Epidermal Growth Factor

Dysregulation of human epidermal growth factor receptor pathways by over-expression or constitutive activation can promote lung tumor processing including angiogenesis and metastasis and is associated with poor prognosis in non-small cell lung cancers. Ganoderma also known as Lingzhi has been one of the most popular chemoprevention mushrooms in East Asia for centuries. Among many bioactive components identified from Ganoderma, an immunomodulatory protein is the major ingredient for the treatment of lung cancer. Recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. However, knowledge on the pharmacological and molecular mechanisms in suppressing EGF-mediated tumor invasion and metastasis is poorly understood. The goal of study is investigate in suppressing tumor invasion and metastasis activity of GMI. GMI exhibited an inhibitory effect on EGF-induced migration and invasion. GMI treatment with EGF presented the most potent anti-migration and anti-invasion properties on Boyden chamber assay. GMI inhibited EGF-induced phosphorylation and activation of EGFR and AKT pathway kinases in a dose-dependent manner. Additionally, the EGF-induced activation of Cdc42 GTPase was inhibited by GMI, while GMI had little effect on the EGF-induced activation of Rac1 GTPase. GMI also inhibited the EGF-induced microfilament depolymerization. These findings are the first to reveal the novel functions of GMI in tumor anti-metastasis and the molecular basis for its anticancer action.

Growth factors are low molecular peptides active in the stimulation of cell proliferation and in the regulation of embryonic development and cellular differentiation. Significant progress has been made in developing effective strategies to treat human malignancies with new chemical compounds based on a rationale directed against various components of signaling pathways. Many of these drugs target a growth factor receptor-for instance, in the form of monoclonal antibodies or inhibitors of tyrosine kinases, such as monoclonal antibodies against epidermal growth factor receptors used in treating certain types of breast cancer. Imatinib mesylate [Gleevec]) is an excellent example of mediators of signal transduction, such as tyrosine kinases. Growth factors proper are used to ameliorate various and sometimes fatal side effects of cytotoxic and/or myelosuppressive chemotherapy. Basic characteristics of several growth families are discussed with therapeutic modalities based on growth factor activity or, more often, inhibition of such activity.

Purpose: To describe a new drilling system that allows the surgeon to obtain autologous living bone that, when associated with a plasma rich in growth factors (PRGF), can be used in bone grafting. Materials and Methods: Bone particles collected using both conventional and new drilling systems were analyzed by means of optic and electronic microscopy in 10 patients. Blood was collected from 43 volunteers and used to prepare PRGF. Quantitative aspects of the PRGF, including number of platelets and concentration of growth factors (insulin growth factor [IGF-I], transforming growth factor [TGF-␤1], platelet-derived growth factor [PDGF-AB], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], and epidermal growth factor [EGF]) were assessed. A demonstrative case study was presented. Results: Microscopic examination showed that the bone structure and the presence of living cells in the bone chips were conserved in all samples obtained from drilling at low speed, whereas material obtained by conventional drilling did not maintain these qualities. Mean counts for TGF-␤1 (55.27 ± 16.23 ng/mL), PDGF-AB (27.96 ± 12.13 ng/mL), VEGF (421.09 ± 399.0 pg/mL), EGF (455.49 ± 210.04 pg/mL), and HGF (605.70 ± 269.20 pg/mL) were significantly correlated with the number of platelets (590,000 ± 197,000 platelets/μL; P < .05). Discussion and Conclusion: The new drilling procedure was developed based on biologic criteria. The method may reduce damage to the host tissue and can be used to obtain a mass of living bone for subsequent grafting in association with autologous growth factors. This new procedure may present new possibilities for enhanced bone healing and needs to be evaluated in a clinical trial. (Technical Note) INT J ORAL MAX-ILLOFAC IMPLANTS 2007;22:138-145

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the ␥-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII a /tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX a . The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158 -169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII a and IX a . The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II a /thrombomodulin interaction.

