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PubMed1999

The BH3 domain of Bcl-x(S) is required for inhibition of the antiapoptotic function of Bcl-x(L).

bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-x(L), is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-x gene codes for the protein Bcl-x(S), which lacks 63 amino acids present in Bcl-x(L) and has proapoptotic activity. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-x(S) does not seem to induce cell death in the absence of an additional death signal. However, Bcl-x(S) does interfere with the ability of Bcl-x(L) to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-x(S) was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-x(S) which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-x(L). Bcl-x(S) was able to form heterodimers with Bcl-x(L) in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-x(L). Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-x(L). The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-x(L) survival function.

Apoptosis↗
PubMed2002

DNA-dependent adenosine triphosphatase (helicaselike transcription factor) activates beta-globin transcription in K562 cells.

Correct developmental regulation of beta-like globin gene expression is achieved by preferential transcription of a gene at a given developmental stage, silencing of other beta-like gene promoters, and competition among these promoters for interaction with the locus control region (LCR). Several evolutionarily conserved DNA elements in the promoters of the beta-like genes and LCR have been studied in detail, and the role of their binding factors has been investigated. However, the beta-globin promoter includes additional evolutionarily conserved sequences of unknown function. The present study examined the properties of a 21-base pair (bp) promoter-conserved sequence (PCS) located at positions -115 to -136 bp relative to the transcription start site of the beta-globin gene. A helicaselike transcription factor (HLTF) belonging to the SWI2/SNF2 family of proteins binds to the PCS and a partly homologous sequence in the enhancer region of the LCR hypersensitive site 2 (HS2). Elevation of the level of HLTF in K562 erythroleukemic cells increases beta-promoter activity in transient transfection experiments, and mutations in the PCS that remove HLTF-binding regions abolish this effect, suggesting that HLTF is an activator of beta-globin transcription. Overexpression of HLTF in K562 cells does not affect the endogenous levels of gamma- and epsilon-globin message, but it markedly activates beta-globin transcription. In conclusion, this study reports a transcription factor belonging to the SWI2/SNF2 family, which preferentially activates chromosomal beta-globin gene transcription and which has not previously been implicated in globin gene regulation.

Animals↗
PubMed1998

N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

The functional importance of the three oligosaccharide chains linked to Asn35, Asn80 and Asn108, of the long form of the PRL receptor (PRLR) was investigated by individual or multiple substitutions of asparagyl residues using site-directed mutagenesis and transient transfection of these mutated forms of PRLR in monkey kidney cells, Chinese hamster ovary, and human 293 fibroblast cells that exhibit different levels of protein expression. Scatchard analysis performed on monkey kidney cells revealed that the mutants possess the same affinity for PRL as compared with wild-type PRLR. A strong reduction (90%) of the aglycosylated PRLR expression at the cell surface of monkey kidney or human 293 cells was observed. Immunohistochemistry experiments using an anti-PRLR monoclonal antibody showed an accumulation of the deglycosylated receptor in the Golgi area of transfected monkey kidney cells. Upon PRL stimulation, the aglycosylated PRLR associated with Janus kinase 2 was phosphorylated and was able to activate a beta-casein gene promoter in transfected 293 fibroblast cells. The active form of the PRLR was thus acquired independently of glycosylation. By contrast, no functional activity was detectable in transfected Chinese hamster ovary cells that expressed low levels of PRLR. These studies demonstrate that the glycosylation on the asparagyl residues of the extracellular domain of the PRLR is crucial for its cell surface localization and may affect signal transduction, depending on the cell line.

Animals↗
PubMed1994

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling.

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

3T3 Cells↗
PubMed2003

The Th2 cell cytokines IL-4 and IL-13 regulate found in inflammatory zone 1/resistin-like molecule alpha gene expression by a STAT6 and CCAAT/enhancer-binding protein-dependent mechanism.

The onset of allergic inflammation in the lung is driven by a complex genetic program. This study shows that found in inflammatory zone (FIZZ)1 and FIZZ2, but not FIZZ3, gene expression was up-regulated 6 h after Ag challenge in a mouse model of acute pulmonary inflammation. Induction of both genes was abolished in allergen-challenged STAT6-deficient mice. FIZZ1, but not FIZZ2, mRNA was up-regulated upon incubation of the myeloid cell line BMnot with IL-4. The promoter region of FIZZ1 contains functional binding sites for STAT6 and C/EBP. FIZZ1 promoter reporter gene constructs responded to IL-4 and IL-13 stimulation in transiently transfected cells. Point mutations in the STAT6 or the C/EBP site led to loss of cytokine responsiveness indicating that IL-4-mediated induction of murine FIZZ1 is orchestrated by the coordinate action of STAT6 and C/EBP. It is concluded that the expression of the genes encoding FIZZ1 and FIZZ2, but not FIZZ3, is induced in allergen-challenged lungs in a STAT6-dependent fashion. STAT6 directly regulates IL-4- and IL-13-triggered induction of FIZZ1 expression at the transcriptional level by cooperation with C/EBP. Induction of FIZZ2 gene expression most likely occurs independent of a direct effect by these cytokines and may be due to indirect STAT6-driven mechanisms.

