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PubMed1991

Analysis of constitutive and noninducible mutations of the PUT3 transcriptional activator.

The Saccharomyces cerevisiae PUT3 gene encodes a transcriptional activator that binds to DNA sequences in the promoters of the proline utilization genes and is required for the basal and induced expression of the enzymes of this pathway. The sequence of the wild-type PUT3 gene revealed the presence of one large open reading frame capable of encoding a 979-amino-acid protein. The protein contains amino-terminal basic and cysteine-rich domains homologous to the DNA-binding motifs of other yeast transcriptional activators. Adjacent to these domains is an acidic domain with a net charge of -17. A second acidic domain with a net charge of -29 is located at the carboxy terminus. The midsection of the PUT3 protein has homology to other activators including GAL4, LAC9, PPR1, and PDR1. Mutations in PUT3 causing aberrant (either constitutive or noninducible) expression of target genes in this system have been analyzed. One activator-defective and seven activator-constitutive PUT3 alleles have been retrieved from the genome and sequenced to determine the nucleotide changes responsible for the altered function of the protein. The activator-defective mutation is a single nucleotide change within codon 409, replacing glycine with aspartic acid. One activator-constitutive mutation is a nucleotide change at codon 683, substituting phenylalanine for serine. The remaining constitutive mutations resulted in amino acid substitutions or truncations of the protein within the carboxy-terminal 76 codons. Mechanisms for regulating the activation function of the PUT3 protein are discussed.

Amino Acid Sequence↗
PubMed1994

Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae.

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Amino Acid Sequence↗
PubMed1994

Constitutive NF-kappa B activity in neurons.

NF-kappa B is inducible transcription factor present in many cell types in a latent cytoplasmic form. So far, only immune cells including mature B cells, thymocytes, and adherent macrophages have been reported to contain constitutively active forms of NF-kappa B in the nucleus. A recent study showed that the human immunodeficiency virus type 1 (HIV-1) promoter is highly active in several brain regions of transgenic mice (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clements, Science 258:1804-1807, 1992). Since the activity of this viral enhancer is governed mainly by two binding sites for NF-kappa B, we were prompted to investigate the state of NF-kappa B activity in neurons. Primary neuronal cultures derived from rat hippocampus and cerebral cortex showed a high constitutive expression of an HIV-1 long terminal repeat-driven luciferase reporter gene, which was primarily dependent on intact NF-kappa B binding sites and was abolished upon coexpression of the NF-kappa B-specific inhibitor I kappa B-alpha. Indirect immunofluorescence and confocal laser microscopy showed that the activity of NF-kappa B correlated with the presence of the NF-kappa B subunits p50 and RelA (p65) in nuclei of cultured neurons. NF-kappa B was also constitutively active in neurons in vivo. As investigated by electrophoretic mobility shift assays, constitutive NF-kappa B DNA-binding activity was highly enriched in fractions containing neuronal nuclei prepared from rat cerebral cortex. Nuclear NF-kappa B-specific immunostaining was also seen in cryosections from mouse cerebral cortex and hippocampus. Only a subset of neurons was stained. Activated NF-kappa B in the brain is likely to participate in normal brain function and to reflect a distinct state of neuronal activity or differentiation. Furthermore, it may explain the high level of activity of the HIV-1 enhancer in neurons, an observation potentially relevant for the etiology of the AIDS dementia complex caused by HIV infection of the central nervous system.

Animals↗
PubMed1996

Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha.

IkappaBalpha is a phosphoprotein that sequesters the NF-kappaB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IkappaBalpha is further phosphorylated and degraded, allowing NF-kappaB/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IkappaBalpha and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IkappaBalpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IkappaBalpha, in that unassociated Mutf has a half-life two times longer than wild-type IkappaBalpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the Mutf phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IkappaBalpha, and this phosphorylation is required for rapid proteolysis of the unassociated protein.

Amino Acid Sequence↗
PubMed1997

Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

Alleles↗
PubMed1998

Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.

Animals↗
PubMed1982

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.

