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PubMed2002

Constitutive activation of met kinase in non-small-cell lung carcinomas correlates with anchorage-independent cell survival.

Lung cancer is currently the most frequent cause of cancer death in North America. Hepatocyte growth factor (HGF) and its receptor Met are frequently over-expressed in non-small-cell lung carcinomas (NSCLC), but their potential role in tumor progression is not clearly known. To assess the role of HGF/Met signaling in lung carcinomas, we have examined the expression, activation status, and function of Met in NSCLC cell lines (n = 7), established from primary tumors or pleural fluids of cancer patients. We observed Met expression in three NSCLC cell lines, two of which exhibited constitutive tyrosine-phosphorylation of Met, and Met kinase activity. In addition, the observed constitutive activation of Met was sustained under anchorage-independent conditions, and correlated with phosphatidyl inositol 3-kinase-dependent cell survival. Immunoreactive HGF-like protein was secreted by two Met-positive and two Met-negative NSCLC cell lines. However HGF activity, as determined by the ability to induce cell scattering and tyrosine-phosphorylation of Met in reporter cell lines, was detected in conditioned medium from only one Met-negative NSCLC cell line: none of the conditioned media from Met-expressing NSCLC cell lines showed detectable HGF activity. Thus, constitutive activation of Met in NSCLC cell lines may occur at least in part through intracrine, or HGF-independent mechanisms. Interestingly, additional paracrine stimulation with exogenous recombinant HGF was required for DNA synthesis and correlated with increased activation of ERK1/2 in all Met-positive NSCLC cell lines, regardless of the basal activation status of Met. These findings indicate that a medium level of constitutive activation of Met occurs in some NSCLC cell lines, and correlates with survival of detached carcinoma cells; whereas additional paracrine stimulation by recombinant HGF is required for DNA synthesis. Thus constitutive and paracrine activation of Met may provide complementary signals that promote survival and proliferation, respectively, during tumor progression of NSCLC.

Animals↗
PubMed2004

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells.

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.

Benzopyrenes↗
PubMed2004

Histone modification in constitutive heterochromatin versus unexpressed euchromatin in human cells.

Histone modifications are implicated in regulating chromatin condensation but it is unclear how they differ between constitutive heterochromatin and unexpressed euchromatin. Chromatin immunoprecipitation (ChIP) assays were done on various human cell populations using antibodies specific for acetylated or methylated forms of histone H3 or H4. Analysis of the immunoprecipitates was by quantitative real-time PCR or semi-quantitative PCR (SQ-PCR). Of eight tested antibodies, the one for histone H4 acetylated at lysine 4, 8, 12, or 16 was best for distinguishing constitutive heterochromatin from unexpressed euchromatin, but differences in the extent of immunoprecipitation of these two types of chromatin were only modest, although highly reproducible. With this antibody, there was an average of 2.5-fold less immunoprecipitation of three constitutive heterochromatin regions than of four unexpressed euchromatic gene regions and about 15-fold less immunoprecipitation of these heterochromatin standards than of two constitutively expressed gene standards (P <0.001). We also analyzed histone acetylation and methylation by immunocytochemistry with antibodies to H4 acetylated at lysine 8, H3 trimethylated at lysine 9, and H3 methylated at lysine 4. In addition, immunocytochemical analysis was done with an antibody to heterochromatin protein 1alpha (HP1alpha), whose preferential binding to heterochromatin has been linked to trimethylation of H3 at lysine 9. Our combined ChIP and immunocytochemical results suggest that factors other than hypoacetylation of the N-terminal tails of H4 and hypermethylation of H3 at lysine 9 can play an important role in determining whether a chromatin sequence in mammalian cells is constitutively heterochromatic.

Acetylation↗
PubMed2004

Constitutive apical membrane recycling in Aplysia enterocytes.

In Aplysia californica enterocytes, alanine-stimulated Na+ absorption increases both apical membrane exocytosis and fractional capacitance (fCa; a measure of relative apical membrane surface area). These increases are thought to reduce membrane tension during periods of nutrient absorption that cause the enterocytes to swell osmotically. In the absence of alanine, exocytosis and fCa are constant. These findings imply equal rates of constitutive endocytosis and exocytosis and constitutive recycling of the apical plasma membrane. Thus, the purpose of this study was to confirm and determine the relative extent of constitutive apical membrane recycling in Aplysia enterocytes. Biotinylated lectins are commonly used to label plasma membranes and to investigate plasma membrane recycling. Of fourteen biotinylated lectins tested, biotinylated wheat germ agglutinin (bWGA) bound preferentially to the enterocytes apical surface. Therefore, we used bWGA, avidin D (which binds tightly to biotin), and the UV fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated avidin D to assess the extent of constitutive apical membrane recycling. A temperature-dependent (20 vs. 4 degrees C) experimental protocol employed the use of two tissues from each of five snails and resulted in a approximately 60% difference in apical surface fluorescence intensity. Because the extent of membrane recycling is proportional to the difference in surface fluorescence intensity, this difference reveals a relatively high rate of constitutive apical membrane recycling in Aplysia enterocytes.

