictly aerobic strains designated AK24^T and AK26 were isolated from water and sediment samples of Lonar Lake, Buldhana district, Maharahstra, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids were iso-C15:0 (41.5%), anteiso-C15:0 (9.7%), iso-C17:0 3OH (9.6%), iso-C17:1 ω9c (10.2%) and C16:1 ω7c/C16:1 ω6c/iso-C15:0 2OH (summed feature 3) (14.4%). The strains contained MK-7 as the major respiratory quinone, and phosphatidylethanolamine and five unidentified lipids as the polar lipids. Blast analysis of the 16S rRNA gene sequence of strain AK24^T showed that it was closely related to Aquiflexum balticum, with a pair-wise sequence similarity of 91.6%, as well as to Fontibacter ferrireducens, Belliella baltica and Indibacter alkaliphilus (91.3, 91.2 and 91.2% pair-wise sequence similarity, respectively), but it only had between 88.6 and 91.0% pair-wise sequence similarity to the rest of the family members. The MALDI-TOF assay reported no significant similarities for AK24^T and AK26, since they potentially represented a new species. A MALDI MSP dendrogram showed close similarity between the two strains, but they maintained a distance from their phylogenetic neighbors. The genome of AK24^T showed the presence of heavy metal tolerance genes, including the genes providing resistance to arsenic, cadmium, cobalt and zinc. A cluster of heat shock resistance genes was also found in the genome. Two lantibiotic producing genes, LanR and LasB, were also found in the genome of AK24^T. Strains AK24^T and AK26 were very closely related to each other with 99.5% pair-wise sequence similarity. Phylogenetic analysis indicated that the strains were members of the family Cyclobacteriaceae and they clustered with the genus Mariniradius, as well as with the genera Aquiflexum, Cecembia, Fontibacter, Indibacter, and Shivajiella. DNA–DNA hybridization between strains AK24^T and AK26 showed a relatedness of 82% and their rep-PCR banding patterns were very similar. Based on data from the current polyphasic study, it is proposed that the isolates be placed in a new genus and species with the name Lunatimonas lonarensis gen. nov., sp. nov. The type strain of Lunatimonas lonarensis is AK24^T (=JCM 18822^T = MTCC 11627^T).     

Infrared-to-visible conversion in strontium sulphate through a defect-based infrared stimulated visible emission phenomenon

Strontium sulphate (SrSO₄) is a defect-based photoluminescence material, generally used in thermoluminescence applications, and has been studied for infrared (IR) stimulated visible emission. The SrSO₄ particles were synthesized using a precipitation method. The orthorhombic phase of SrSO₄ was confirmed from the X-ray diffraction pattern and the formation of micron-sized particles was authenticated from field emission scanning electron micrographs. The elemental composition of oxygen and strontium was determined using energy-dispersive X-ray analysis measurement that confirmed the presence of $V_O^{••}$ and $V_{\mathit{Sr}}^{\prime \prime }$ intrinsic defects in the material. Photoluminescence investigations showed the presence of various defect bands in the band gap giving rise to intrinsic luminescence in SrSO₄. The emission in the visible region was attributed to the defect band arising due to $V_O^{••}$. Photoluminescence lifetime measurement confirmed the presence of stable defect states with a lifetime in microseconds. The SrSO₄ sample was tested using IR lasers and a red–orange emission spot was observed from the powder sample when excited with IR lasers. The underlying principle for IR-to-visible conversion in the material is a defect-mediated phenomenon that has been described through the energy level diagram of the material.     

Development of chickpea EST-SSR markers and analysis of allelic variation across related species

Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.