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PubMed1994

Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides.

Messenger RNA levels of the cytochrome P450 gene CYP6A1 were measured in the insecticide resistant Diazinon-R 'Rutgers' strain and in the susceptible strain sbo of the house fly with a cloned cDNA probe. The constitutive expression of the CYP6A1 gene was at least 10 times higher in the Rutgers strain than in the sbo strain. In both strains, CYP6A1 was inducible by phenobarbital treatment of the flies. Analysis of genomic DNA from the two strains indicated that there was no amplification of the CYP6A1 gene in the Rutgers strain. A developmental analysis revealed that CYP6A1 is expressed larvae and adults, and very low levels of CYP6A1 mRNA were detected in eggs and pupae. The constitutive overexpression of CYP6A1 in the Rutgers strain was observed in both larvae and adults. Crosses between the Rutgers strain and the multiply marked sbo strain revealed that the high constitutive expression of CYP6A1 in the Rutgers strain is controlled by one or more loci located on chromosome II.

Animals↗
PubMed1994

ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR zeta in murine thymocytes and lymph node T cells.

Studies with T cell lines and clones have shown that engagement of the TCR results in the tyrosine phosphorylation of the TCR subunits. This leads to the recruitment of the ZAP-70 protein tyrosine kinase, an interaction involving the two SH2-domains of ZAP-70 with tyrosine-phosphorylated zeta and CD3. However, as previously described, murine thymocytes and lymph node T cells express a constitutively tyrosine-phosphorylated zeta subunit in the basal state. Here, we show that a fraction of ZAP-70 molecules are constitutively associated with tyrosine-phosphorylated zeta. TCR ligation promotes a large increase in the tyrosine phosphorylation of ZAP-70 as well as other TCR subunits. Genetic studies reveal that the constitutive ZAP-70 association with tyrosine-phosphorylated zeta does not absolutely require either TCR or coreceptor interactions with MHC molecules.

Animals↗
PubMed2003

Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts.

The nuclear factor-E2 p45-related factor (Nrf) 2 is a transcription factor for the antioxidant responsive element-mediated induction of enzymes responsible for conjugation. Since multidrug resistance-associated protein1 (Mrp1/Abcc1) is an ATP-binding cassette transporter which plays an important role in the cellular extrusion of conjugated metabolites and, therefore, acts synergistically with conjugating enzymes for the cellular detoxification of xenobiotics, we examined the possibility that Nrf2 is also involved in the expression of Mrp1 in mouse embryo fibroblasts. The constitutive expression levels of Mrp1 mRNA and protein were significantly lower in Nrf2 (-/-) cells compared with those in wild type cells. In addition, significant induction by diethyl maleate was observed in wild type, but not in Nrf2 (-/-), cells, suggesting the involvement of Nrf2 in both the constitutive and inducible mRNA and protein expression of Mrp1. In addition, the uptake of [3H]2,4-dinitrophenyl-S-glutathione, a typical substrate of Mrp1, into isolated membrane vesicles also demonstrated that Nrf2 regulates the transport activity of glutathione conjugates in mouse fibroblasts. This is the first demonstration that Nrf2 is required for the constitutive and inducible expression of Mrp family proteins.

Adenosine Triphosphate↗
PubMed2005

Constitutive activity of endogenous receptors by inducible Gq overexpression.

We have developed an inducible cell line that transiently expresses Gq alpha G protein subunits in response to doxycycline. HEK293/Tet-On pBI(Gq alpha) cells worked consistently, achieving high and tightly regulated levels of Gq alpha overexpression (38-fold increase compared with non-induced cells). We investigated the possibility of using an inducible system to increase the proportion of constitutively active endogenously expressed G protein-coupled receptors (GPCRs) by overexpressing Gq alpha. Not only did we observe an increase in basal activity following doxycycline treatment, but also increased intrinsic activity of agonists such as carbachol, endothelin, lysophosphatidic acid (LPA), and bradykinin. Furthermore, carbachol and LPA potency increased following Gq alpha overexpression, as did the intrinsic activity of the partial agonist pilocarpine, observations indicative of constitutive activity. An inducible cell line, transiently expressing G proteins, can therefore be employed to induce constitutive activity of endogenously expressed GPCRs. This model system could be used to identify clinically important inverse agonists.

Animals↗
PubMed2006

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells.

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.

Animals↗
PubMed2006

ADAM19 is tightly associated with constitutive Alzheimer's disease APP alpha-secretase in A172 cells.

To elucidate whether new proteases are involved in the processing of amyloid precursor protein (APP), we examined catalytically active ADAM12 and ADAM19 as candidates alpha-secretases. The overexpression of ADAM19 in HEK293 cells resulted in an increase in sAPPalpha. Therefore, we suggest that ADAM19 has a constitutive alpha-secretase activity. We examined regulated alpha-secretase activity by adding phorbol 12-myristate 13-acetate (PMA), but no regulated activity was found. To verify that endogenous ADAM19 has an APP alpha-secretase activity, we examined whether the constitutive level of alpha-secretase activity was reduced by RNA interference with ADAM19 in A172 cells. The amount of secreted sAPPalpha decreased by about 21% following RNAi. These results suggest that ADAM19 has a constitutive alpha-secretase activity for APP.

