Neuritogenesis: the prion protein controls β1 integrin signaling activity

Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of β1 integrins at the plasma membrane. β1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.     

Synchrotron radiation in life sciences

Synchrotron Radiation (SR) presents itself as a "play-ground" with a large range of methods and techniques suitable to unveil the mysteries of life. Here we attempt to present a few of these methods that complement those employed in the home laboratory. SR diffraction, spectroscopy and imaging methods relevant to the atomic structure determination and characterization of the properties and function of chemical compounds and macromolecules of biological relevance, are introduced.     

Pathways affected by asbestos exposure in normal and tumour tissue of lung cancer patients

Background:

The early detection of prostate cancer has resulted in an increase in the number of patients with localized prostate cancer and has paralleled the reported reduction in prostate cancer mortality. The increased rate of detection of patients with localized prostate cancer may also increase the risk of potentially morbid therapy in a patient with indolent cancer. Defining the biomarker correlates of prostate cancer virulence will facilitate the appropriate application and development of therapy for patients with early disease.

Methods:

A 255 core prostate cancer tissue microarray (TMA) from 47 prostatectomy specimens with organ confined tumor was constructed. Prostate cancer foci of transition and peripheral zone origin were represented on the TMA. Further, replicate cores of the two Gleason grades comprising the Gleason score, representative of Gleason scores 5-9, were arrayed from each prostatectomy specimen. Standard immunohistochemical techniques were used to assess expression of nine, cell death and cell cycle regulatory proteins implicated in the pathogenesis of prostate cancer (bax, bcl-2, bcl-x_L, bin1, CD95, mdm2, p21, p53, and NFkB).

Results:

The Spearman correlation coefficient revealed a strong correlation of bax, bin1, FAS, p65 and p21 expression with Gleason grade. Spearman correlation coefficients showed that expression of, bax and bin1, bax and MDM2, Bax and p21, and bax and p65 NFkB was highly associated. Other significant associations were identified between bin1 and p21, bin1 and MDM2, bin1 and p65 NFkB and between p21 and p65 NFκB. A model for predicting the biological potential of Gleason score 7 prostate cancer using multivariable logistic regression methods was developed. The findings also indicate that the profile of specific markers for Gleason grade 3 prostate cancer correlates with the overall context of the Gleason score.

Conclusion:

These data support the view that important molecular differences exist among and between the Gleason scores. Furthermore, there is significant molecular heterogeneity among prostatectomy specimens containing Gleason grade 3 cancer. This observation may have broader implications regarding the determination of risk among patients with prostate cancer that is currently considered to be of either good prognosis or unclear prognosis, i.e. Gleason score 7 tumors.     

New perspectives on the cardioprotective phosphonate effect of the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: an hemodynamic and 31P NMR study in rat hearts.

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) is an improved ESR probe to assess superoxide (O2*-) formation in the postischemic heart. We recently found that DEPMPO pretreatment improves recovery of cardiac function with the concomitant inhibition of postischemic O2*- production. By perfusing diethyl methylphosphonate MeP(O)(OEt)2 to ischemic-reperfused isolated rat hearts, we provide hemodynamic evidence that this preservation, which exerts during ischemia, is in fact specific to the phosphonate group. Using 31P NMR on intact rat hearts, it was also found that the "phosphonate effect" of DEPMPO is related to the preservation of ATP levels during ischemia, when compared to 5,5-dimethyl-1-pyrroline N-oxide. This mechanism may be a means of reducing the potency of cardiac tissue to produce O2*- during reperfusion.     

The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo

The ontogeny of neural crest cells (NCC) involves a number of orchestrated variety of derivatives, including components of the peripheral nervous system and melanocytes. Thus, it represents an excellent model system to investigate mechanisms controlling epithelial-mesenchymal transitions, cell migration and differentiation, as well as cell proliferation and death. We have established a new transgenic line expressing the Cre recombinase under the control of the human tissue plasminogen activator promoter (Ht-PA). The activity of the reporter in the Ht-PA-Cre/R26R embryos is observed as early as Theiler stage 12 in the cephalic mesenchyme. Later, the targeted cells include all the known derivatives of cranial, vagal, and trunk NCC, including craniofacial structures and cranial ganglia, cardiac and endocrine derivatives, melanocytes, peripheral, and enteric nervous system. At the vagal level, the location of presumptive enteric NCC differs from their avian counterparts in their ability to invade the mesenchyme lateral to the neural tube. In contrast to the Wnt1-Cre line, the Ht-PA-Cre line does not target the central nervous system and therefore renders it more specific for NCC. Our Ht-PA-Cre mice represent a novel model to specifically target conditional mutations in migratory NCC.     

High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli. High yields of fully functional holoprotein synthesis in the BLi5 E. coli strain

Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity. Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide. Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein. The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose. The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks. UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size. Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1.     

Functional Characterization of the Purified Holo Form of Hemoglobin I from Lucina pectinata Overexpressed in Escherichia coli

The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.     

ATR-FTIR spectroscopy of the P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase

The structures of P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the "fast" oxidized state to the P(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H(2)O(2). Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film. For both bovine and P. denitrificans oxidase, the major features of the IR difference spectrum of P(M) were similar when produced by CO/oxygen or by H(2)O(2) treatments. These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H(2)O(2). Some IR bands could be assigned tentatively to perturbations of heme a(3) ring modes and substituents, and these perturbations were greater in P(M) than in F. Other bands could be assigned to surrounding protein changes. Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P(M) and relaxed back in F. A second redox-sensitive carboxylic acid was also perturbed in the bovine P(M) intermediate. Further consistent signatures of P(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm(-1) in bovine oxidase (1542 and 1314 cm(-1) in P. denitrificans) and a positive band at approximately 1519 cm(-1). From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu(B). These findings are discussed in relation to the oxidase catalytic cycle.     

Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.     

Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1

Tumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease.