Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

Activation of V(1a) receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. We found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V(1a) receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and beta-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways.     

Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2_t, residues 196–464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2_t production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2_t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2_t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2_t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2_t promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2_t and of MBP–recROP2_t led respectively to the complete degradation or to the truncation of the recROP2_t moiety. However, recROP2_t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.     

Identifying the membrane proteome of HIV-1 latently infected cells

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.     

Effect of ankle joint position and electrode placement on the estimation of the antagonistic moment during maximal plantarflexion.

During maximal efforts, antagonistic activity can significantly influence the joint moment. During maximal voluntary "isometric" contractions, certain joint rotation can not be avoided. This can influence the estimation of the antagonistic moment from the EMG activity. Our study aimed to quantify the influence on the calculated agonistic moment produced during maximal voluntary isometric plantarflexions (a) when estimating antagonistic moments at different ankle angles and (b) when placing the EMG electrodes at different portions over the m. tibialis anterior. Ten subjects performed maximal voluntary isometric plantarflexions at 90 degrees ankle angle. In order to estimate the antagonistic moment, submaximal isometric dorsiflexions were performed at various ankle angles. Moment and EMG signals from mm. triceps surae and tibialis anterior were measured. The RMS differences between plantarflexors moment calculated considering the antagonistic cocontraction estimated at the same ankle angle at which the maximal plantarflexion moment was achieved and at different ankle angles ranged from 0.10 to 2.94 Nm. The location of the electrodes led to greater RMS differences (2.35-5.18 Nm). In conclusion, an angle 10 degrees greater than the initial plantarflexion angle is enough to minimize the effect of the change in length of the m. tibialis anterior during the plantarflexion on the estimation of the plantarflexors moment. The localisation of the electrodes over the m. tibialis anterior can influence the estimation of its cocontraction during maximal plantarflexion efforts.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997