EGF activates NF-kB, and constitutively activated NF-kB contributes to EGFR mutation-associated tumorigenesis, but it remains unclear precisely how EGFR signaling leads to NF-kB activation. Here we report that CARMA3, a caspase recruitment domain (CARD)-containing scaffold molecule, is required for EGF-induced NF-kB activation. CARMA3 deficiency impaired the activation of the IKK complex following EGF stimulation, resulting in a defect of EGF-induced IkBa phosphorylation and NF-kB activation. We found that CARMA3 and Bcl10 contributed to several characteristics of EGFR-associated malignancy, including proliferation, survival, migration, and invasion. Most importantly, CARMA3 contributed to tumor growth in vivo. Our findings elucidate a crucial link between EGFR-proximal signaling components and the downstream IKK complex, and they suggest a new therapeutic target for treatment of EGFR-driven cancers. Cancer Res; 71(6); 2183-92. Ó2011 AACR.

Mutations in the gene encoding the heavy chain subunit (DYNC1H1) of cytoplasmic dynein cause spinal muscular atrophy with lower extremity predominance, Charcot-Marie-Tooth disease and intellectual disability. We used the legs at odd angles (Loa) (DYNC1H1 F580Y) mouse model for spinal muscular atrophy with lower extremity predominance and a combination of live-cell imaging and biochemical assays to show that the velocity of dynein-dependent microtubule minus-end (towards the nucleus) movement of EGF and BDNF induced signalling endosomes is significantly reduced in Loa embryonic fibroblasts and motor neurons. At the same time, the number of the plus-end (towards the cell periphery) moving endosomes is increased in the mutant cells. As a result, the extracellular signal-regulated kinases (ERK) 1/2 activation and c-Fos expression are altered in both mutant cell types, but the motor neurons exhibit a strikingly abnormal ERK1/2 and c-Fos response to serum-starvation induced stress. These data highlight the cell-type specific ERK1/2 response as a possible contributory factor in the neuropathological nature of Dync1h1 mutations, despite generic aberrant kinetics in both cell types, providing an explanation for how mutations in the ubiquitously expressed DYNC1H1 cause neuron-specific disease.

Purpose Platelet-rich plasma (PRP) harbors high concentrations of growth factors related to the promotion of wound healing. We evaluated the efficacy of PRP eyedrops in the treatment of persistent epithelial defects (PEDs). Methods Autologous PRP and autologous serum (AS) were prepared from whole blood. The concentrations of transforming growth factor (TGF)-b1, TGF-b2, epidermal growth factor (EGF), vitamin A and fibronectin in the PRP and AS were analyzed and compared. The corneal epithelial healing efficacy of PRP was compared with that of AS in patients with PED induced by post-infectious inflammation.

property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. Furthermore, the smooth optical properties of the corneal epithelial surface are sustained through this renewal process. The rate of renewal is dependent on a highly integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. One experimental approach to characterize these three aspects of the renewal process has been to study the kinetics and dynamics of corneal re-epithelialization in a wound-healing model. This effort has employed in vivo and in vitro studies. From such studies it is evident that the appropriate integration and coordination of corneal epithelial proliferation, adhesion, migration, and cell demise is dependent on the actions of a myriad of cytokines. Our goal here is to provide an overview into how these mediators and environmental factors elicit control of cellular proliferation, adhesion, migration, and apoptosis. To this end we review the pertinent literature dealing with the receptor and the cell signaling events that are responsible for mediating cytokine control of corneal epithelial renewal. It is our hope that a better appreciation can be obtained about the complexity of the control processes that are responsible for assuring continuous corneal epithelial renewal in health and disease.

