Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

Chaperonins are a class of molecular chaperones that assemble into a large double ring architecture with each ring constituting seven to nine subunits and enclosing a cavity for substrate encapsulation. The well-studied Escherichia coli chaperonin GroEL binds non-native substrates and encapsulates them in the cavity thereby sequestering the substrates from unfavorable conditions and allowing the substrates to fold. Using this mechanism, GroEL assists folding of about 10–15 % of cellular proteins. Surprisingly, about 30 % of the bacteria express multiple chaperonin genes. The presence of multiple chaperonins raises questions on whether they increase general chaperoning ability in the cell or have developed specific novel cellular roles. Although the latter view is widely supported, evidence for the former is beginning to appear. Some of these chaperonins can functionally replace GroEL in E. coli and are generally indispensable, while others are ineffective and likewise are dispensable. Additionally, moonlighting functions for several chaperonins have been demonstrated, indicating a functional diversity among the chaperonins. Furthermore, proteomic studies have identified diverse substrate pools for multiple chaperonins. We review the current perception on multiple chaperonins and their physiological and functional specificities.     

Degradation of pentachlorophenol by <Emphasis Type="Italic">Kocuria</Emphasis> sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600 mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.     

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K_i = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k_inact) of (0.73 ± 0.07) × 10^? 3 μM^? 1 min^? 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.     

Theoretical studies on nitrogen rich energetic azoles

Different nitro azole isomers based on five membered heterocyclics were designed and investigated using computational techniques in order to find out the comprehensive relationships between structure and performances of these high nitrogen compounds. Electronic structure of the molecules have been calculated using density functional theory (DFT) and the heat of formation has been calculated using the isodesmic reaction approach at B3LYP/6-31G* level. All designed compounds show high positive heat of formation due to the high nitrogen content and energetic nitro groups. The crystal densities of these energetic azoles have been predicted with different force fields. All the energetic azoles show densities higher than 1.87 g/cm³. Detonation properties of energetic azoles are evaluated by using Kamlet-Jacobs equation based on the calculated densities and heat of formations. It is found that energetic azoles show detonation velocity about 9.0 km/s, and detonation pressure of 40GPa. Stability of the designed compounds has been predicted by evaluating the bond dissociation energy of the weakest C-NO₂ bond. The aromaticity using nucleus independent chemical shift (NICS) is also explored to predict the stability via delocalization of the π-electrons. Charge on the nitro group is used to assess the impact sensitivity in the present study. Overall, the study implies that all energetic azoles are found to be stable and expected to be the novel candidates of high energy density materials (HEDMs).     

Computational design and structure–property relationship studies on heptazines

This study aimed to design novel nitrogen-rich heptazine derivatives as high energy density materials (HEDM) by exploiting systematic structure–property relationships. Molecular structures with diverse energetic substituents at varying positions in the basic heptazine ring were designed. Density functional techniques were used for prediction of gas phase heat of formation by employing an isodesmic approach, while crystal density was assessed by packing calculations. The results reveal that nitro derivatives of heptazine possess a high heat of formation and further enhancement was achieved by the substitution of nitro heterocycles. The crystal packing density of the designed compounds varied from 1.8 to 2 g cm⁻³, and hence, of all the designed molecules, nitro derivatives of heptazine exhibit better energetic performance characteristics in terms of detonation velocity and pressure. The calculated band gap of the designed molecules was analyzed to establish sensitivity correlations, and the results reveal that, in general, amino derivatives possess better insensitivity characteristics. The overall performance of the designed compounds was moderate, and such compounds may find potential applications in gas generators and smoke-free pyrotechnic fuels as they are rich in nitrogen content.     

Individual Management of Recurrent Intracranial Aneurysms: The Wuxi Experience

Recurrent intracranial aneurysms can occur after either surgical clipping or endovascular therapy. In this article, we present a consecutive series of 18 patients who underwent individual treatment for recurrent aneurysms after primary coil embolization or surgical clipping. During an 8-year period between May 1997 and December 2005, 18 patients underwent individual treatment for recurrent aneurysms. Clinical data and imaging studies of the patients were analyzed retrospectively. Out of the 18 patients, 13 had recurrent aneurysms located in the anterior circulation, and 5 had aneurysms of the posterior circulation. Treatment consisted of coiling in 16 patients and clipping in two patients. Of the 18 patients, 15 achieved a good or excellent recovery, two were paralyzed, and one died post-treatment. Both the surgical clipping and endovascular embolization for the treatment of recurrent intracranial aneurysms can achieve very good radiological results with low mortality rates. One of the key points for the successful treatment of this kind of lesions is the proper, individual, and interdisciplinary patient selection.     

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.     

Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM

Aims: To optimize the medium components for the production of indole‐3‐acetic acid (IAA) by isolated bacterium Pantoea agglomerans strain PVM. Methods and Results: Present study deals with the production of an essential plant hormone IAA by a bacterial isolate P. agglomerans strain PVM identified by 16S rRNA gene sequence analysis. The medium containing 8 g l^?1 of meat extract and 1 g l^?1 of l ‐tryptophan (precursor) at optimum pH 7, 30°C and 48‐h incubation gave the maximum production of IAA (2·191 g l^?1). Effect of IAA synthesized on in vitro root induction in Nicotiana tobacum (leaf) explants was compared with that of control. IAA was characterized by high‐performance thin‐layer chromatography, high‐performance liquid chromatography and gas chromatography–mass spectroscopy. Conclusions: Pantoea agglomerans strain PVM was a good candidate for the inexpensive and utmost production of IAA in short period, as it requires simple medium (meat extract and l ‐tryptophan). Significance and Impact of the Study: The present report first time showed the rapid, cost‐effective and maximum production of IAA. No reports are available on the optimization of particular medium components for the production of IAA. This study demonstrates a novel approach for in vitro root induction in N. tobacum (leaf) explants.     

GTI: a novel algorithm for identifying outlier gene expression profiles from integrated microarray datasets

Background

Meta-analysis of gene expression microarray datasets presents significant challenges for statistical analysis. We developed and validated a new bioinformatic method for the identification of genes upregulated in subsets of samples of a given tumour type (‘outlier genes’), a hallmark of potential oncogenes.

Methodology

A new statistical method (the gene tissue index, GTI) was developed by modifying and adapting algorithms originally developed for statistical problems in economics. We compared the potential of the GTI to detect outlier genes in meta-datasets with four previously defined statistical methods, COPA, the OS statistic, the t-test and ORT, using simulated data. We demonstrated that the GTI performed equally well to existing methods in a single study simulation. Next, we evaluated the performance of the GTI in the analysis of combined Affymetrix gene expression data from several published studies covering 392 normal samples of tissue from the central nervous system, 74 astrocytomas, and 353 glioblastomas. According to the results, the GTI was better able than most of the previous methods to identify known oncogenic outlier genes. In addition, the GTI identified 29 novel outlier genes in glioblastomas, including TYMS and CDKN2A. The over-expression of these genes was validated in vivo by immunohistochemical staining data from clinical glioblastoma samples. Immunohistochemical data were available for 65% (19 of 29) of these genes, and 17 of these 19 genes (90%) showed a typical outlier staining pattern. Furthermore, raltitrexed, a specific inhibitor of TYMS used in the therapy of tumour types other than glioblastoma, also effectively blocked cell proliferation in glioblastoma cell lines, thus highlighting this outlier gene candidate as a potential therapeutic target.

Conclusions/Significance

Taken together, these results support the GTI as a novel approach to identify potential oncogene outliers and drug targets. The algorithm is implemented in an R package (Text S1).