Allantoin↗
PubMed2000

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

Animals↗
PubMed2000

Thyroglobulin repression of thyroid transcription factor 1 (TTF-1) gene expression is mediated by decreased DNA binding of nuclear factor I proteins which control constitutive TTF-1 expression.

Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp -264 and -153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp -264 to -153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.

Amino Acid Sequence↗
PubMed2004

Constitutive expression of tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice.

Here we describe a new mouse model with constitutive expression of the catalytic subunit of telomerase (Tert) targeted to thymocytes and peripheral T cells (Lck-Tert mice). Two independent Lck-Tert mouse lines showed higher incidences of spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls, indicating that constitutive expression of Tert promotes lymphoma. Interestingly, T-cell lymphomas in Lck-Tert mice were more disseminated than those in wild-type controls and affected both lymphoid and nonlymphoid tissues, while nonlymphoid tissues were never affected with lymphoma in age-matched wild-type controls. Importantly, these roles of Tert constitutive expression in promoting tumor progression and dissemination were independent of the role of telomerase in telomere length maintenance, since telomere length distributions on a single-cell basis were identical in Lck-Tert and wild-type thymocytes. Finally, Tert constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes, although it resulted in greater chromosomal instability upon gamma irradiation in Lck-Tert primary lymphocytes than in controls, suggesting that Tert overexpression may interfere with the cellular response to DNA damage.

Animals↗
PubMed2004

Regulation of constitutive p50/c-Rel activity via proteasome inhibitor-resistant IkappaBalpha degradation in B cells.

Constitutive NF-kappaB activity has emerged as an important cell survival component of physiological and pathological processes, including B-cell development. In B cells, constitutive NF-kappaB activity includes p50/c-Rel and p52/RelB heterodimers, both of which are critical for proper B-cell development. We previously reported that WEHI-231 B cells maintain constitutive p50/c-Rel activity via selective degradation of IkappaBalpha that is mediated by a proteasome inhibitor-resistant, now termed PIR, pathway. Here, we examined the mechanisms of PIR degradation by comparing it to the canonical pathway that involves IkappaB kinase-dependent phosphorylation and beta-TrCP-dependent ubiquitylation of the N-terminal signal response domain of IkappaBalpha. We found a distinct consensus sequence within this domain of IkappaBalpha for PIR degradation. Chimeric analyses of IkappaBalpha and IkappaBbeta further revealed that the ankyrin repeats of IkappaBalpha, but not IkappaBbeta, contained information necessary for PIR degradation, thereby explaining IkappaBalpha selectivity for the PIR pathway. Moreover, we found that PIR degradation of IkappaBalpha and constitutive p50/c-Rel activity in primary murine B cells were maintained in a manner different from B-cell-activating-factor-dependent p52/RelB regulation. Thus, our findings suggest that nonconventional PIR degradation of IkappaBalpha may play a physiological role in the development of B cells in vivo.

Animals↗
PubMed1986

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms.

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

Base Sequence↗
PubMed2005

Novel approach of molecular genetic understanding of iridology: relationship between iris constitution and angiotensin converting enzyme gene polymorphism.

Iridology is the study of the iris of the eye to detect the conditions of the body and its organs, genetic strengths and weaknesses, etc. Although iridology is not widely used as a scientific tool for healthcare professionals to get to the source of people's health conditions, it has been used as a supplementary source to help the diagnosis of medical conditions by noting irregularities of the pigmentation in the iris among some Korean Oriental medical doctors. Angiotensin converting enzyme (ACE) gene polymorphism is one of the most well studied genetic markers of vascular disease. We investigated the relationship between iridological constitution and ACE polymorphism in hypertensives. We classified 87 hypertensives and 79 controls according to iris constitution and determined the ACE genotype of each individual. DD genotype was more prevalent in patients with a neurogenic constitution than in controls. This finding supports the hypothesis that D allele is a candidate gene for hypertension and demonstrates the association among ACE genotype, Korean hypertensives and iris constitution.

Adult↗
PubMed2006

Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function.

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.

Capillary Permeability↗
PubMed2002

Constitutive properties of hypertrophied myocardium: cellular contribution to changes in myocardial stiffness.