Ureteral obstruction (UO) is one of the most common problems confronting the urologist. Although large amounts of animal and clinical research have been done, the pathophysiologic mechanisms accompanying UO are not fully elucidated. Most of our knowledge on UO has been derived from experimental studies in a variety of animal models. Both antenatal and postnatal UO models have been developed mainly by ligation of the ureter or by burying the ureter into the psoas muscle. Most experimental studies have focused on short-term complete ureteral obstruction. The long-term effects of partial ureteral obstruction have been less intensively studied. It is now clear that obstructive nephropathy is not a simple result of mechanical impairment to urine flow but a complex syndrome resulting in alterations of both glomerular hemodynamics and tubular function caused by the interaction of a variety of vasoactive factors and cytokines that are activated in response to UO. Leukocyte infiltration appears to play an important role in obstructive nephropathy suggesting that UO also has an immunological component. Growth factors such as platelet-derived growth factor, transforming growth factor-beta, epidermal growth factor and insulin-like growth factor I may all play a role in the development and progression of fibrotic and sclerotic changes in the obstructed kidney. At present, the selection of patients with congenital hydronephrosis for operative treatment is controversial. Studies in animals and patients have shown that partial unilateral UO does not always cause a loss of renal function or progression in urinary tract dilation during long-term follow-up. The implications of UO continue to raise many questions and further work is necessary to achieve a better understanding of the pathogenesis in obstructive nephropathy.

As cancer therapy becomes more effective, allowing patients to live longer, the long-term morbidity of treatment assumes greater significance. Trastuzumab (Herceptin 1 ), a monoclonal antibody that targets the epidermal growth factor, has proven efficacy in breast cancer patients but is also known to be cardiotoxic. Left ventricular ejection fraction (LVEF), a measure of cardiac function, is commonly used to evaluate patients receiving trastuzumab by Multiple Gated Acquisition (MUGA) isotope scans. In this paper, we have assessed the utility of previously published ventricular synchrony parameters in patients undergoing trastuzumab therapy. In addition, we apply Approximate Entropy (ApEn) to MUGA images, as a new measure of cardiac dysfunction and have evaluated its utility in the same patients. A significant change in LVEF (p = 0.015) and ApEn (p = 0.020) but not ventricular synchrony measures were observed over the course of treatment in these patients. The results suggest that ApEn provides a useful measure of cardiac function and synchrony. ß

Necrotizing enterocolitis (NEC) is the leading cause of death for preterm infants resulting from gastrointestinal disease. This review will focus on several components of human breast milk that may be beneficial in the prevention and treatment of NEC. The severe pathological features of NEC include inflammation, mucosal ulceration and disruption of the intestinal barrier. Despite maximal neonatal intensive care, the incidence and mortality rate of the disease remains high. Administration of breast milk, as well as donor breast milk, to preterm infants has been shown to reduce the incidence of NEC. Beyond this, there is no disease specific treatment for NEC. The immunomodulatory and protective properties of human breast milk have been evaluated in search of key components that may be utilized for the effective prevention and treatment of NEC.

This study has assessed the amounts of insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), transforming growth factor b (TGF-b), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) and their binding to extracellular matrix components of Wharton's jelly. Studies were performed on the umbilical cords taken from human newborns delivered by healthy mothers. Wharton's jelly was separated and submitted to homogenisation and extraction with acetic acid and TriseHCl buffer. The assays of growth factors were carried out with the use of ELISA commercial kits, together with SDS/polyacrylamide gel electrophoresis of tissue extracts followed by Western immunoblotting. Several growth factors, viz. acidic FGF, basic FGF, EGF, IGF-I, PDGF and TGF-b were detected in Wharton's jelly. The amounts of these factors per gram of tissue vary from about 40 pg (EGF, PDGF) to about 200 ng (IGF-I). The amounts of peptide growth factors calculated per microgram of DNA are distinctly higher in Wharton's jelly in comparison to the umbilical cord artery. Western blot analysis demonstrated that almost the entire amount of these factors is bound to high molecular weight components. Since the number of cells in Wharton's jelly is very low and the amounts of extracellular matrix components are very high, it is concluded that the cells are strongly stimulated by peptide growth factors to produce large amounts of collagen and glycosaminoglycans. Placenta (2005), 26, 747e752

