Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

Background

Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure.

Methods

We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells.

Results

Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested.

Conclusions

We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific.     

Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔH^cal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.     

LINC01101 and LINC00277 expression levels as novel factors in HPV‐induced cervical neoplasia

Recently long non‐coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7‐siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7‐siRNA‐transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV‐induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV‐positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV‐induced cervical cancer, enriching our understanding of their potential role in this pathology.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity. Ca(2+) binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na(+) concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca(2+) activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP?) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na(+) concentrations also induce an inactivated state of the exchanger (Na(+)-dependent inactivation) that becomes dominant when cytosolic pH and PIP? levels fall. Na(+)-dependent inactivation may provide a means of protecting cells from Ca(2+) overload due to NCX-mediated Ca(2+) influx during ischemia.     

Radiation dose reduction and image enhancement in biological imaging through equally-sloped tomography

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3-5nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.     

Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V. cholerae O1 were exposed to 5°C directly from 35°C. Also, cultures of V. cholerae first exposed to 15°C for 2 h and then maintained at 5°C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15°C after first being exposed to 15°C for 2 h, followed by transfer to 5°C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35°C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15°C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V. cholerae O1.     

Chromium-induced lymph node histiocytic proliferation after hip replacement. A case report

BACKGROUND: Prosthetic joint replacement is frequently used for the treatment of degenerative joint disease, rheumatoid arthritis, bone tumors and traumatic lesions. The prostheses contain such materials as titanium, cobalt and chromium. We describe a patient who, after total hip arthroplasty, developed an inguinal-pelvic mass. Fine needle aspiration revealed metallic particles, also seen on light microscopy in reactive pelvic lymph nodes. Ultrastructure was consistent with the presence of foreign particles, while energy dispersive x-ray microanalysis established the presence of chromium. To our knowledge, this is the first report of chromium-related lymph node metallosis diagnosed by fine needle aspiration. CASE: Eight years after total hip arthroplasty, a 78-year-old woman developed a right pelvic cystic mass. Aspiration drainage was performed. Smears from fine needle aspiration showed numerous macrophages with abundant, foamy cytoplasm and round nuclei without atypia. Small, birefringent particles were seen in the cytoplasm. Histopathology showed fibroconnective tissue with chronic inflammation and marked lymph node sinus histiocytosis. Within histiocytes, numerous particles were present, identical to those seen in the smears. Their nature as "foreign bodies" was confirmed by electron microscopy, and the presence of chromium was shown by energy dispersive x-ray analysis. CONCLUSION: Fine needle aspiration and polarized microscopy are excellent techniques to evaluate foreign materials in lymph nodes draining the sites of joint prostheses, thus precluding confusion with other conditions, such as metastatic carcinoma.     

Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteome

Proteome analysis in the central nervous system area represents a large and important challenge in drug discovery. One major problem is to obtain representative and well characterized tissues of high quality for analysis. We have used brain tissues from normal mice to study the effect of post mortem time (up to 32 h) and temperature (4 degrees C and room temperature) on protein expression patterns. A number of proteins were identified using mass spectrometry and potential markers were localized. One of the proteins identified, dihydropyrimidinase related protein-2 (DRP-2), occurs as multiple spots in two-dimensional electrophoresis gels. The ratio between the truncated form of DRP-2 (fDRP-2) and full length DRP-2 is suggested as an internal control that can be used as a biomarker of post mortem time and post mortem temperature between unrelated brain protein samples. Results of this study may be useful in future efforts to detect disease specific alterations in proteomic studies of human post mortem brain tissues.