Animals↗
PubMed1999

[A case of congenital plasminogen abnormality with recurrent cerebrovascular ischemic attacks].

We reported a 10-year-old boy with congenital plasminogen abnormality resulting in recurrent cerebrovascular ischemic attacks. He suddenly developed dystonia of the left upper limb at 9 years of age. MRI demonstrated small infarcts in the right thalamus and caudate nucleus. He then had a transient ischemic attack at 10 years. He complained of headache, vertigo, diplopia, and unsteady gait. DNA analysis revealed that he was heterozygotic for abnormal plasminogen. Thus, congenital plasminogen abnormality was suspected to be a cause of recurrent cerebrovascular ischemic attacks in this case.

Cerebral Infarction↗
PubMed1991

Role of src-like protooncogenes in lymphocyte proliferation.

Cross-linking of membrane-bound immunoglobulins, which are B cell antigen receptors, causes proliferation and differentiation of B cells or the inhibition of their growth. The receptor-mediated signalling involves tyrosine phosphorylation of cellular proteins. The Src-like protein-tyrosine kinase Lyn is expressed preferentially in B cells and is an intracytoplasmic constituent of the B cell antigen receptor complex. Cross-linking of membrane-bound immunoglobulin M with antibody induced rapid increases in the kinase activities of Lyn and Lyn-associated phosphatidylinositol-3 kinase. Cross-linking of B-cell antigen receptor also induced association of Lyn with an 85 kDa non-catalytic subunit of phosphatidylinositol-3 kinase. Thus, Lyn is functionally associated with membrane-bound immunoglobulin M, and seems to participate in B cell antigen receptor-mediated signalling. On the other hand, accumulating evidence shows that Fyn and Lck are involved in T-cell activation. Co-immunoprecipitation experiments showed that Fyn was physically associated with T cell antigen receptor-CD3 complex. Transient expression of actively mutated Fyn, having Phe-528 instead of Tyr-528, in Jurkat T cells stimulated the fos promoter and serum response element (SRE), suggesting that the Fyn kinase stimulates c-fos expression through SRE. An analogous set of experiments showed that introduction of the active Fyn alone had little effect on IL-2 promoter. However, it could stimulate transcription from this promoter when transfected cells were stimulated by a combination of Concanavalin A (ConA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Under the same conditions, normal Lck and Lyn could not stimulate IL-2 promoter.

1-Phosphatidylinositol 4-Kinase↗
PubMed1985

[Population and demographic study of the Turkmen Tekke].

Genetical and demographic studies of typical rural population of Turkomen teke in Yangi-Kala village of the Ashkhabad region (about 5.000 inhabitants) were performed through the examination of a sample of 3528 persons. The population is characterised by intensive growth (mean number of children per one woman is 6.17), a high level of endogamy (the total coefficient of inbreeding being 0.010912), preserving the traditional marriage system (91.9 and 8.1% of intrageneric and intergeneric marriages, respectively). Migration does not significantly influence endogamy, because it takes place among kin families.

Adolescent↗
PubMed1994

Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression.

Many tumor cells produce vascular endothelial growth factor (VEGF), which is thought to be a pivotal mediator of tumor neoangiogenesis. Expression of the VEGF gene can be induced by tumor promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate protein kinase C (PKC). Here we show that in transient transfection assays a mutated form of the murine p53 tumor suppressor gene (ala135-->val) induces expression of VEGF mRNA and potentiates TPA stimulated VEGF mRNA expression. In NIH 3T3 cells which stably overexpress the temperature sensitive p53 (ala135-->val), displaying mutant phenotype at 37 degrees C and wildtype phenotype at 32.5 degrees C, induction of VEGF mRNA and protein by activated PKC is strongly synergistic with mutant, but not wildtype p53. Mutant p53 specifically increases TPA induction of VEGF without affecting the expression of other TPA inducible genes. TPA dependent VEGF expression is also enhanced by human p53 mutated at amino acid 175. Thus, our data link PKC and p53, the gene most frequently altered in human tumors, with the regulation of tumor angiogenesis.

3T3 Cells↗
PubMed1993

Functional and physical interaction of protein-tyrosine kinases Fyn and Csk in the T-cell signaling system.

