Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.     

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods

Aim: To evaluate whole cell protein profiling vis‐à‐vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. Methods and Results: Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS–PAGE. Twenty‐one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30–6,31 strains were clustered into two discrete subgroups. Conclusions: Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. Significance and impact of the study: Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.     

Synapsin I is a major endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.     

Growth and nutrient removal of three macrophytes in response to concentrations and ratios of N and P

Wastewater from different sources shows great differences in concentrations and ratios of N and P. In order to choose suitable plant species to remove excess N and/or P from polluted waters, it is important to know the performances of these plants under different N and P concentrations. In this study, we investigated the growth and N and P removal rate of three macrophytes, Coix lacryma-jobi, Iris wilsonii, and Arundo donax under six N and P combination treatments. C. lacryma-jobi preferred higher N and P concentrations (16 mg N L^?1 and 3.2 mg P L^?1), and increasing N supply could increase its P removal rate. I. wilsonii exhibited a growth preference at a combination of moderate N and P concentrations (8 mg N L^?1 and 0.8 mg P L^?1). A. donax could grow well at all combinations of N and P and had significantly higher relative growth rate and N and P removal rates than the other two species. These results showed A. donax is a promising species to treat various polluted waters and the other two species can be used specifically to treat certain types of wastewater.     

A unified framework for the restoration of Southeast Asian mangroves—bridging ecology, society and economics

The effect of intensive human intervention, poor socio-economic conditions and little knowledge on mangrove ecology pose enormous challenges for mangrove restoration in Southeast Asia. We present a framework for tropical mangrove restoration. Our proposed restoration framework addresses the ecology, economy and social issues simultaneously by considering the causes of mangrove degradation. We provide a step by step guideline for its restoration. We argue that although, ecological issues are of prime importance, economic and social issues must be considered in the restoration plan in order for it to be successful. Since mangrove ecology is not adequately studied in this region, local ecological knowledge can be used to fill the baseline information gaps. Unwanted human disturbance can be minimized by encouraging community participation. This can be ensured and sustained by facilitating the livelihood of the coastal community. We translated the restoration paradigm into a readily available practical guideline for the executors of the plans. We provide an example of mangrove restoration project that is closely related to our proposed framework. We are optimistic that this framework has the potential for universal application with necessary adjustments.     

Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme

Sirtuins are emerging as the key regulators of metabolism and aging, and their potential activators and inhibitors are being explored as therapeutics for improving health and treating associated diseases. Despite the global structural similarity among all seven isoforms of sirtuins (of which most of them catalyze the deacetylation reaction), SIRT5 is the only isoform that catalyzes the cleavage of negatively charged acylated substrates, and the latter feature appears to be encoded by the presence of Tyr102 and Arg105 residues at the active site pocket of the enzyme. To determine the contributions of the above residues in SIRT5 (vis a vis the corresponding residues of SIRT1) on substrate selectivity, inhibition by EX527 and nicotinamide, secondary structural features and thermal stability of the enzymes, we created single and double mutations (viz. Y102A, R105l, and Y102A/R105I) in SIRT5. The kinetic data revealed that while Y102A mutant enzyme catalyzed both deacetylation and desuccinylation reactions with comparable efficiencies, R105I and Y102A/R105I mutant enzymes favored the deacetylase reaction. Like SIRT1, the nicotinamide inhibition of SIRT5 double mutant (Y102A/R105I) exhibited the mixed non-competitive behavior. On the other hand, the desuccinylation reaction of both wild-type and Y102A mutant enzymes conformed to the competitive inhibition model. The inhibitory potency of EX527 progressively increased from Y102A, R105I, to Y102A/R105 mutant enzymes in SIRT5, but it did not reach to the level obtained with SIRT1. The CD spectroscopic data for the wild-type and mutant enzymes revealed changes in the secondary structural features of the enzymes, and such changes were more pronounced on examining their thermal denaturation patterns. A cumulative account of our experimental data reveal mutual cooperation between Y102 and R105 residues in promoting the desuccinylation versus deacetylation reaction in SIRT5, and the overall catalytic feature of the enzyme is manifested via the mutation induced modulation in the protein structure.     

Identification of gene signatures from RNA-seq data using Pareto-optimal cluster algorithm

Background

Gene signatures are important to represent the molecular changes in the disease genomes or the cells in specific conditions, and have been often used to separate samples into different groups for better research or clinical treatment. While many methods and applications have been available in literature, there still lack powerful ones that can take account of the complex data and detect the most informative signatures.

Methods

In this article, we present a new framework for identifying gene signatures using Pareto-optimal cluster size identification for RNA-seq data. We first performed pre-filtering steps and normalization, then utilized the empirical Bayes test in Limma package to identify the differentially expressed genes (DEGs). Next, we used a multi-objective optimization technique, “Multi-objective optimization for collecting cluster alternatives” (MOCCA in R package) on these DEGs to find Pareto-optimal cluster size, and then applied k-means clustering to the RNA-seq data based on the optimal cluster size. The best cluster was obtained through computing the average Spearman’s Correlation Score among all the genes in pair-wise manner belonging to the module. The best cluster is treated as the signature for the respective disease or cellular condition.

Results

We applied our framework to a cervical cancer RNA-seq dataset, which included 253 squamous cell carcinoma (SCC) samples and 22 adenocarcinoma (ADENO) samples. We identified a total of 582 DEGs by Limma analysis of SCC versus ADENO samples. Among them, 260 are up-regulated genes and 322 are down-regulated genes. Using MOCCA, we obtained seven Pareto-optimal clusters. The best cluster has a total of 35 DEGs consisting of all-upregulated genes. For validation, we ran PAMR (prediction analysis for microarrays) classifier on the selected best cluster, and assessed the classification performance. Our evaluation, measured by sensitivity, specificity, precision, and accuracy, showed high confidence.

Conclusions

Our framework identified a multi-objective based cluster that is treated as a signature that can classify the disease and control group of samples with higher classification performance (accuracy 0.935) for the corresponding disease. Our method is useful to find signature for any RNA-seq or microarray data.

     

Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.     

Inhibition of a Mitotic Motor Compromises the Formation of Dendrite-like Processes from Neuroblastoma Cells

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.