Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding

The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

II Encuentro Institucional del Consejo Escolar de la Regi??n de Murcia, Consejos Escolares Municipales y Consejos Escolares de Centro : la participaci??n como factor de mejora de la calidad educativa

Esta publicaci??n recoge los estudios, ponencias y conclusiones de un trabajo realizado a lo largo de todo un curso escolar y que ha cristalizado en el II Encuentro de consejos escolares (regional, municipales y de centros) en el que se llev?? a cabo un debate sobre la participaci??n en los centros en los centros que abord?? los siguientes temas: la participaci??n en la UE, programa para la mejora de la participaci??n en la Comunidad Valenciana y en la de Madrid y an??lisis de la participaci??n en Consejos Escolares en las Comarcas del Noroeste y R??o Mula de la Regi??n de Murcia.

MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1. (C) 2010 Elsevier Inc. All rights reserved.

Absence of transforming growth factor-beta type II receptor is associated with poorer prognosis in HER2-negative breast tumours

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Angiotensin Ii As A General Growth Factor

Angiotensin II (Ang II), a key protein in the renin-angiotensin system, can induce cardiac hypertrophy through an intracrine system as well as affect gene transcription. The receptor to Ang II responsible for this effect, AT1, has been localized to the nucleus of cell types in addition to cardiomyocytes. In this study, we induced expression of Ang II in MC3T3 osteoblasts and K7M2 osteosarcomas and measured changes in protein expression of Annexin V and matrix metalloproteinase 2 (MMP2), proteins identified previously through mass spectrometry analysis as being regulated by Ang II. Annexin V is downregulated in both immortalized murine bone (MC3T3) cells and in cancerous immortalized murine (K7M2) cells induced to express Ang II. MC3T3 cells which express Ang II show a downregulation of MMP2 expression, but Ang II-expressing K7M2 cells show an upregulation of MMP2. The differential regulation of MMP2 between the cancerous cells and noncancerous cells implicates a role for Ang in in tumor metastasis, as MMP2 is a metastatic protein. Annexin V is used as a marker for apoptosis, but nothing is known of the function of the endogenous protein. That Annexin V is potentially regulated by Ang II provides more information with which to characterize the protein and could suggest a function for Annexin V as part of a signal transduction pathway inside of the cell.

Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Role of plerixafor in autologous stem cell mobilization with vinorelbine chemotherapy and granulocyte-colony stimulating factor in patients with myeloma: a phase II study (PAV-trial)

Current practice in Switzerland for the mobilization of autologous stem cells in patients with myeloma is combining vinorelbine chemotherapy and granulocyte-colony stimulating factor (G-CSF) cytokine stimulation. We prospectively investigated adding intravenous plerixafor to the vinorelbine/G-CSF combination (VGP), and compared it with vinorelbine/plerixafor (VP) and G-CSF/plerixafor (GP) combinations. In a final cohort (VP-late), plerixafor was given on the first day of CD34 + cells increasing to > 15 000/mL peripheral blood. Four consecutive cohorts of 10 patients with myeloma were studied. We observed that intravenously administered plerixafor can be safely combined with vinorelbine/G-CSF. VGP was superior in mobilizing peripheral stem and progenitor cells compared to the three double combinations (VP, GP and VP-late), and GP mobilized better than VP. Our data indicate that the triple combination of VGP is an efficient strategy to collect autologous CD34 + cells, with G-CSF contributing predominantly in this concept. Plerixafor can be safely added to G-CSF and/or vinorelbine chemotherapy.

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.