The Src-like protein-tyrosine kinase Fyn is associated with T-cell antigen receptor. Transient expression of actively mutated Fyn, having Phe-528 instead of Tyr-528 or Thr-338 instead of Ile-338, in Jurkat T-cells stimulated the serum response element (SRE), 12-O-tetradecanoyl-phorbol-13-acetate response element, cyclic AMP response element, and c-fos promoter. The stimulation of SRE was particularly prominent not only with active Fyn but also with normal (wild-type) Fyn. SRE was also stimulated by both normal and active Lck. Furthermore, normal and active Fyn stimulated transcription from the IL-2 gene promoter when transfected cells were stimulated by concanavalin A plus 12-O-tetradecanoylphorbol-13-acetate. Under the same conditions, Lck did not stimulate IL-2 promoter unless it was activated by mutation. Interestingly, a mutant Fyn, which has deletions within the SH2 region and so is able to transform chicken embryo fibroblasts, did not stimulate either the c-fos or IL-2 promoter, suggesting the importance of this region in T-cell signaling. Csk, which phosphorylates tyrosine residues in the negative regulatory sites of Src family kinases, down-regulated Fyn- and Lck-mediated stimulation of the serum response element and Fyn-mediated enhancement of IL-2 promoter activity. These data suggest that Fyn and Lck, whose activities are regulated by Csk, are involved in different phases of T-cell activation.

Amino Acid Sequence↗
PubMed1993

Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

The CDK inhibitor p21WAF1/Cip1 is induced through a p300-dependent mechanism during NGF-mediated neuronal differentiation of PC12 cells.

The block of cell proliferation elicited by the addition of nerve growth factor (NGF) to exponentially-growing PC12 cells results, in part, from the inhibition of cyclin D1-associated kinase activity by p21WAF1/CIP1. NGF treatment of PC12 cells provokes the accumulation of p21 mRNA, due to transcriptional activation of the p21 promoter in a p53-independent manner. Transient expression of a mutated form of the adenovirus E1A protein (E1A dCR2), which retains its capacity to bind the transcriptional co-activator p300, completely abolishes the NGF-mediated stimulation of p21 promoter activity. This phenomenon can be reversed by ectopic expression of p300, suggesting that p300 is necessary for the induction of p21 by NGF. In addition, stable expression of E1A dCR2 in PC12 cells results in the inhibition of the NGF response, i.e. it prevents activation of the p21 promoter, cell cycle arrest, and neuronal differentiation. The signalling pathway from the TrkA receptor via the MAP kinase pathway is not altered in these cells. Together, these data indicate that p300 could play a pivotal role in the triggering of the anti-mitogenic effect of NGF and of neuronal differentiation.

Animals↗
PubMed1992

Kinds of mutations induced by aflatoxin B1 in a shuttle vector replicating in human cells transiently expressing cytochrome P4501A2 cDNA.

Transient expression of rat liver cytochrome P450lA2 cDNA was combined with the use of a shuttle vector as a mutational target to determine the frequency and types of mutation caused by the conversion of aflatoxin B1 into genotoxic metabolites within human cells. Ad293 cells were first transfected with p91-lA2, a rat liver P450lA2 cDNA expression vector, or with p91-lA2(i) (a control vector that has the P450 cDNA in the inverted orientation) and incubated for 24 h to permit P450lA2 accumulation. Cells were then transfected with the pS189 shuttle-vector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B1 to permit promutagen activation and pS189 replication. In shuttle vectors replicated in p91-lA2-transfected cells, the supF point-mutation frequency increased with increasing concentration of aflatoxin B1. This frequency was nine to 23 times greater than the background point-mutation frequency obtained with aflatoxin B1-treated control (p91-lA2(i)-transfected) cells. The large majority of the aflatoxin B1-induced supF point mutations were base substitutions, mostly G:C----T:A transversions. This mutagenesis system permits the molecular analysis of mutations induced by specific P450/promutagen pairs in a shuttle vector replicating in human cells and will permit the investigation of host cell mechanisms involved in the generation of these mutations.

Aflatoxins↗
PubMed2001

Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia.

The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.

Alkaline Phosphatase↗
PubMed1999

Clinical and functional effects of mutations in the DAX-1 gene in patients with adrenal hypoplasia congenita.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder caused by mutations in a gene referred to as DAX-1. AHC is characterized by adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. The DAX-1 protein is structurally related to orphan nuclear receptors, although it lacks the characteristic zinc finger DNA-binding domain that is highly conserved in other members of this family. In this report, we describe the clinical features and genetic alterations in six families with AHC. These patients reveal the variable clinical presentation of adrenal insufficiency in AHC and underscore the importance of considering this diagnosis. Nonsense mutations that introduce a stop codon were found in three cases (W171X, W171X, Y399X). Frameshift mutations (405delT, 501delA, and 702delC), each of which resulted in a premature stop codon at amino acid 263, were found in the other three families. Three of these mutations (Y399X, 405delT, 702delC) are novel. Using transient gene expression assays to assess DAX-1 function, these mutations were shown to eliminate the ability of DAX-1 to repress the transcription of genes that are stimulated by a related nuclear receptor, steroidogenic factor-1. These studies reveal the variable clinical presentation of DAX-1 mutations and emphasize the value of genetic testing in boys with primary adrenal insufficiency and suspected X-linked AHC.

