Mediator structure and conformation change

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Denaturing Reversed-Phase HPLC Using a Mobile Phase Containing Urea for Oligodeoxynucleotide Analysis

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.     

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.     

Activation and mechanism of Clostridium septicum alpha toxin

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (AT^pro) which could be activated via a carboxy-terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy-terminus. Proteolytically activated AT^pro had a specific activity of approximately 1.5 × 10⁶ haemolytic units mg^-1. The trypsin-activated toxin (AT^act) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of M_r >210000 on erythrocyte membranes. AT^pro did not exhibit these properties. AT^act formed pores with a diameter of at least 1.3-1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.     

Cyclase‐associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells

Adenylyl cyclase‐associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K‐mTOR/SREBP‐1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.     

Secretion of the STA3 heat-stable enterotoxin of Escherichia coli: extracellular delivery of Pro-STA is accomplished by either Pro or STA

The methanol-soluble, heat-stable enterotoxin of Escherichia coli is a protease-resistant extracellular peptide which is synthesized as a 72-amino-acid precursor Pre-Pro-STA3. The specific roles of Pre (19 amino acids), Pro (34 amino acids) and STA3 (19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short-lived cell-associated molecule with an M(r) equivalent to that of Pro-STA3. Deletion of Pro (i.e., Pre-STA3) resulted in the rapid extracellular accumulation of STA3; the periplasmic intermediate found in the secretion of the wild-type toxin was undetected. Deletion of the STA3 domain resulted in a cell-associated Pre-Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro-STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected. The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STA3 domain to become susceptible to intracellular proteases. The rapid degradation of intracellular STA3 indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre-STA3 and Pre-Pro, the STA3 and Pro peptides can exit to the culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)