The total synthesis of the epidermal growth factor inhibitor reveromycin B (2) is described. A novel, convergent, and stereoselective reaction sequence was utilized to construct the 5,6-spiroketal system 10 which was converted into the natural product 2 by a 16-step sequence. The reveromycins A (1) and B (2) are members of a novel family of bioactive spiroketal-containing natural products isolated from a soil actinomycete belonging to the Streptomyces genus. 1,2 These compounds were found to act as inhibitors of the mitogenic activity of epidermal growth factor (EGF) and may represent a new class of antitumor agents. 3 The structures of 1 and 2 were deduced by spectroscopic analysis while the absolute configuration depicted was inferred from chiroptical and spectroscopic analysis of various degradation products. 4 Synthetic studies toward these compounds have been initiated by several groups, 5,6 and the first total synthesis of reveromycin B (2) was recently reported by Theodorakis and Drouet. 7 A second total synthesis of 2 has also been communicated by Shimizu, Nakata, and co-workers. 8 We now report the asymmetric total

CAIR-1/BAG-3 forms an EGF-regulated ternary complex with Hsp70/Hsc70 and latent phospholipase C-g (PLC-g). The expression of CAIR-1, CAI stressed-1, was induced in A2058 human melanoma cells by continuous exposure to CAI, an inhibitor of nonvoltage-gated calcium in¯ux. CAIR-1 sequence is identical, save 2 amino acids, to BAG-3 also cloned recently as Bis, a member of the bcl-2-associated athanogene family. We show that CAIR-1/BAG-3 binds to Hsp70/Hsc70 in intact cells and this binding is increased by short term exposure to CAI (P50.007). CAIR-1/BAG-3 is phosphorylated in vivo in the absence of stimulation. Basal phosphorylation is inhibited by treatment with d-erythrosphingosine (d-ES), a broad inhibitor of the protein kinase C family. CAIR-1/BAG-3 contains several PXXP SH3 binding domains leading to the hypothesis that it is a partner protein of phospholipase C-g. PLC-g is bound to CAIR-1/BAG-3 in unstimulated cells. It is increased by CAI or d-ES (P=0.05) treatment, and abrogated by EGF (r 2 =0.99); d-ES treatment blocks the EGFmediated dissociation. We show that CAIR-1/BAG-3 binds to PLC-g and Hsp70/Hsc70 through separate and distinct domains. Hsp70/Hsc70 binds to the BAG domain of BAGs-1 and-3. CAIR-1/BAG-3 from control and EGF-treated cell lysates bound selectively to the SH3 domain of PLC-g, but not its N-SH2 or C-SH2 domains. Con®rming the SH3 interaction, PLC-g was pulled down by CAIR-1/BAG-3 PXXP-GST fusions, but GST-PXXP constructs confronted with lysates from EGF-treated cells did not bind PLC-g as was seen in intact cells. Hsp70/Hsc70 was brought down by the PLC-g SH3 construct equally from native and EGFtreated cells, but did not bind the PXXP construct under either condition. We propose that CAIR-1/BAG-3 may act as a multifunctional signaling protein linking the Hsp70/Hsc70 pathway with those necessary for activation of the EGF receptor tyrosine kinase signaling pathways. Oncogene (2000) 19, 4385 ± 4395.

Epidermal growth factor receptors (EGFR) and transferrin receptors (TFR) are known to be involved in cell proliferation and to be expressed in normal human epidermis. To date little is known about EGFR and TRF expression in human skin during embryonic and fetal development. In the present work, we studied skin specimens from 30 aborted embryos and fetuses ranging from 7 to 31 weeks estimated gestational age. Monoclonal antibodies to EGFR and TFR were applied on frozen skin sections using an amplification biotin-streptavidin-fluorescein technique. TFR was faintly expressed on epidermal basal cells throughout embryonic and fetal development, as it is in adult epidermis. Up to week 12, EGFR was uniformly expressed on cells of the basal, intermediate and periderm cell layers. From the midfetal period onwards, the suprabasal cell layers showed a decreased staining compared with the basal layer. During the third trimester the cornified cell layer was completely negative. The hair germ and heir peg cells were positive. Later, the outer root sheath and hair bulb remained labelled, with less staining of the hair cone. The sebaceous and eccrine sweat glands were also labelled. These results suggest that in embryonic and fetal epidermis, TFR expression is not correlated with cellular proliferation, whereas EGFR appear to be associated with proliferating and undifferentiated cells.

