Cloning and Expression of Catalytic Subunit of MLIII, the Ribosome-Inactivating Protein from Viscum album

We have cloned the gene encoding a precursor of mistletoe (Viscum album) toxin MLIII. Analyses of nucleotide and deduced amino acid sequences of this gene revealed significant differences between MLI and MLIII preprotoxin genes. Immunochemical properties of recombinant A-subunit expressed in Escherichia coli and renatured were investigated using a panel of monoclonal antibodies raised against three mistletoe toxins (MLI, MLII, and MLIII). Ribosome-inactivating activity of recombinant MLIII A-subunit was detected in cell-free lysate of rabbit reticulocytes.     

Fragment assembly with short reads

MOTIVATION: Current DNA sequencing technology produces reads of about 500-750 bp, with typical coverage under 10x. New sequencing technologies are emerging that produce shorter reads (length 80-200 bp) but allow one to generate significantly higher coverage (30x and higher) at low cost. Modern assembly programs and error correction routines have been tuned to work well with current read technology but were not designed for assembly of short reads. RESULTS: We analyze the limitations of assembling reads generated by these new technologies and present a routine for base-calling in reads prior to their assembly. We demonstrate that while it is feasible to assemble such short reads, the resulting contigs will require significant (if not prohibitive) finishing efforts. AVAILABILITY: Available from the web at http://www.cse.ucsd.edu/groups/bioinformatics/software.html     

The phenoptosis problem: What is causing the death of an organism? Lessons from acute kidney injury

Programmed execution of various cells and intracellular structures is hypothesized to be not the only example of elimination of biological systems — the general mechanism can also involve programmed execution of organs and organisms. Modern rating of programmed cell death mechanisms includes 13 mechanistic types. As for some types, the mechanism of actuation and manifestation of cell execution has been basically elucidated, while the causes and intermediate steps of the process of fatal failure of organs and organisms remain unknown. The analysis of deaths resulting from a sudden heart arrest or multiple organ failure and other acute and chronic pathologies leads to the conclusion of a special role of mitochondria and oxidative stress activating the immune system. Possible mechanisms of mitochondria-mediated induction of the signaling cascades involved in organ failure and death of the organism are discussed. These mechanisms include generation of reactive oxygen species and damage-associated molecular patterns in mitochondria. Some examples of renal failure-induced deaths are presented with mechanisms and settings determined by some hypothetical super system rather than by the kidneys themselves. This system plays the key role in the process of physiological senescence and termination of an organism. The facts presented suggest that it is the immune system involved in mitochondrial signaling that can act as the system responsible for the organism’s death.     

Protein Identification Using Top-Down Spectra

In the last two years, because of advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. However, computational tools for database search of top-down spectra against protein databases are still in their infancy. We describe MS-Align+, a fast algorithm for top-down protein identification based on spectral alignment that enables searches for unexpected post-translational modifications. We also propose a method for evaluating statistical significance of top-down protein identifications and further benchmark various software tools on two top-down data sets from Saccharomyces cerevisiae and Salmonella typhimurium. We demonstrate that MS-Align+ significantly increases the number of identified spectra as compared with MASCOT and OMSSA on both data sets. Although MS-Align+ and ProSightPC have similar performance on the Salmonella typhimurium data set, MS-Align+ outperforms ProSightPC on the (more complex) Saccharomyces cerevisiae data set.In the past two decades, proteomics was dominated by bottom-up mass spectrometry that analyzes digested peptides rather than intact proteins. Bottom-up approaches, although powerful, do have limitations in analyzing protein species, e.g. various proteolytic forms of the same protein or various protein isoforms resulting from alternative splicing. Top-down mass spectrometry focuses on analyzing intact proteins and large peptides (1–10) and has advantages in localizing multiple post-translational modifications (PTMs)¹ in a coordinated fashion (e.g. combinatorial PTM code) and identifying multiple protein species (e.g. proteolytically processed protein species) (11). Until recently, most top-down studies were limited to single purified proteins (12–15). Top-down studies of protein mixtures were restricted by difficulties in separating and fragmenting intact proteins and a shortage of robust computational tools.In the last two years, because of advances in protein separation and top-down instrumentation, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples containing hundreds and even thousands of proteins (16–21). Because algorithms for interpreting top-down spectra are still in their infancy, many recent developments include computational innovations in protein identification.Because top-down spectra are complex, the first step in top-down spectral interpretation is usually spectral deconvolution, which converts a complex top-down spectrum to a list of monoisotopic masses (a deconvolved spectrum). Every protein (possibly with modifications) can be scored against a top-down deconvoluted spectrum, resulting in a Protein-Spectrum-Match (PrSM). The top-down protein identification problem is finding a protein in a database with the highest scoring PrSM for a top-down spectrum and further output the PrSM if it is statistically significant. There are several software tools for top-down protein identification (

