Pockets of short-range transient order and restricted topological heterogeneity in the guanidine-denatured state ensemble of GED of dynamin

The nature and variety in the denatured state of a protein, a non-native state under a given set of conditions, has been a subject of intense debate. Here, using multidimensional NMR, we have characterized the 6 M Gdn-HCl-denatured state of GED, the assembly domain of dynamin. Even under such strongly denaturing conditions, we detected the presence of conformations in slow exchange on the NMR chemical shift time scale. Although the GED oligomer as well as the SDS-denatured monomeric GED were seen to be predominantly helical [Chugh et al. (2006) FEBS J. 273, 388-397], the 6 M Gdn-HCl-denatured GED has largely beta-structural preferences. However, against such a background, we could detect the presence of a population with a short helical stretch (Arg42-Ile47) in the ensemble. The 1H-1H NOEs suggested presence of pockets of transient short-range order along the chain. Put together these segments may lead to a rather small number of interconverting topologically distinguishable ensembles. Spectral density analysis of 15N relaxation rates and {1H}-15N NOE, measured at 600 and 800 MHz, and comparison of J(0) with hydrophobic patches calculated using AABUF approach, indicated presence of four domains of slow motions. These coincided to a large extent with those showing significant Rex. Additionally, a proline residue in the connection between two of these domains seems to cause a fast hinge motion. These observations help enhance our understanding of protein denatured states, and of folding concepts, in general.     

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl₂. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd²⁺ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd²⁺. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and plasmid profile of soil and clinical isolates of Nocardia.

The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for generic and species-specific differentiation of Nocardia from other morphologically similar bacterial pathogens. To examine the utility of the PCR-RFLP approach in species identification, genomic DNA was prepared from 40 soil isolates, 10 clinical isolates and 8 reference strains of Nocardia. A set of oligonucleotide primers was designed from the consensus sequence of the highly conserved groEL gene that encodes the 65-kDa heat shock protein (hsp 65). The primers selectively amplified 422 bp DNA from the genomic DNA of all Nocardia species and isolates. The digestion of the amplicons with the restriction enzyme MspI produced DNA fragments that could differentiate between different Nocardia species regardless of their origin. Additionally, the RFLP patterns obtained with restriction enzymes MspI and BsaHI resulted in the differentiation of six Nocardia species which were earlier identified by biochemical tests. Apart from soil isolates of N. asteroides, which had shown some degree of genotypic polymorphism with BsaHI, the remaining taxa yielded more consistent results. Our results on the isolation of plasmids indicated that their occurrence is not a consistent feature in Nocardia species. It is neither related to the source of origin (clinical versus saprobic), nor to virulence, anti-microbial resistance or species specificity.     

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P₂ region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure–activity relationship, and selectivity against related proteases are delineated.     

NMR insights into a megadalton-size protein self-assembly

Protein self-association is critical to many biological functions. However, atomic-level structural characterization of these assemblies has remained elusive. In this report we present insights into the mechanistic details of the process of self-association of the 136-residue GTPase effector domain (GED) of the endocytic protein dynamin into a megadalton-sized soluble mass. Our approach is based on NMR monitoring of regulated folding and association through Gdn-HCl titration. The results suggest the evolution of a sequence–self-association paradigm. Equally significantly, the study demonstrates an elegant bottom-up strategy that can render large protein self-assemblies accessible to NMR investigations that have remained difficult to date.     

Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat₂, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat₂ (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat₂-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat₂-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat₂ can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.     

Role of antioxidant and anaerobic metabolism enzymes in providing tolerance to maize (Zea mays L.) seedlings against waterlogging

The present investigation was undertaken to identify the possible mode of mechanism that could provide tolerance to maize (Zea mays L.) seedlings under waterlogging. Using cup method, a number of maize genotypes were screened on the basis of survival of the seedlings kept under waterlogging. Two tolerant (LM5 and Parkash) and three susceptible (PMH2, JH3459 and LM14) genotypes were selected for the present study. Activities of antioxidant and ethanolic fermentation enzymes and content of hydrogen peroxide (H2O2), glutathione and ascorbic acid were determined in roots of these genotypes after 72 h of waterlogging. Waterlogging treatment caused decline in activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in all the genotypes. However, only susceptible genotypes showed slight increase in glutathione reductase (GR) activity. Significant reduction in APX/GR ratio in susceptible genotypes might be the cause of their susceptibility to waterlogging. The tolerant seedlings had higher GR activity than susceptible genotypes under unstressed conditions. Stress led to decrease in H202 and increase in glutathione content of both tolerant and susceptible genotypes, but only tolerant genotypes exhibited increase in ascorbic acid under waterlogging conditions. In the tolerant genotypes, all the enzymes of anaerobic metabolism viz. alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and pyruvate decarboxylase (PDC) were upregulated under waterlogging, whereas in susceptible genotypes, only ADH was upregulated, suggesting that efficient upregulation of entire anaerobic metabolic machinery is essential for providing tolerance against waterlogging. The study provides a possible mechanism for waterlogging tolerance in maize.     

Toll-Like Receptor-3 Is Dispensable for the Innate MicroRNA Response to West Nile Virus (WNV)

The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.