Long-term clinical observations and ongoing studies have shown significant antitumor effect of external Qi of Yan Xin Qigong which originated from traditional Chinese medicine. In order to understand the molecular and cellular mechanisms underlying the antitumor effect of external Qi of Yan Xin Qigong, we have examined its cytotoxic effect on BxPC3 pancreatic cancer cells and its effect on the Akt and extracellular signal-regulated kinase pathways. We found that external Qi of Yan Xin Qigong dramatically inhibited basal phosphorylation levels of Akt and extracellular signal-regulated kinases, epidermal growth factor-mediated phosphorylation of extracellular signal-regulated kinases, and phosphatidylinositol 3-kinase activity. External Qi of Yan Xin Qigong also inhibited constitutive and inducible activities of nuclear factor-kappa B, a target of the Akt and epidermal growth factor receptor pathways. Furthermore, a single 5 min exposure of BxPC3 cells to external Qi of Yan Xin Qigong induced apoptosis, accompanied by a dramatic increase of the sub-G1 cell population, DNA fragmentation, and cleavage of caspases 3, 8 and 9, and poly(ADP-ribose) polymerase. Prolonged treatment with external Qi of Yan Xin Qigong caused rapid lysis of BxPC3 cells. In contrast, treatment of fibroblasts with external Qi of Yan Xin Qigong induced transient activation of extracellular signal-regulated kinases and Akt, and caused no cytotoxic effect. These findings suggest that external Qi of Yan Xin Qigong may differentially regulate these survival pathways in cancer versus normal cells and exert cytotoxic effects preferentially on cancer cells, and that it could potentially be a valuable approach for therapy of pancreatic carcinomas.

The implantation process involves complex and synchronized molecular and cellular events between the uterus and the implanting embryo. These events are regulated by paracrine and autocrine factors. Trophoblast invasion and migration through the uterine wall is mediated by molecular and cellular interactions, controlled by the trophoblast and the maternal microenvironment. This review is focused on the molecular constituents of the human trophoblast, their actions and interactions, including interrelations with the uterine endometrium.

In this paper the results of investigations on the effect of interferon-alpha (IFN-alpha) on the growth of meningioma cells in culture is reported. A consecutive series of six meningiomas and one meningioma/neurofibroma derived from a patient with neurofibromatosis type 2 was investigated and it was found that the growth of all seven tumours in response to mitotic stimuli (fetal bovine serum or epidermal growth factor) is strongly inhibited by IFN-alpha. Maximal response varied between 100% and 70% inhibition of the incorporation of tritiated thymidine. In some cases an inhibitory response was obtained already at very low doses (less than or equal to 10 U of IFN-alpha per ml). These results indicate that further clinical investigation of the application of IFN-alpha to the treatment of meningioma is warranted.

It has become increasingly evident that growth cone guidance depends on the concerted actions of cytoskeletal proteins, molecular motors and cell adhesion molecules. Recent studies suggest that modulation of coupling between extracellular substrates and intracellular cytoskeletal networks via cell surface receptors is an important mechanism for regulating directed neuronal growth.

onstrates that both CNTF-induced STAT and ERK activation are involved in promoting Müller cell production. Moreover, absorbing epidermal growth factor (EGF) signals with a neutralizing antibody did not affect CNTF-induced Müller glial genesis, indicating that the effect of CNTF is not mediated by the known Müller-enhancing activity of EGF. Together, these results support a novel function of CNTF-like cytokines in retinal gliogenesis.

Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion.

▼ Introduction: Here, we present a stem-cell based study on the de-novo generation of beta-IIItubulin-positive neurons after treatment with the classic antipsychotic drug haloperidol or after treatment with the second-generation antipsychotic (SGA) ziprasidone. Methods: Adult neural stem cells (ANSC) dissociated from the adult mouse hippocampus were expanded in cell culture with basic fi broblast growth factor (bFGF) and epidermal growth factor (EGF). ANSC diff erentiated upon withdrawal of EGF and bFGF.