Adrenal Insufficiency↗
PubMed1990

Mutational analysis identifies a new functional domain of the thyroid hormone receptor.

We studied the functional significance of a region of the thyroid hormone receptor ligand binding domain which is conserved in all members of the erbA superfamily. The homologous region of the glucocorticoid receptor has been implicated in the binding of heat shock protein 90, and deletions of the glucocorticoid receptor that include this region result in constitutive activity. Both deletion and point mutations were made in this area of the rat beta 1 thyroid hormone receptor (amino acids 286-305), and the functional consequences of these mutations were analyzed in JEG cells by transient cotransfection along with a T3 responsive reporter gene. All mutations studied resulted in significant inhibition of ligand-dependent transcriptional regulation without inducing significant constitutive activity. For some mutations, the lack of transcriptional response correlated with a diminished ability to bind ligand. However, point mutations of amino acids 288, 290, and 300 resulted in impaired transcriptional regulation despite wild type T3 binding affinity. In addition, mutations of these three amino acids failed to impair localization of the receptor to the nucleus or binding to DNA in vitro. Cotransfection of plasmids expressing the wild type and mutant T3 receptor proteins resulted in inhibition of the wild type T3 receptor function. We conclude that this region of the rat beta 1 T3 receptor is essential for full transcriptional activity, but this is not due to a role in T3 binding, DNA binding, or nuclear localization. We postulate that nuclear factors may need to bind to this region for full transcriptional activity.

Amino Acid Sequence↗
PubMed1995

Crypt fission in the small intestine and colon. A mechanism for the emergence of G6PD locus-mutated crypts after treatment with mutagens.

In the small intestine and colon, administration of mutagens leads to the emergence of crypts populated by cells with a different, mutated phenotype. This is preceded by a transient rise in the frequency of crypts with a partially mutated phenotype, and the disappearance of these partially mutated crypts occurs contemporaneously with the attainment of a plateau value of the wholly mutated crypts. Here, using the mutagen ethyl nitrosourea and loss of glucose-6-phosphate dehydrogenase staining as a marker, we show that the plateau is reached at between 4.6 and 7 weeks in the colon and at 12 weeks in the small intestine of the same mice. Explanations for this difference have included differences in the stem cell cycle time of a single "master" stem cell or multiple stem cells occupying a stem cell "niche" with random loss after stem cell division. However, we demonstrate that the crypt fission index, or the incidence of crypts in fission, is some four times higher in the colon than in the small intestine at the time of ethyl nitrosourea injection, and propose an alternative hypothesis based on crypt fission as the mechanism for the more rapid evolution of wholly mutated crypts in the colon. The hypothesis should enable us to predict the results of future experiments, namely that the emergence of wholly mutated crypts is proportional to the crypt fission index.

Animals↗
PubMed2006

Computational simulation of hypertrophic cardiomyopathy mutations in troponin I: influence of increased myofilament calcium sensitivity on isometric force, ATPase and [Ca2+]i.

Familial hypertrophic cardiomyopathy (FHC) is an inherited disease that is characterized by ventricular hypertrophy, cardiac arrhythmias and increased risk of premature sudden death. FHC is caused by autosomal-dominant mutations in genes for a number of sarcomeric proteins; many mutations in Ca(2+)-regulatory proteins of the cardiac thin filament are associated with increased Ca(2+) sensitivity of myofilament function. Computational simulations were used to investigate the possibility that these mutations could affect the Ca(2+) transient and mechanical response of a myocyte during a single cardiac cycle. We used existing experimental data for specific mutations of cardiac troponin I that exhibit increased Ca(2+) sensitivity in physiological and biophysical assays. The simulated Ca(2+) transients were used as input for a three-dimensional half-sarcomere biomechanical model with filament compliance to predict the resulting force. Mutations with the highest Ca(2+) affinity (lowest K(m)) values, exhibit the largest decrease in peak Ca(2+) assuming a constant influx of Ca(2+) into the cytoplasm; they also prolong Ca(2+) removal but have little effect on diastolic Ca(2+). Biomechanical model results suggest that these cTnI mutants would increase peak force despite the decrease in peak [Ca(2+)](i). There is a corresponding increase in net ATP hydrolysis, with no change in tension cost (ATP hydrolyzed per unit of time-integrated tension). These simulations suggest that myofilament-initiated hypertrophic signaling could be associated with decreased [Ca(2+)](i), increased stress/strain, and/or increased ATP flux.

Actin Cytoskeleton↗