The Mitochondria-Targeted Antioxidants and Remote Kidney Preconditioning Ameliorate Brain Damage through Kidney-to-Brain Cross-Talk

Background

Many ischemia-induced neurological pathologies including stroke are associated with high oxidative stress. Mitochondria-targeted antioxidants could rescue the ischemic organ by providing specific delivery of antioxidant molecules to the mitochondrion, which potentially suffers from oxidative stress more than non-mitochondrial cellular compartments. Besides direct antioxidative activity, these compounds are believed to activate numerous protective pathways. Endogenous anti-ischemic defense may involve the very powerful neuroprotective agent erythropoietin, which is mainly produced by the kidney in a redox-dependent manner, indicating an important role of the kidney in regulation of brain ischemic damage. The goal of this study is to track the relations between the kidney and the brain in terms of the amplification of defense mechanisms during SkQR1 treatment and remote renal preconditioning and provide evidence that the kidney can generate signals inducing a tolerance to oxidative stress-associated brain pathologies.

Methodology/Principal Findings

We used the cationic plastoquinone derivative, SkQR1, as a mitochondria-targeted antioxidant to alleviate the deleterious consequences of stroke. A single injection of SkQR1 before cerebral ischemia in a dose-dependent manner reduces infarction and improves functional recovery. Concomitantly, an increase in the levels of erythropoietin in urine and phosphorylated glycogen synthase kinase-3β (GSK-3β) in the brain was detected 24 h after SkQR1 injection. However, protective effects of SkQR1 were not observed in rats with bilateral nephrectomy and in those treated with the nephrotoxic antibiotic gentamicin, indicating the protective role of humoral factor(s) which are released from functional kidneys. Renal preconditioning also induced brain protection in rats accompanied by an increased erythropoietin level in urine and kidney tissue and P-GSK-3β in brain. Co-cultivation of SkQR1-treated kidney cells with cortical neurons resulted in enchanced phosphorylation of GSK-3β in neuronal cells.

Conclusion

The results indicate that renal preconditioning and SkQR1-induced brain protection may be mediated through the release of EPO from the kidney.     

Systemic response to Campylobacter jejuni infection by profiling gene transcription in the spleens of two genetic lines of chickens

Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial enteritis worldwide with poultry products being a major source of C. jejuni contamination. The chicken is the natural reservoir of C. jejuni where bacteria colonize the digestive tract of poultry, but rarely cause symptoms of disease. To understand the systemic molecular response mechanisms to C. jejuni infection in chickens, total splenic RNA was isolated and applied to a whole genome chicken microarray for comparison between infected (I) and non-infected (N) chickens within and between genetic lines A and B. There were more total splenic host genes responding to the infection in resistant line A than in susceptible line B. Specifically, genes for lymphocyte activation, differentiation and humoral response, and Ig light and heavy chain were upregulated in the resistant line. In the susceptible line, genes for regulation of erythrocyte differentiation, hemopoiesis, and RNA biosynthetic process were all downregulated. An interaction analysis between genetic lines and treatment demonstrated distinct defense mechanisms between lines: the resistant line promoted apoptosis and cytochrome c release from mitochondria, whereas the susceptible line responded with a downregulation of both functions. This was the first time that such systemic defensive mechanisms against C. jejuni infection have been reported. The results of this study revealed novel molecular mechanisms of the systemic host responses to C. jejuni infection in chickens that warrant further investigation.     