ADAM Proteins↗
PubMed2006

Constitutive activity and inverse agonism at the alpha1adrenoceptors.

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2004

An augmented hybrid constitutive model for simulation of unloading and cyclic loading behavior of conventional and highly crosslinked UHMWPE.

Ultra-high molecular weight polyethylene (UHMWPE) is extensively used in total joint replacements. Wear, fatigue, and fracture have limited the longevity of UHMWPE components. For this reason, significant effort has been directed towards understanding the failure and wear mechanisms of UHMWPE, both at a micro-scale and a macro-scale, within the context of joint replacements. We have previously developed, calibrated, and validated a constitutive model for predicting the loading response of conventional and highly crosslinked UHMWPE under multiaxial loading conditions (Biomaterials 24 (2003) 1365). However, to simulate in vivo changes to orthopedic components, accurate simulation of unloading behavior is of equal importance to the loading phase of the duty cycle. Consequently, in this study we have focused on understanding and predicting the mechanical response of UHMWPE during uniaxial unloading. Specifically, we have augmented our previously developed constitutive model to also allow for accurate predictions of the unloading behavior of conventional and highly crosslinked UHMWPE during cyclic loading. It is shown that our augmented hybrid model accurately captures the experimentally observed characteristics, including uniaxial cyclic loading, large strain tension, rate-effects, and multiaxial deformation histories. The augmented hybrid constitutive model will be used as a critical building block in future studies of fatigue, failure, and wear of UHMWPE.

Biocompatible Materials↗
PubMed2005

A case of myelodysplastic syndrome with acquired monosomy 7 in a child with a constitutional t(1;19) and a mosaicism for trisomy 21.

A 3-year-old patient presented with anemia, thrombocytopenia, and blasts in the peripheral blood. A bone marrow aspirate revealed a myelodysplastic syndrome (MDS). A mosaic abnormal female karyotype 46,XX, t(1;19)(q42; p13.1)c[12]/ 47,idem,+21c[3]/ 47,idem,-7,+21c,+mar[7] was obtained on G-banded metaphases from unstimulated bone marrow aspirate cell culture. To rule out constitutional abnormalities, we performed a cytogenetic analysis on the patient's phytohemagglutinin-stimulated peripheral blood and cultured skin fibroblasts. A karyotype of 46,XX,t(1;19) (q42;p13.1)c was found in all 20 peripheral lymphocytes analyzed, confirming the constitutional origin of the translocation. In addition, 5 out of 50 cells from two separate cultures of the skin fibroblasts contained an extra chromosome 21. The presence of two cell lines in multiple cultures indicates that the patient is a true low-level mosaic for trisomy 21. Because of the finding of monosomy 7 and a marker chromosome only in the trisomy 21 clone, we conclude that the leukemic clone arose from a hematopoietic precursor with constitutional trisomy 21. It is also possible that the t(1;19) played some role in the development of the MDS. Because acute myelogenous leukemia (AML) and MDS with Down syndrome (DS) have distinct biologic and clinical features, the identification of DS patients with a mild or normal phenotype in the AML/MDS population is of fundamental importance for clinical diagnosis and management.

Child, Preschool↗
PubMed2005

The constitution of mineral trioxide aggregate.

OBJECTIVES: The aim of this study was to determine the constitution of a commercially available root-end filling material, mineral trioxide aggregate, (MTA) (ProRoot MTA, Tulsa Dental, Tulsa, OK, USA). The surface morphology of the material with various treatment conditions was also evaluated. METHODS: The constitution of two commercial versions of MTA was determined before and after mixing with water. The unset material was analysed using Energy Dispersive Analysis by X-ray (EDAX) in a scanning electron microscope (SEM) and X-ray diffraction (XRD). The first technique identified the constituent elements while XRD analysis identified the compounds or phases present. The set material was evaluated using EDAX. The surface morphology of the material stored under various conditions (100% humidity, immersion in water, or immersion in phosphate solution) was evaluated using SEM. RESULTS: The EDAX showed the white MTA to be composed primarily of calcium, silicon, bismuth and oxygen, with the gray MTA also having small peaks for iron and aluminum. The XRD analysis showed gray MTA to be composed primarily of tricalcium silicate and dicalcium silicate. The surface morphology of the materials differed under the various conditions, particularly following immersion in phosphate solution with crystal formation. SIGNIFICANCE: The commercial versions of MTA were shown to have broadly similar constitution to ordinary Portland cement except for the addition of bismuth compounds. The white MTA did not contain iron.

Aluminum↗
PubMed2005

A "locked-on," constitutively active mutant of the adenosine A1 receptor.

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or "constitutively" active in a [35S]GTPgammaS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar "locked-on" phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPgammaS and the cAMP assay, unlike the wild-type and the two other mutant receptors.

Adenosine↗
PubMed2005

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells.

