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PubMed2005

Finite element implementation of a generalized Fung-elastic constitutive model for planar soft tissues.

Numerical simulations of the anisotropic mechanical properties of soft tissues and tissue-derived biomaterials using accurate constitutive models remain an important and challenging research area in biomechanics. While most constitutive modeling efforts have focused on the characterization of experimental data, only limited studies are available on the feasibility of utilizing those models in complex computational applications. An example is the widely utilized exponential constitutive model proposed by Fung. Although present in the biomechanics literature for several decades, implementation of this model into finite element (FE) simulations has been limited. A major reason for limited numerical implementations are problems associated with inherent numerical instability and convergence. To address this issue, we developed and applied two restrictions for a generalized Fung-elastic constitutive model necessary to achieve numerical stability. These are (1) convexity of the strain energy function, and (2) the condition number of material stiffness matrix set lower than a prescribed value. These constraints were implemented in the nonlinear regression used for constitutive model parameter estimation to the experimental biaxial mechanical data. We then implemented the generalized Fung-elastic model into a commercial FE code (ABAQUS, Pawtucket, RI, USA). Single element and multi-element planar biaxial test simulations were conducted to verify the accuracy and robustness of the implementation. Results indicated that numerical convergence and accurate FE implementation were consistently obtained. The present study thus presents an integrated framework for accurate and robust implementation of pseudo-elastic constitutive models for planar soft tissues. Moreover, since our approach is formulated within a general FE code, it can be straightforwardly adopted across multiple software platforms.

Anisotropy↗
PubMed2004

Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-kappaB in human HPV-18 E6-positive HEp-2 cells.

Because the role of nuclear factor kappaB (NF-kappaB) is in cellular growth control and neoplasia, we explored the status of NF-kappaB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-kappaB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-kappaB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IkappaBalpha, IkappaBalpha super repressor (IkappaBalpha-SR), suppressed constitutive NF-kappaB activity as well as proliferation, suggesting that constitutive NF-kappaB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-kappaB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IkappaBalpha phosphorylation leading to stabilization of IkappaBalpha protein in the cells. Carboplatin inhibited NF-kappaB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-kappaB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-kappaB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.

Antineoplastic Agents↗
PubMed2005

Curcumin (diferuloylmethane) inhibits constitutive NF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma.

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-kappaB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-kappaB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-kappaB and IkappaB kinase and phosphorylated forms of IkappaBalpha and p65. This correlated with expression of TNF, IkappaBalpha, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-kappaB. On treatment of cells with curcumin, however, downregulated constitutive active NF-kappaB and inhibited the consitutively active IkappaBalpha kinase (IKK), and phosphorylation of IkappaBalpha and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-kappaB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-kappaB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-kappaB complex and inhibited the proliferation of MCL cells. Constitutive NF-kappaB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-kappaB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-kappaB and IKK leading to suppression of expression of NF-kappaB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.

Active Transport, Cell Nucleus↗
PubMed2006

Active Ras-induced effects on skeletal myoblast differentiation and apoptosis are independent of constitutive PI3-kinase activity.

23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts. Surprisingly, while expression of either G12V:H-Ras or F156L:H-Ras results in constitutive signaling through PI3-kinase, only cells expressing G12V:H-Ras additionally possess constitutive signaling through MAPK, and NFkappaB. Pharmacological abrogation of the Ras-induced constitutive PI3-kinase signal, however, is not responsible for the impaired differentiation or apoptosis in either A2:G12V:H-Ras myoblasts or A2:F156L:H-Ras myoblasts. Thus, our data suggest that a pathway distinct from those that signals through MAPK, NFkappaB or PI3-kinase is responsible for the impaired differentiation and apoptosis in 23A2 skeletal myoblasts expressing constitutively active Ras.

Animals↗
PubMed2006

Constitutive and IFNgamma-induced activation of MHC2TA promoter type III in human melanoma cell lines is governed by separate regulatory elements within the PIII upstream regulatory region.