The expression of certain growth factors in the epidermal growth factor (EGF) family is altered in response to renal injury. Recent studies have demonstrated that heparin binding EGF-like growth factor (HB-EGF) expression may be cytoprotective in response to apoptotic signals. The purpose of this study was to investigate the potential role of HB-EGF in the upper urinary tract following unilateral ureteral obstruction. We present evidence that:

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C.

A human salivary intercalated duct cell line (HSG) is capable of morphological change to acinar-type cells, and of salivary amylase (AMY1) expression, by culturing on basement membrane extracts (BME). The aim of this study was to determine the critical conditions for functional and morphological differentiation of HSG cells and to establish if the processes are related. Cells were grown on BMEs that had different protein concentrations and growth factor content, and then examined with respect to morphology and AMY1 expression. To investigate the role of intracellular calcium in amylase expression, a pcDNA3.1-TRPC1alpha construct was used to overexpress htrp1alpha, which mediates the store-operated calcium entry in HSG cells. Expression of the AMY1, TRPC1alpha and beta genes was quantified by means of real time RT-PCR. Growth factor-reduced BME (12.8 mg/ml) induced multicellular acinar structures with lumen formation but without stimulation of either AMY1 or TRPC1. HSG cells cultured on higher concentration BME (17.5 or 16.4 mg/ml) formed reticular networks. AMY1 expression increased both on growth factor-reduced BME (17.5 mg/ml: 3.0-fold, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) and on regular BME (16.4 mg/ml: 3.7-fold, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) accompanied by a slight increase in expression of TRPC1alpha and TRPC1beta. Overexpression of htrp1alpha did not cause any significant changes in AMY expression, though it attenuated the BME (17.5 mg/ml)-induced AMY1 upregulation. Overall, the higher protein concentration BME favors amylase expression in HSG cells, whereas the lower concentration causes marked morphological changes.

Epidermal growth factor (EGF) is a common mitogenic factor that stimulates the proliferation of different types of cells, especially fibroblasts and epithelial cells. EGF activates the EGF receptor (EGFR/ErbB), which initiates, in turn, intracellular signaling. EGFR family is also expressed in neurons of the hippocampus, cerebellum, and cerebral cortex in addition to other regions of the central nervous system (CNS). EGF enhances the differentiation, maturation and survival of a variety of neurons. Transgenic mice lacking the EGFR developed neurodegenerative disease and die within the first month of birth. EGF acts not only on mitotic cells but also on postmitotic neurons, and many studies have indicated that EGF has neuromodulatory effect on various types of neurons in the CNS. This review highlights some of the major recent findings pertinent to the EGF and ErbB family with special references to elucidating their roles in the regulation of neurogenesis, signal transduction and trafficking and degradation.

Endocrinol Nutr. 2007;54(Supl 1):51-7 Palabras clave: Análogos de somatostatina. Radionúclido. Angiogénesis. Factor de crecimiento epidérmico. Correspondencia: Dra. M.A. Tomé Martínez de Retuerto. Castelo, s/n. 15229 Ames. La Coruña. España. Correo electrónico: [email protected]

The development of the mouse mammary gland requires the interaction between several different ovarian and pituitary hormones such as estrogen, progesterone and prolactin as well as several locally-derived growth factors in the mammary gland such as epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin (AR) and heregulin (HRG). The focus of this study was to investigate the degree of mammary growth and differentiation in the adult, virgin mammary gland of wild type (wt) and estrogen receptor knockout (ERKO) females that lack estrogen receptor α (ERα) after reciprocal transplantation into the cleared mammary fat pad of virgin wt or ERKO mice. In addition, we assessed the local response of ERKO mammary tissue to TGFα or HRGβ1 delivered from slow release-Elvax pellets. Our initial results indicated that when we transplanted virgin wt mammary tissue into ERKO mammary fat pads, mammary morphogenesis failed to occur. However, when transplanted virgin ERKO mammary tissue was transplanted into fat pads of virgin or pregnant wt mice, the development and differentiation of lobuloalveoli was readily observed. In addition, treatment of the virgin ERKO mammary gland with TGFα or HRGβ1 stimulated ducts to undergo localized branching and growth and both growth factors induced secretory differentiation as evidenced by the production of milk proteins, caseins and/or whey acidic protein (WAP). The results from this study imply that in ERKO mammary tissue, ERKO ductal epithelium has the capacity to proliferate and differentiate in response to non-estrogenic, morphogenic stimuli.