Animals↗
PubMed2002

Mechanisms of constitutive NF-kappaB activation in human prostate cancer cells.

BACKGROUND: Activation of the NF-kappaB transcription factor has been previously demonstrated in two androgen receptor negative prostate cancer cell lines. We wished to extend this work to additional prostate cancer cells and to characterize the mechanisms responsible for constitutive NF-kappaB activation. METHODS: Electrophoretic mobility shift assays were performed to measure NF-kappaB DNA-binding activity in prostate cancer cell lines, and immunohistochemistry was performed to detect nuclear localization of NF-kappaB in prostate cancer tissues. Western blot analysis was used to study the status of IkappaBalpha. Transient transfection assays were employed to characterize the contributions of IkappaB kinase (IKK), MAPK kinase kinases (MAPKKKs), androgen receptor (AR), and tyrosine phosphorylation to the constitutive activation of NF-kappaB in the prostate cancer cell lines. RESULTS: Constitutive NF-kappaB activity was observed in AR-negative cell lines as well as in the prostate cancer patient samples, but was not present in AR positive cells. A "super-repressor" IkappaBalpha, as well as dominant negative forms of IKKbeta and NF-kappaB-inducing kinase (NIK), and tyrosine kinase inhibition were able to suppress NF-kappaB activity in the cells with constitutive activation. CONCLUSIONS: The constitutive activation of NF-kappaB observed in prostate cancer cells is likely due to a signal transduction pathway involving tyrosine kinases, NIK, and IKK activation.

DNA-Binding Proteins↗
PubMed1999

The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene.

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.

Aldehyde Dehydrogenase↗
PubMed1997

A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity.

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the secretin and parathyroid hormone (PTH) family of seven transmembrane-spanning receptors. Point mutations of a histidine at the junction between the first intracellular loop and the second membrane-spanning domain and a threonine in the sixth membrane-spanning domain of the human PTH-receptor have been reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia. In this study, we explored whether such mutations in the GIP-R might similarly induce constitutive, ligand-independent activation of the receptor. Single amino acid substitutions in the GIP receptor were made by site-directed mutagenesis and receptor binding and cAMP levels were measured in transfected human embryonal kidney cell line (L293). Mutation of the threonine at position 340 in the sixth transmembrane spanning domain to proline (T340P) led to agonist-independent constitutive activity and exhibited a four-fold increase in basal cAMP level as compared to the wild-type GIP-R. The increase in cAMP level in T340P mutant was proportional to the amount of transfected plasmid and corresponded to the receptor number on the cell surface. Despite its high basal cAMP level, the T340P mutant could be further stimulated by GIP, with maximal cAMP generation comparable to the wild-type receptor. The change of amino acid histidine at position 169 to arginine (H169R), however, behaved like the wild type receptor and did not possess constitutive activity. These results illustrate that a point mutation of threonine to proline at position 340 results in constitutive activation of the GIP receptor, without affecting its sensitivity to agonist stimulation.

Amino Acid Sequence↗
PubMed1997

Cloning of a cDNA encoding a constitutively expressed rat liver cytosolic aldehyde dehydrogenase.

The presence of a constitutively expressed aldehyde dehydrogenase (ALDH) in the rat liver cytosol is controversial (Tottmar et al., 1973; Lindahl and Evces, 1984; Berger and Weiner, 1977; Tank et al., 1981; Truesdale-Mahoney et al., 1981; Cao et al., 1989). A cDNA encoding a constitutively expressed rat liver cytosolic class 1 ALDH was cloned using a PCR-based strategy. The open reading frame consisted of 1503 nucleotides which encoded a protein of 501 amino acids. In order to compare the rat and human nucleotide sequences, we sequenced the entire open reading frame of a human liver cytosolic ALDH cDNA clone (Zheng et al., 1993). Rat liver constitutively expressed cytosolic ALDH was 99.7, 91.8, 89.0, and 83.8% identical to rat kidney, mouse liver, rat liver phenobarbital-inducible, and human liver cytosolic class 1 ALDH cDNAs, respectively. Northern blot analysis indicated that constitutively expressed rat cytosolic ALDH mRNA is expressed in lung, kidney, liver, skeletal muscle, and testis, with weak expression in heart and brain. These results strongly suggest that a constitutively expressed ALDH is present in rat liver cytosol.

Aldehyde Dehydrogenase↗
PubMed1997

Polarity of constitutive and regulated von Willebrand factor secretion by transfected MDCK-II cells.

Von Willebrand factor (vWF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel-Palade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly multimerized vWF. Highly multimerized vWF acts as a more effective adhesive ligand than the lower molecular weight forms that are constitutively secreted. We employed the extensively characterized polar Madin-Darby Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length vWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. Immunofluorescence analysis and metabolic labeling experiments revealed that multimeric "wild-type" vWF is stored in MDCK-II cells and released upon stimulation with phorbol esters. Furthermore, we show that 62.0 +/- 3.8% of constitutively secreted and 83.2 +/- 6.6% of the regulated secreted wild-type vWF is encountered at the apical side of the cell. The polarity of the constitutive secretion of deletion mutant vWFdelD'D3 is similar to that of constitutively secreted wild-type vWF, whereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 +/- 5.7% apical).

Animals↗
PubMed1987

Relative position of constitutive heterochromatin and of nucleolar structures during mouse spermiogenesis.

In this study, the selective fluorochrome staining of constitutive heterochromatin and a specific ultrastructural silver-staining of nucleolar material (i.e., the nucleolus organizing regions) were undertaken to be used as indicators for the chromosomal arrangement during mouse spermiogenesis. Since in mice all somatic chromosomes are telocentric and the constitutive heterochromatin and nucleolar organizing regions are closely associated to the centromeres, this combination of techniques provided for the first time ultrastructural evidence 1) for the dispersion of the constitutive heterochromatic chromocentre and a centrifugal migration to the postacrosomal portion of the nuclear envelope where constitutive heterochromatin seems to mediate the assembling of microtubules in the so-called manchette. As elongation continues, the constitutive heterochromatin migrates back into central position and forms the "focous of earlier condensing chromatin", which initiates further chromatin condensation. 2) The fate of the nucleolus during spermiogenesis could also be further clarified: The nucleolus is first associated with the chromocentre, but starts to disintegrate during elongation phase. However, argyrophilic remnants are still visible in the centre of the nucleus, pointing to an ongoing transcriptional activity. When they final disappear, they leave behind "nuclear vacuoles" in the dense chromatin mass of the mature sperm nucleus.

Animals↗
PubMed1986

Insertion of non-homologous DNA sequences into a regulatory gene cause a constitutive maltase synthesis in yeast.

Two maltase constitutive alleles MAL1-1c and MAL1-2c were obtained as revertants from a defective mall-1 mutant allele not promoting maltose fermentation. Classical genetical analysis showed that the mutations were linked or allelic to the MAL1 locus. Dominance relations were established by testing alpha-glucosidase activities in diploids containing various allele combinations. The maltose regulatory genes belonging to the MAL1, MAL1-1c and MAL1-2c alleles were cloned. Differences in restriction sites were found between the wild type MAL1 and the derived MAL1-constitutive alleles. The MAL1 regulatory gene was located in a 1.15 kb EcoRI fragment (Rodicio and Zimmermann 1985a, b). An EcoRI fragment of this size was found in plasmids containing the MAL1 regulatory wild type allele but was absent from plasmids carrying the constitutive alleles. The genomic organization of the MAL loci in the constitutive mutants was confirmed by Southern analysis. Various fragments containing sequences of the different MAL1 alleles were used to probe genomic digests of MAL1, MAL1-1c and MAL1-2c strains. The results obtained support the conclusion that the constitutive mutations had arisen by a rearrangement between the original mal1-1 mutant allele and sequences with different location in the genome.

DNA Restriction Enzymes↗
PubMed1989

Selection of constitutively resistant mutants of inducible clindamycin-resistant Bacteroides vulgatus.