Cell lines established from tumor tissue of cutaneous melanoma biopsies often display constitutive and IFNgamma-inducible expression of MHC class II molecules. The expression of MHC class II molecules in melanoma is associated with an overall poor prognosis and unfavorable clinical outcome. We have analyzed the DNA elements and interacting transcription factors that control the constitutive and IFNgamma-inducible expression of the class II transactivator (CIITA), a co-activator essential for transcription of all MHC class II genes. Our studies reveal the activation of multiple CIITA promoter regions (CIITA-PII, -PIII and -PIV) in melanoma cell lines for both the constitutive and IFNgamma-inducible expression of MHC class II molecules. Furthermore, we show that constitutive and IFNgamma-inducible expression of the CIITA-PIII isoform is governed by separate regulatory elements within the PIII upstream regulatory region (PURR). Similarly constitutive activation in melanoma of CIITA-PII, CIITA-PIII, and CIITA-PIV does not require components of the IFNgamma signaling pathway. However, these components are readily recruited to the PURR and CIITA-PIV after exposure of cells to IFNgamma and account for the IFNgamma-induced expression of CIITA. Together, our data reveal the contribution of distinct elements and factors in the constitutive and IFNgamma-inducible expression of CIITA in melanoma cell lines of the skin.

Antiviral Agents↗
PubMed2004

Constitutional chromosome aberrations as pathogenetic events in hematologic malignancies.

A predisposition to tumor development is associated with some constitutional chromosomal abnormalities. Investigations of families with an apparent hereditary cancer and constitutional chromosome rearrangements have led to the molecular identification of tumor suppressor genes. Under the somatic mutation theory for the development of cancer, two mutational events are required. The first step may be a constitutional event and the second an acquired genetic mutation. Cytogenetic studies were performed on 5633 bone marrow specimens from patients with hematologic malignancies from a single institution. Fifty cases of constitutional chromosome aberrations were detected. Data collected from the literature and from our series are reviewed and compared with the incidence of specific constitutional chromosome aberrations in the newborn population. Possible mechanisms that may predispose individuals with constitutional chromosome aberrations to the development of a hematologic malignancy are reviewed.

Adolescent↗
PubMed1998

Constitutive and interferon-gamma-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1.

Proteasomes generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. During viral infection, interferon-gamma (IFN-gamma) acts as a cytokine altering the catalytic specificity of proteasomes by inducing the synthesis of the three proteasome subunits, low molecular weight protein (LMP) 2, LMP7 and multicatalytic endopeptidase complex-like 1 (MECL1). LMP2 and LMP7 have been shown to favour the presentation of certain antigenic peptides. These subunits are constitutively expressed in cell lines related to the immune system and IFN-gamma-inducible in other cell lines. Less is known about MECL1. To reveal the extent of constitutive and IFN-gamma-induced expression of MECL1, we studied MECL1 in different cell lines by Northern and Western blotting. The two B cell lines IM9 and Reh showed high constitutive expression of MECL1, only slightly induced by IFN-gamma stimulation. The B cell line Daudi and the monocyte cell line THP-1 expressed MECL1 constitutively at an intermediate level. The MECL1 protein level in the THP-1 cells increased markedly in response to IFN-gamma. In cells unrelated to the immune system, a very low constitutive expression of MECL1 was detected, highly inducible by IFN-gamma. These results indicate that, similar to LMP2 and LMP7, MECL1 is constitutively expressed at high levels only in certain cell lines and can be induced by IFN-gamma in other cell lines. The differential expression of MECL1 may be of importance for which antigenic peptides are presented by different cells as well as by the same cells at different IFN-gamma levels.

Adenocarcinoma↗
PubMed2001

A set of constitutive relationships accounting for residual NAPL in the unsaturated zone.

Although laboratory experiments show that non-aqueous phase liquid (NAPL) is retained in the unsaturated zone, no existing multiphase flow model has been developed to account for residual NAPL after NAPL drainage in the unsaturated zone. We developed a static constitutive set of saturation-capillary pressure relationships for water, NAPL and air that accounts for both this residual NAPL and entrapped NAPL. The set of constitutive relationships is formulated similarly to the set of scaled relationships that is frequently applied in continuum models. The new set consists of three fluid-phase systems: a three-phase system and a two-phase system, that both comply with the original constitutive model, and a newly introduced residual NAPL system. The new system can be added relatively easily to the original two- and three-phase systems. Entrapment is included in the model. The constitutive relationships of the non-drainable residual NAPL system are based on qualitative fluid behavior derived from a pore scale model. The pore scale model reveals that the amount of residual NAPL depends on the spreading coefficient and the water saturation. Furthermore, residual NAPL is history-dependent. At the continuum scale, a critical NAPL pressure head defines the transition from free, mobile NAPL to residual NAPL. Although the Pc-S relationships for water and total liquid are not independent in case of residual NAPL, two two-phase Pc-S relations can represent a three-phase residual system of Pc-S relations. A newly introduced parameter, referred to as the residual oil pressure head, reflects the mutual dependency of water and oil. Example calculations show consistent behavior of the constitutive model. Entrapment and retention in the unsaturated zone cooperate to retain NAPL. Moreover, the results of our constitutive model are in agreement with experimental observations.