Kidney cell death in inflammation: The role of oxidative stress and mitochondria

Pyelonephritis is an infectious disease, and common treatment strategy is based on antibiotic therapy directed at the elimination of a pathogen. However, urinary tract infections are accompanied by inflammation and oxidative stress, which are major damaging factors, and therefore can serve as a target for therapeutic intervention. The goal of this study was to clarify the role of the mitochondrial reactive oxygen species (ROS) in kidney cell damage under experimental pyelonephritis. We investigated the mechanisms of inflammation and the role of mitochondria and oxidative stress in inflammation in kidney tissue using in vivo and in vitro models of pyelonephritis. We observed the development of oxidative stress in renal tubular epithelium in vitro, and resulting apoptotic cell death. This oxidative damage was caused by the leukocytes producing ROS after interaction with bacterial antigens. The essential role of mitochondria-mediated oxidative stress was confirmed using an experimental model of pyelonephritis in vivo. We revealed increased levels of malonic dialdehyde in kidneys of rats with experimental pyelonephritis that pointed to lipid peroxidation. Besides, high ROS levels were observed in blood leukocytes from rats with pyelonephritis. The mitochondria-targeted antioxidant SkQ1 significantly reduced the signs of kidney inflammatory injury, in particular the infiltration of neutrophils. Summarizing the data obtained, we assume the importance of mitochondrial ROS in different phases of acute pyelonephritis onset. Protection of kidney cells from infection-mediated damage can be attained by the induction of tolerance mechanisms and by antioxidant treatment.     

Lithium salts — Simple but magic

For many decades pharmacological drugs based on lithium salts have been successfully used in psychiatry to treat bipolar disorder, and they remain the “gold standard” of pharmacological therapy of patients with this disease. At the same time, over recent years in experiments in vitro and in vivo a plethora of evidence has accumulated on a positive effect of lithium ions in other areas including their neuro-, cardio-, and nephroprotective properties, regulation of stem cells functions, regulation of inflammation, and others. Numerous studies have shown that the effect of lithium ions involves several mechanisms; however, one of its main targets in the implementation of most of the effects is glycogen synthase kinase 3β, a key enzyme in various pathological and protective signaling pathways in cells. However, one of the main limitations of the use of lithium salts in clinics is their narrow therapeutic window, and the risk of toxic side effects. This review presents the diversity of effects of lithium ions on the organism emphasizing their potential clinical applications with minimal undesirable side effects. In the end, we present a schematic “Lithiometer”, comparing the range of Li⁺ concentrations that might be used for the treatment of acute pathologies with possible toxic effects of Li⁺.     

Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches

Protein identifications with the borderline statistical confidence are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in the reliable and reproducible characterization of proteomes. Here, we present a method for rapid validation of borderline hits that circumvents the need in, often biased, manual inspection of raw MS/MS spectra. The approach takes advantage of the independent interpretation of corresponding MS/MS spectra by PepNovo de novo sequencing software followed by mass spectrometry-driven BLAST (MS BLAST) sequence-similarity database searches that utilize all partially inaccurate, degenerate and redundant candidate peptide sequences. In a case study involving the identification of more than 180 Caenorhabditis elegans proteins by nanoLC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, the approach enabled rapid assignment (confirmation or rejection) of more than 70% of Mascot hits of borderline statistical confidence.     

How many signal peptides are there in bacteria?

Over the last 5 years proteogenomics (using mass spectroscopy to identify proteins predicted from genomic sequences) has emerged as a promising approach to the high‐throughput identification of protein N‐termini, which remains a problem in genome annotation. Comparison of the experimentally determined N‐termini with those predicted by sequence analysis tools allows identification of the signal peptides and therefore conclusions on the cytoplasmic or extracytoplasmic (periplasmic or extracellular) localization of the respective proteins. We present here the results of a proteogenomic study of the signal peptides in Escherichia coli K‐12 and compare its results with the available experimental data and predictions by such software tools as SignalP and Phobius. A single proteogenomics experiment recovered more than a third of all signal peptides that had been experimentally determined during the past three decades and confirmed at least 31 additional signal peptides, mostly in the known exported proteins, which had been previously predicted but not validated. The filtering of putative signal peptides for the peptide length and the presence of an eight‐residue hydrophobic patch and a typical signal peptidase cleavage site proved sufficient to eliminate the false‐positive hits. Surprisingly, the results of this proteogenomics study, as well as a re‐analysis of the E. coli genome with the latest version of SignalP program, show that the fraction of proteins containing signal peptides is only about 10%, or half of previous estimates.