The rate of spontaneous mutation to constitutive clindamycin resistance and the kinetics of selection of such mutants in preinduced and non-preinduced cells was evaluated in the Bacteroides vulgatus strain RYC18F6 (original MIC less than or equal to 0.25 micrograms/ml), which shows inducible resistance to clindamycin and erythromycin. Mutants demonstrating a high level of constitutive resistance to clindamycin (64 micrograms/ml) occurred at a frequency of 10(-7). Culture in broth containing either subinhibitory or inhibitory levels of clindamycin resulted in induction of clindamycin resistance. This permitted survival of a part of the population that seemed to facilitate the further growth of constitutively resistant mutants (up to a frequency of 10(-1)). At both low and high clindamycin concentrations mutants appeared to be selected over merely induced cells. Preincubation of cultures with erythromycin gave rise to an apparently higher level of induction. Under these circumstances, the selection of constitutively resistant mutants was less effective (10(-4). The use of erythromycin or clindamycin against populations of Bacteroides spp. exhibiting inducible resistance may contribute to selection of mutants showing constitutive clindamycin resistance.

Bacteroides↗
PubMed2003

Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome.

Intracellular proteins are degraded largely by proteasomes. In cells stimulated with gamma interferon, the active proteasome subunits are replaced by "immuno" subunits that form immunoproteasomes. Phylogenetic analysis of the immunosubunits has revealed that they evolve faster than their constitutive counterparts. This suggests that the immunoproteasome has evolved a function that differs from that of the constitutive proteasome. Accumulating experimental degradation data demonstrate, indeed, that the specificity of the immunoproteasome and the constitutive proteasome differs. However, it has not yet been quantified how different the specificity of two forms of the proteasome are. The main question, which still lacks direct evidence, is whether the immunoproteasome generates more MHC ligands. Here we use bioinformatics tools to quantify these differences and show that the immunoproteasome is a more specific enzyme than the constitutive proteasome. Additionally, we predict the degradation of pathogen proteomes and find that the immunoproteasome generates peptides that are better ligands for MHC binding than peptides generated by the constitutive proteasome. Thus, our analysis provides evidence that the immunoproteasome has co-evolved with the major histocompatibility complex to optimize antigen presentation in vertebrate cells.

Animals↗
PubMed1994

A comparative study of constitutive and induced alkoxyresorufin O-dealkylation and individual cytochrome P450 forms in cynomolgus monkey (Macaca fascicularis), human, mouse, rat and hamster liver microsomes.

The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). In monkeys there were no sex-differences in untreated, phenobarbitone (PB)- or beta-naphthoflavone (BNF)-treated animals in AROD activities, or in individual P450 proteins detected by immunoblotting. Basal MROD and EROD activities varied by less than 7-fold between the five species, but the comparative pattern of basal MROD, EROD, PROD and BROD activities (the "MEPB profile") was very species-specific, with monkeys being similar to rats but different from man, mouse and hamster. The induction of AROD activities by PB and BNF was also highly species-specific. Monkeys expressed constitutive proteins immunorelated to the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A sub-families (human CYP2A6 cross-reacted with the anti-rat CYP2B1 antibodies used, and so CYP2A and CYP2B forms could not be separately identified in the monkey). Single constitutive immunoblot bands were identified in monkey for CYP1A (54 kDa), CYP2A/CYP2B (51 kDa) and CYP3A (51 kDa), respectively, but two strong (51 and 52 kDa) plus two weak (49 and 49.5 kDa) bands were shown for CYP2C. Human liver expressed CYP1A2 (54 kDa), CYP2A6 (51 kDa), CYP3A4 (50.5 kDa) and three CYP2C9-immunorelated protein bands (48, 50 and 54 kDa). In monkeys BNF induced the 54 kDa CYP1A protein and CYP1A-dependent MROD, EROD and PROD activities (18-, 15- and 6-fold increases in activity, respectively), whereas PB strongly induced the 51 kDa CYP2A/CYP2B protein but did not induce PROD activity. PB also induced non-constitutive CYP2A/CYP2B protein bands at 49 and 52 kDa in some monkeys. BROD activity was induced less that four-fold by either PB or BNF in monkeys. In conclusion, cynomolgus monkeys expressed a range of constitutive CYP1A, CYP2A or CYP2B, CYP2C and CYP3A proteins similar to man, and a range of AROD monooxygenase reaction rates similar to both man and rat, but the basal MEPB profile of AROD activities in monkeys was more similar to rat than to man. MROD and EROD were good measures of CYP1A induction by polycyclic aromatic hydrocarbons in cynomolgus monkeys, but neither PROD nor BROD were indices of CYP2B induction by PB.

Adult↗
PubMed1983

C-banding of Peromyscus constitutive heterochromatin persists following histone hyperacetylation.