A pregnancy rate of approximately 15% per cycle renders the process of human reproduction inefficient. The cycle-dependent expression of molecules involved in the embryo-endometrial dialogue has lead to the identification of a 'window of implantation'. This is the unique temporal and spatial expression of factors that allows the embryo to implant (via signalling, appositioning, attachment and invasion) in a specific time frame of 48 h, 7-10 days after ovulation. Integrin molecules, L-selectin ligands, mucin-1, heparin-binding epidermal growth factor and pinopodes are involved in appositioning and attachment. The embryo produces cytokines and growth factors [interleukins, prostaglandins, vascular endothelial growth factor (VEGF)] and receptors for endometrial signals (leukaemia inhibitory factor receptor, colony stimulating factor receptor, insulin-like growth factors and heparin binding epidermal growth factor receptor). The immune system plays an important role. Immunomodulatory factors such as glycodelin, inhibin and interleukin prevent a graft-versus-host reaction. Angiogenesis controlled by VEGF and prostaglandins is needed for formation of a receptive endometrium and a placenta. Identification of these factors has led to their use as markers of implantation that may identify defects causing subfertility. An ideal marker of implantation is sensitive and specific, and easy to obtain without disturbing implantation. Glycodelin and leukaemia inhibitory factor (serum) and integrins and pinopodes (biopsies) are promising candidates.

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range of overlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aortic features. The factors that modulate phenotypical severity, both among and within families, remain to be determined. The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to perform the largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the nature and severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular, ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in 1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with a missense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients with an FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframe mutation. Mutations in exons 24-32 were associated with a more severe and complete phenotype, including younger age at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation, aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicated even when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinical manifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsufficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGFb signalling). Exon 24-32 mutations define a high-risk group for cardiac manifestations associated with severe prognosis at all ages.

There is now extensive evidence to indicate that components of the cAMP signaling pathway are up-regulated in the human myometrium during pregnancy so as to potentiate the maintenance of uterine quiescence until term. In many tissue and cell types, increased signaling of the cAMP pathway results in profound changes in gene expression that are catalyzed via stimulation of PKA and activation of cAMP-dependent transcription factors that bind cAMP response elements (CREs) within the promoter regions of affected genes. In the myometrium, these CRE containing genes include ␤2-adrenoceptor, cyclo-oxygenase 2, oxytocin receptor, and connexin-43. In preliminary investigations, we reported the differential expression of members of the cAMP bZIP protein family in the myometrium during pregnancy and labor. In this present study, we have now identified and functionally characterized these proteins with respect to myometrial gene expression. We report the identification of a 39,000 mol wt CRE response element modulator protein (CREM)2␣ protein having both

The superfamily of G-protein-coupled receptors (GPCRs) could be subclassified into 7 families (A, B, large N-terminal family B-7 transmembrane helix, C, Frizzled/Smoothened, taste 2, and vomeronasal 1 receptors) among mammalian species. Cloning and functional studies of GPCRs have revealed that the superfamily of GPCRs comprises receptors for chemically diverse native ligands including (1) endogenous compounds like amines, peptides, and Wnt proteins (i.e., secreted proteins activating Frizzled receptors); (2) endogenous cell surface adhesion molecules; and (3) photons and exogenous compounds like odorants. The combined use of site-directed mutagenesis and molecular modeling approaches have provided detailed insight into molecular mechanisms of ligand binding, receptor folding, receptor activation, G-protein coupling, and regulation of GPCRs. The vast majority of family A, B, C, vomeronasal 1, and taste 2 receptors are able to transduce signals into cells through G-protein coupling. However, G-protein-independent signaling mechanisms have also been reported for many GPCRs. Specific interaction motifs in the intracellular parts of these receptors allow them to interact with scaffold proteins. Protein engineering techniques have provided information on molecular mechanisms of GPCR-accessory protein, GPCR-GPCR, and GPCR-scaffold protein interactions. Site-directed mutagenesis and molecular dynamics simulations have revealed that the inactive state conformations are stabilized by specific interhelical and intrahelical salt bridge interactions and hydrophobic-type interactions. Constitutively activating mutations or agonist binding disrupts such constraining interactions leading to receptor conformations that associates with and activate G-proteins. .no (K. Kristiansen).

