Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5β

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially mediated by preservation of hepatic ATP contents. However, the precise molecular mechanisms of these events remain poorly elucidated. In this study, liver proteomes of the mice subjected to I/R injury pretreated with or without IPC were analyzed using 2‐DE combined with MALDI‐TOF/TOF mass analysis. Twenty proteins showing more than 1.5‐fold difference were identified in the livers upon I/R injury. Among these proteins, four proteins were further regulated by IPC when compared with nonpretreated controls. One of these proteins, ATP synthase β subunit (ATP5β) catalyzes the rate‐limiting step of ATP formation. The expression level of ATP5β, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Change pattern of hepatic ATP corresponded with that of ATP5β expression, indicating that increasing hepatic ATP5β expression might be a reason for ATP‐preserving effect of IPC. In summary, this study provided new clues for understanding the mechanisms of IPC against I/R injury. The protective role of ATP5β might give evidences for developing new therapeutic approaches against hepatic I/R injury.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Using the protein chip interface with quadrupole time‐of‐flight mass spectrometry to directly identify peaks in SELDI profiles – initial evaluation using low molecular weight serum peaks

Mass spectrometric profiling, particularly in the form of SELDI, has been used in many studies, particularly in attempts to generate diagnostic serum profiles. Several studies have generated promising results but one of the limitations is the inability to identify easily potential discriminatory peaks. This may enable specific assays to be developed and increased biological insight. We describe the first systematic technical evaluation of the ProteinChip interface coupled to a tandem mass spectrometer which allows direct sequencing of peptides <6000 Da, and describe the direct sequence identification of 21 peaks commonly observed in serum samples. Additionally we describe for the first time the use of on‐chip acetylation to assist in the validation of sequence identification.     

NSOM‐ and AFM‐based nanotechnology elucidates nano‐structural and atomic‐force features of a Y. pestis V immunogen‐containing particle vaccine capable of eliciting robust response

It is postulated that unique nanoscale proteomic features of immunogen on vaccine particles may determine immunogen‐packing density, stability, specificity, and pH‐sensitivity on the vaccine particle surface and thus impact the vaccine‐elicited immune responses. To test this presumption, we employed near‐filed scanning optical microscopy (NSOM)‐ and atomic force microscopy (AFM)‐based nanotechnology to study nano‐structural and single‐molecule force bases of Yersinia pestis (Y. pestis) V immunogen fused with protein anchor (V‐PA) loaded on gram positive enhancer matrix (GEM) vaccine particles. Surprisingly, the single‐molecule sensitive NSOM revealed that ～90% of V‐PA immunogen molecules were packed as high‐density nanoclusters on GEM particle. AFM‐based single‐molecule force analyses indicated a highly stable and specific binding between V‐PA and GEM at the physiological pH. In contrast, this specific binding was mostly abrogated at the acidic pH equivalent to the biochemical pH in phagolysosomes of antigen‐presenting‐cells in which immunogen protein is processed for antigen presentation. Intranasal mucosal vaccination of mice with such immunogen loaded on vaccine particles elicited robust antigen‐specific immune response. This study indicated that high‐density, high‐stability, specific, and immunological pH‐responsive loading of immunogen nanoclusters on vaccine particles could readily be presented to the immune system for induction of strong antigen‐specific immune responses.     

PBOND： Web Server for the Prediction of Proline and Non-Proline cis / trans Isomerization

PBOND is a web server that predicts the conformation of the peptide bond between any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide (cis-Pro), cis amide (cis-nonPro), trans imide (trans-Pro) and trans amide (trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages: (1) feature extraction, (2) feature selection and (3) peptide bond clas- sification. PBOND can handle both s...     

Significant Deviations in the Configurations of Homologous Tandem Repeats in Prokaryotic Genomes

We explored the possibilities of whole-genome duplication (WGD) in prokaryotic species,where we performed statistical analyses of the configurations of the central angles between homologous tandem repeats (TRs) on the circular chromosomes.At first,we detected TRs on their chromosomes and identified equivalent tandem repeat pairs (ETRPs); here,an ETRP is defined as a pair of tandem repeats sequentially similar to each other.Then we carried out statistical analyses of the central angle distributions of the de...     

A Modified Ant Colony Optimization Algorithm for Tumor Marker Gene Selection

Microarray data are often extremely asymmetric in dimensionality,such as thousands or even tens of thousands of genes but only a few hundreds of samples or less.Such extreme asymmetry between the dimensionality of genes and samples can lead to inaccurate diagnosis of disease in clinic.Therefore,it has been shown that selecting a small set of marker genes can lead to improved classification accuracy.In this paper,a simple modified ant colony optimization (ACO) algorithm is proposed to select tumor-related ma...