Approx. 35% of the DNA of cultured cells from the cactus mouse, Peromuscus eremicus, is contained in highly condensed constitutive heterochromatin which can be visualized in metaphase chromosomes stained by the C-band technique. Previous studies have shown this constitutive heterochromatin to contain a large proportion of underacetylated, arginine-rich histones, the majority of which can be hyperacetylated when cells are treated with butyrate. In order to determine whether this simulation of the acetylated state of euchromatin alters the cytological properties of constitutive heterochromatin as well, chromosomes from butyrate-treated cells have been examined. Because of the paucity of cells in butyrate-treated cultures, prematurely condensed chromosomes (PCCs) were produced from butyrate-treated cells by fusion with mitotic cells. In these PCCs, both the highly condensed nature and the ability to C-band were preserved in the hyperacetylated constitutive heterochromatin, suggesting that the subset of arginine-rich histones which is refractory to acetylation in the presence of butyrate may be responsible for the maintenance of the heterochromatic state. In addition, PCC analyses indicated that butyrate arrests Peromyscus cells in both the G1 and G2 phases of the cell cycle and confirmed the late-replicating pattern of constitutive heterochromatin.

Acetylation↗
PubMed1990

Immunological characterization of constitutive isozymes of cytochrome P-450 from rainbow trout. Evidence for homology with phenobarbital-induced rat P-450s.

Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.

Animals↗
PubMed2005

Relationship between constitutive nuclear factor-kappaB (NF-kappaB) and inhibitor kappaB-alpha (IkappaB-alpha) in an interferon-alpha-sensitive human Burkitt lymphoma cell line.

The human Burkitt lymphoma Daudi cell line expresses constitutively active nuclear factor kappaB (NF-kappaB) in the nucleus in spite of high levels of inhibitor kappaB-alpha (IkappaB-alpha) in the cytoplasm. The antiproliferative response of these cells to interferon-alpha (IFN-alpha) correlated with the inhibition of the constitutive NF-kappaB activity by the cytokine. The present study shows that IFN-alpha caused an increase in p53 level, inhibited cell proliferation by [(3)H]thymidine incorporation, and stimulated cytotoxicity and apoptosis by PARP-cleavage in the Daudi cells. In order to study the relationship between the constitutively active NF-kappaB and IkappaB-alpha, a dominant negative mutant IkappaB-alpha (IkappaB-alphaDN), lacking the N-terminal 36 amino acids required for the activation of NF-kappaB by tumor necrosis factor-alpha (TNF-alpha), was expressed in the Daudi cells. The expression of IkappaB-alphaDN protein did not inhibit the constitutive NF-kappaB activity, but it inhibited cell proliferation, antiproliferative response to IFN-alpha, and phosphorylated mitogen activated protein kinase (p-MAPK) level. Thus, our results suggest that constitutive NF-kappaB activity in the human Burkitt lymphoma Daudi cells is maintained by a mechanism independent of IkappaB-alpha degradation, and that the IkappaB-alpha is involved in the proliferation of these cells, possibly through the MAP kinase pathway. Therefore, in addition to IFN-alpha treatment, both NF-kappaB and IkappaB-alpha may be used as drug targets for inhibiting cell proliferation in the lymphomas.

Apoptosis↗
PubMedDate not supplied

The use of simplified constitutional indications for self-prescription of homeopathic medicine.

OBJECTIVES: To develop simplified constitutional indications for homeopathic medicines so that parents of children with recurrent upper respiratory tract infection (URTI) could choose homeopathic medicines for their children more "accurately", and to subsequently evaluate if these choices match the prescriptions of trained homeopaths. DESIGN AND SETTING: To initially select the most commonly used homeopathic medicines for URTI, data from a survey of 1097 patients visiting 80 different Norwegian homeopaths were used. A simplified constitutional indication was then developed for the three homeopathic medicines most frequently prescribed for recurrent URTI and otitis media. The constitutional indications were developed by a group of five homeopaths and were then sent to 20 homeopaths for further evaluation. To evaluate the parents' choice of homeopathic medicines compared to the prescription by trained homeopaths, a group of 11 randomly selected homeopaths were asked to participate. They recruited parents of 70 child patients. RESULT: By using simplified constitutional indications for the three most commonly prescribed remedies, Calcarea carb, Pulsatilla and Sulphur, parents were able to choose the same homeopathic medicine as homeopaths' prescribed for 55% (95% CI 43-67) of children with URTI. There was excellent agreement between parents' choice and homeopaths' prescription for the three medicines (Kappa of 0.77, p<0.001). CONCLUSION: Simplified constitutional indications can be used to improve the quality of the choice of homeopathic medicines purchased over the counter (OTC) for self-treatment.

Child, Preschool↗