Guided bone regeneration is an accepted surgical method employed in implant dentistry to increase the quantity and quality of the host bone in areas of localized alveolar defects. The lack of predictability in osseous regenerative procedures with various grafting materials suggests that improvement in the osteoinductive properties of these materials is highly desirable. Platelet-rich plasma (PRP), a modification of fibrin glue made from autologous blood, is being used to deliver growth factors in high concentration to sites requiring osseous grafting. Growth factors released from the platelets include platelet-derived growth factor, transforming growth factor beta, platelet-derived epidermal growth factor, platelet-derived angiogenesis factor, insulin-like growth factor 1, and platelet factor 4. These factors signal the local mesenchymal and epithelial cells to migrate, divide, and increase collagen and matrix synthesis. PRP has been suggested for use to increase the rate of bone de...

The intestine has an inherent ability to adapt morphologically and functionally in response to internal and external environmental changes. The functional adaptations encompass modifications of the brush border membrane fluidity and permeability, as well as up-or down-regulation of carrier-mediated transport. Intestinal adaptation improves the nutritional status following the loss of a major portion of the small intestine, following chronic ingestion of ethanol, following sublethal doses of abdominal irradiation, in diabetes, in pregnancy and lactation, with ageing, and with fasting and malnutrition. Following intestinal resection, morphological and functional changes occur depending upon the extent of the intestine removed, the site studied, and the lipid content of the diet. Therefore, intestinal adaptation has important implications in the survival potential and welfare of the host. An understanding of the mechanisms of, and signals for, intestinal adaptation in the experimental setting forms the basis for the use of management strategies in humans with the short-bowel syndrome.

Liposomes are of interest as drug delivery tools for therapy of cancer and infectious diseases. We investigated conjugation of epidermal growth factor, EGF, to liposomes using the micelle-transfer method. EGF was conjugated to the distal end of PEG-DSPE lipid molecules in a micellar solution and the EGF-PEG-DSPE lipids were then transferred to preformed liposomes, either empty or containing the DNA-binding compound, water soluble acridine, WSA. We found that the optimal transfer conditions were a 1-h incubation at 60°C. The final conjugate, 125 I-EGF-liposome-WSA, contained approximately 5 mol % PEG, 10-15 EGF molecules at the liposome surface, and 10 4 to 10 5 encapsulated WSA molecules could be loaded. The conjugate was shown to have EGF-receptor-specific cellular binding in cultured human glioma cells.

We have previously shown that epidermal growth factor (EGF) augments CAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism . More recently, employing an antibody against the carboxy-terminus decapeptide of G , , ,

Colorectal cancer (CRC) is common in North America. Metastatic disease is present at diagnosis in 30% of the patients, and approximately half of early-stage patients will eventually present with metastatic disease. Until recently, few chemotherapy options were available to treat metastatic CRC. The authors review the results of recent clinical trials and the design of ongoing trials in the management of patients with metastatic colorectal cancer. Fluorouracil (5-FU) with leucovorin (LV) modulation has a marginal but positive effect on survival in those patients. The recent incorporation of irinotecan (CPT-11) and oxaliplatin for the management of advanced CRC has generated further improvement in survival. The development of oral fluoropyrimidines, mimicking continuous infusion 5-FU, is convenient. In randomized trials, capecitabine was equally effective to bolus 5-FU and LV in the management of metastatic CRC. Recently completed or ongoing clinical trials to study novel targeting ag...

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro-matrix metalloproteinase (MMP)-2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP-2 was not in its active form, we used Western blot to study the expression of members of the MMP-2 activation pathway (membrane type 1-MMP and tissue inhibitor of metalloproteinase-2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol-12-myristate-13-acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP-2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.