Isolation and characterization of PERV-C env from domestic pig in Korea

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.     

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.     

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1^561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1^561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1^561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1^561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)_n.     

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.     

A phase synchronization clustering algorithm for identifying interesting groups of genes from cell cycle expression data

Background

Quantitative proteomics holds great promise for identifying proteins that are differentially abundant between populations representing different physiological or disease states. A range of computational tools is now available for both isotopically labeled and label-free liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics. However, they are generally not comparable to each other in terms of functionality, user interfaces, information input/output, and do not readily facilitate appropriate statistical data analysis. These limitations, along with the array of choices, present a daunting prospect for biologists, and other researchers not trained in bioinformatics, who wish to use LC-MS-based quantitative proteomics.

Results

We have developed Corra, a computational framework and tools for discovery-based LC-MS proteomics. Corra extends and adapts existing algorithms used for LC-MS-based proteomics, and statistical algorithms, originally developed for microarray data analyses, appropriate for LC-MS data analysis. Corra also adapts software engineering technologies (e.g. Google Web Toolkit, distributed processing) so that computationally intense data processing and statistical analyses can run on a remote server, while the user controls and manages the process from their own computer via a simple web interface. Corra also allows the user to output significantly differentially abundant LC-MS-detected peptide features in a form compatible with subsequent sequence identification via tandem mass spectrometry (MS/MS). We present two case studies to illustrate the application of Corra to commonly performed LC-MS-based biological workflows: a pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type 2 diabetes, and a study in yeast to identify in vivo targets of the protein kinase Ark1 via phosphopeptide profiling.

Conclusion

The Corra computational framework leverages computational innovation to enable biologists or other researchers to process, analyze and visualize LC-MS data with what would otherwise be a complex and not user-friendly suite of tools. Corra enables appropriate statistical analyses, with controlled false-discovery rates, ultimately to inform subsequent targeted identification of differentially abundant peptides by MS/MS. For the user not trained in bioinformatics, Corra represents a complete, customizable, free and open source computational platform enabling LC-MS-based proteomic workflows, and as such, addresses an unmet need in the LC-MS proteomics field.     

Blockade of chronic high glucose-induced endothelial apoptosis by Sasa borealis bamboo extract

Hyperglycemia is a causal factor in the development of diabetic vascular complications including impaired vascular smooth muscle contractility and increased cell proliferation. The present study was designed to investigate the effects of Sasa borealis water-extract (SBwE) on chronic hyperglycemia-induced oxidative stress and apoptosis in human umbilical endothelial cells (HUVEC). HUVEC were cultured in 5.5 mM low glucose, 5.5 mM glucose plus 27.5 mM mannitol as an osmotic control, or 33 mM high glucose for 5 days in the absence and presence of 1-30 microg/ ml SBwE. Caspase-3 activation and Annexin V staining revealed chronic high glucose-induced endothelial apoptotic toxicity with a generation of oxidants detected by DCF-fluorescence, and these effects were reversed by SBwE at > or =1 microg/ml in a dose-dependent manner. Cytoprotective SBwE substantially reduced the sustained high glucose-induced expression of endothelial nitric oxide synthase and attenuated the formation of peroxynitrite radicals. The suppressive effects of SBwE were most likely mediated through blunting activation of PKC beta 2 and NADPH oxidase promoted by high glucose. In addition, this bamboo extract modulated the high glucose-triggered mitogen-activated protein kinase-dependent upregulation of heat-shock proteins. Our results suggest that SBwE suppressed these detrimental effects caused by PKC-dependent peroxynitrite formation via activation of NADPH oxidase and induction of nitric oxide synthase and heat-shock protein family that may be essential mechanisms responsible for increased apoptotic oxidative stress in diabetic vascular complications. Moreover, the blockade of high glucose-elicited heat-shock protein induction appeared to be responsible for SBwE-alleviated endothelial apoptosis. Therefore, SBwE may be a therapeutic agent for the prevention and treatment of diabetic endothelial dysfunction and related complications.     

Shear deformation kinematics during cartilage articulation: effect of lubrication, degeneration, and stress relaxation

During joint articulation, the biomechanical behavior of cartilage not only facilitates load-bearing and low-friction, but also provides regulatory cues to chondrocytes. Elucidation of cartilage kinematics under combined compression and shearing conditions clarifies these cues in health and disease. The objectives of this study were to elucidate the effects of lubricant, tissue degeneration, and stress relaxation duration on cartilage shear kinematics during articulation. Human osteochondral cores with normal and mildly degenerate surface structures were isolated. Paired blocks from each core were apposed, compressed, allowed to stress relax for 5 or 60 min, and shear tested with a micro-scale video microscopy system using phosphate-buffered saline (PBS) or synovial fluid as lubricant. During applied lateral motion, local and overall shear strain (Exz) of articular cartilage were determined. The applied lateral displacement at which Exz reached 50% of the peak (Deltax(1/2)) was also determined. Quantitatively, surface Exz increased at the onset of lateral motion and peaked just as surfaces detached and slid. With continued lateral motion, surface Exz was maintained. After short stress relaxation, effects of lubrication on Exz and Deltax(1/2) were not apparent. With prolonged stress relaxation, Exz and Deltax(1/2) near the articular surface increased markedly when PBS was used as lubricant. Similar patterns were observed for overall Exz and Deltax(1/2). With degeneration, surface Exz was consistently higher for all cases after the onset of lateral motion. Thus, cartilage shear kinematics is markedly affected by lubricant, cartilage degeneration, and loading duration. Changes in these factors may be involved in the pathogenesis of osteoarthritis.     

Synthetic biology of proteins: tuning GFPs folding and stability with fluoroproline

Background

Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C^γ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C^γ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.

Methodology/Principal Findings

In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C^γ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.

Conclusions/Significance

We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C^γ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.