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.

Amino Acid Sequence↗
PubMed2003

Mesoscale constitutive modeling of magnetic dispersions: material functions for shear flows.

We recently developed a constitutive model for magnetic dispersions by modeling the magnetic particles as rigid dumbbells dispersed in a solvent. The theory yielded a constitutive equation in which the stress tensor could be expressed as a function of the velocity gradient, an orientational order tensor, S, an average alignment vector, J, and any imposed external magnetic field, H. The constitutive equation is used here to predict material functions for steady shear flow (shear-rate dependent viscosity and first normal stress coefficient) as well as those for unsteady shear flows (stress growth upon inception of steady shear and small-amplitude oscillatory shear). The importance of effects of concentration, equilibrium nematic ordering in the dispersion, and anisotropy in the hydrodynamic drag are emphasized. Comparisons with available experimental data on viscosity for magnetic inks under steady shear flow and inception of steady shear flow show reasonably good agreement.

Journal Article↗
PubMed2004

A constitutive model for air-NAPL-water flow in the vadose zone accounting for immobile, non-occluded (residual) NAPL in strongly water-wet porous media.

A hysteretic constitutive model describing relations among relative permeabilities, saturations, and pressures in fluid systems consisting of air, nonaqueous-phase liquid (NAPL), and water is modified to account for NAPL that is postulated to be immobile in small pores and pore wedges and as films or lenses on water surfaces. A direct outcome of the model is prediction of the NAPL saturation that remains in the vadose zone after long drainage periods (residual NAPL). Using the modified model, water and NAPL (free, entrapped by water, and residual) saturations can be predicted from the capillary pressures and the water and total-liquid saturation-path histories. Relations between relative permeabilities and saturations are modified to account for the residual NAPL by adjusting the limits of integration in the integral expression used for predicting the NAPL relative permeability. When all of the NAPL is either residual or entrapped (i.e., no free NAPL), then the NAPL relative permeability will be zero. We model residual NAPL using concepts similar to those used to model residual water. As an initial test of the constitutive model, we compare predictions to published measurements of residual NAPL. Furthermore, we present results using the modified constitutive theory for a scenario involving NAPL imbibition and drainage.

Air↗
PubMed2004

A constitutive model for air-NAPL-water flow in the vadose zone accounting for immobile, non-occluded (residual) NAPL in strongly water-wet porous media.

A hysteretic constitutive model describing relations among relative permeabilities, saturations, and pressures in fluid systems consisting of air, nonaqueous-phase liquid (NAPL), and water is modified to account for NAPL that is postulated to be immobile in small pores and pore wedges and as films or lenses on water surfaces. A direct outcome of the model is prediction of the NAPL saturation that remains in the vadose zone after long drainage periods (residual NAPL). Using the modified model, water and NAPL (free, entrapped by water, and residual) saturations can be predicted from the capillary pressures and the water and total-liquid saturation-path histories. Relations between relative permeabilities and saturations are modified to account for the residual NAPL by adjusting the limits of integration in the integral expression used for predicting the NAPL relative permeability. When all of the NAPL is either residual or entrapped (i.e., no free NAPL), then the NAPL relative permeability will be zero. We model residual NAPL using concepts similar to those used to model residual water. As an initial test of the constitutive model, we compare predictions to published measurements of residual NAPL. Furthermore, we present results using the modified constitutive theory for a scenario involving NAPL imbibition and drainage.

Air↗
PubMed2004

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests.

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.

Amidohydrolases↗
PubMed2006

Arylamine N-acetyltransferase aggregation and constitutive ubiquitylation.

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.

Amino Acid Sequence↗
PubMed2006

Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: the Leishmania genome is constitutively expressed.

Leishmania are protozoan parasites that cause a wide spectrum of clinical diseases in humans and are a major public health risk in several countries. Leishmania life cycle consists of an extracellular flagellated promastigote stage within the midgut of a sandfly vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host. This study reports the use of DNA oligonucleotide genome microarrays representing 8160 genes to analyze the mRNA expression profiles of L. major promastigotes and lesion derived amastigotes. Over 94% of the genes were expressed in both life stages. Advanced statistical analysis identified a surprisingly low degree of differential mRNA expression: 1.4% of the total genes in amastigotes and 1.5% in promastigotes. These microarray results demonstrate that the L. major genome is essentially constitutively expressed in both life stages and suggest that Leishmania is constitutively adapted for survival and replication in either the sandfly vector or macrophage host utilizing an appropriate set of genes for each vastly different environment. Quantitative proteomics, using the isotope coded affinity tag (ICAT) technology and mass spectrometry, was used to identify L. infantum promastigote and axenic amastigote differentially expressed proteins. Of the 91 distinct proteins identified, 8% were differentially expressed in the amastigote stage, 20% were differentially expressed in the promastigote stage, and the remaining 72% were considered constitutively expressed. The differential expression was validated by the identification of previously reported stage specific proteins and identified several amastigote and promastigote novel stage specific proteins.

Adaptation, Physiological↗