Models, Theoretical↗
PubMed1998

Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain.

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.

Alleles↗
PubMed2004

Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta).

Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.

Alanine↗
PubMed2006

Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations.

Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.

Amino Acid Sequence↗
PubMed2001

Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor.

The wild-type beta2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant beta2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant beta2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the beta2-adrenoceptor. Co-expression of the wild-type beta2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant beta2-adrenoceptor-Renilla luciferase and an equivalent mutant of the alpha1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.

Adenylyl Cyclases↗
PubMed1999

Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA.

The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential.

Adenocarcinoma↗
PubMed2001

Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells.

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3beta) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-x(L). Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Apoptosis↗
PubMed2004

Secretion of cytokines and growth factors as a general cause of constitutive NFkappaB activation in cancer.

The constitutive activation of nuclear factor kappaB (NFkappaB) helps a variety of tumors to resist apoptosis and desensitizes them to chemotherapy, but the causes are still largely unknown. We have analysed this phenomenon in eight mutant cell lines derived from human 293 cells, selected for NFkappaB-dependent expression of a marker gene, and also in seven tumor-derived cell lines. Conditioned media from all of these cells stimulated the activation of NFkappaB (up to 30-fold) in indicator cells carrying an NFkappaB-responsive reporter. Therefore, secretion of extracellular factors as the cause of constitutive activation seems to be general. The mRNAs encoding several different cytokines and growth factors were greatly overexpressed in the tumor and mutant cells. The pattern of overexpression was distinct in each cell line, indicating that the phenomenon is complex. Two secreted factors whose roles in the constitutive activation of NFkappaB are not well defined were investigated further as pure proteins: transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 5 (FGF5) were both highly expressed in some mutant clones and tumor cell lines, each activated NFkappaB alone, and the combination was synergistic. Our data indicate that a group of different factors, expressed at abnormally high levels, can contribute singly and synergistically to the constitutive activation of NFkappaB in all of the mutant and tumor cell lines we studied. Since several NFkappaB target genes encode secreted proteins that induce NFkappaB, autocrine loops are likely to be ubiquitously important in the constitutive activation of NFkappaB in cancer. We provide the first evidence of the general, complex, and synergistic activation of NFkappaB in tumor and mutant cell lines through the action of secreted factors and suggest that the same explanation is likely for the constitutive activation of NFkappaB in cancers.

Cell Line, Tumor↗
PubMed2001

Constitutive shedding of the amyloid precursor protein ectodomain is up-regulated by tumour necrosis factor-alpha converting enzyme.

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells.

ADAM Proteins↗
PubMed2000

Functional characterization of five constitutively activating thyrotrophin receptor mutations.

OBJECTIVE: Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments. METHODS: The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined. RESULTS: In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway. CONCLUSIONS: The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.

Animals↗
PubMed2001

Do constitutional types really exist? A further study using grade of membership analysis.

'Constitutional types' are a feature of homeopathy. Constitutions are constellations of mental, physical and general features. We present results of a 152-item 'Constitutional Type Questionnaire' (CTQ), relating to 19 major constitutional remedies. Patients referred to the Royal London Homoeopathic Hospital (RLHH) were recruited from outpatient clinics. Each remedy was assigned eight associated features. Each item is rated as it applies to the subject most of the time on a five-point scale. Grade of Membership analysis (GOM) was used for evaluation of remedy profiles. Four hundred and forty-seven individuals provided usable data. Demographic data were collected. A nine pure type solution provided the best fit to the data. Few subjects had exclusive 'membership' of one pure type. There are some difficulties in interpretation; for instance some groups were characterised by the absence of 'constitutional' features. Some pure types are coherent, recognisable types. The results give some support to the homeopathic concept of constitution. CTQ results correlate quite well with the actual prescribing of homeopaths. Further validation with other cohorts is required and with refinement it might become a useful screening method. It cannot simulate complex information processing performed by homeopathic practitioners.

Female↗