Recent studies have suggested that pressure overload hypertrophy (POH) alters the viscoelastic properties of individual cardiocytes when studied in isolation. However, whether these changes in cardiocyte properties contribute causally to changes in the material properties of the cardiac muscle as a whole is unknown. Accordingly, a selective, isolated, acute change in cardiocyte constitutive properties was imposed in an in vitro system capable of measuring the resultant effect on the material properties of the composite cardiac muscle. POH caused an increase in both myocardial elastic stiffness, from 20.5 +/- 1.3 to 28.4 +/- 1.8, and viscous damping, from 15.2 +/- 1.1 to 19.8 +/- 1.5 s (normal vs. POH, P < 0.05), respectively. Recent studies have shown that cardiocyte constitutive properties could be acutely altered by depolymerizing the microtubules with colchicine. Colchicine caused a significant decrease in the viscous damping in POH muscles (19.8 +/- 1.5 s at baseline vs. 14.7 +/- 1.3 s after colchicine, P < 0.05). Therefore, myocardial material properties can be altered by selectively changing the constitutive properties of one element within this muscle tissue, the cardiocyte. Changes in the constitutive properties of the cardiocytes themselves contribute to the abnormalities in myocardial stiffness and viscosity that develop during POH.

Animals↗
PubMed1996

Prostaglandin G/H synthase-2 is the constitutive and dominant isoform in cultured human lung epithelial cells.

Two isoforms of prostaglandin G/H synthase (PGHS; prostaglandin endoperoxide synthase, cyclooxygenase) have been identified; PGHS-1 is expressed constitutively in most tissues, whereas PGHS-2 is thought to be induced by various proinflammatory cytokines and growth factors. In this study, we determined which isoform of PGHS mRNA, protein, and activity was present constitutively in A549 (a human lung adenocarcinoma cell line) and in untransformed (normal human bronchial epithelial or NHBE) and transformed (16HBE4o-) human bronchial epithelial cells. Two PGHS-2-specific inhibitors, NS-398 and L-745, 337, blocked the release of prostaglandin E2 from A549 cells with mean inhibitory concentrations of 5 and 18 nM, respectively, but did not inhibit its release from human bronchial smooth muscle cells (BSMC) at a concentration of 10 microM. Northern and immunoblot analysis demonstrated that BSMC expressed PGHS-1 mRNA and protein constitutively, whereas epithelial cells expressed PGHS-2 mRNA and protein constitutively with either undetectable (A549, 16HBE4o-) or very low levels (NHBE) of PGHS-1. We conclude that PGHS-2 is the dominant PGHS isoform in unstimulated and stimulated lung epithelial cells in culture.

Base Sequence↗
PubMed2000

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes.

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.

Animals↗
PubMed1999

Subendothelial cells from normal bovine arteries exhibit autonomous growth and constitutively activated intracellular signaling.

The arterial media is comprised of heterogeneous smooth muscle cell (SMC) subpopulations with markedly different growth responses to pathophysiological stimuli. Little information exists regarding the intracellular signaling pathways that contribute to these differences. Therefore, we investigated the growth-related signaling pathways in a unique subset of subendothelial SMCs (L1 cells) from normal, mature, bovine arteries and compared them with those in "traditional" SMCs derived from the middle media (L2 SMCs). Subendothelial L1 cells exhibited serum-independent autonomous growth, not observed in L2 SMCs. Autonomous growth of L1 cells was driven largely by the constitutively activated extracellular signal-regulated kinase (ERK-1/2) cascade. Inhibition of upstream activators of ERKs (MAP kinase kinase-1, p21(ras), receptor tyrosine kinases, and Gi protein-coupled receptors) led to suppression of autonomous growth in these cells. L1 cells also exhibited constitutive activation of important downstream targets of ERKs (cytosolic phospholipase A(2), cyclooxygenase-2) and secreted large amounts of prostaglandins. Importantly, L1 cells secreted potent mitogenic factor(s), which could potentially contribute in an autocrine fashion to the constitutive activation of these cells. Our data suggest that unique arterial cells with autonomous growth potential and constitutively activated signaling pathways exist in normal arteries and may contribute selectively to the pathogenesis of vascular diseases.

Angiotensin II↗