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PubMed1990

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes.

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.

Adolescent↗
PubMed1995

A novel DNA/protein complex interacts with the insulin-like growth factor binding protein-1 (IGFBP-1) insulin response sequence and is required for maximal effects of insulin and glucocorticoids on promoter function.

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic (CAAAACAAACTTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (< or = 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a beta-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with beta-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Animals↗
PubMed2005

Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

Animals↗
PubMed2000

The DNA replication-related element (DRE)-DRE-binding factor (DREF) system may be involved in the expression of the Drosophila melanogaster TBP gene.

The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. Previously, we found that the promoter region of the Drosophila melanogaster TBP gene contains three sequences similar to the DNA replication-related element (DRE) (5'-TATCGATA). In the present study, we found that the DRE-like sequences are also present in the promoter of the Drosophila virilis TBP gene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE of D. melanogaster TBP gene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE-binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE-related sequences at positions -223 and -63 resulted in an extensive reduction of promoter activity. Thus, the DRE-DREF system appears to be involved in the expression of the D. melanogaster TBP gene.

Animals↗
PubMed1999

Characterization of the transactivation domain of the equine herpesvirus type 1 immediate-early protein.

Equine herpesvirus type 1 (EHV-1) possesses a sole diploid immediate early gene (IE) that encodes a major regulatory protein of 1487 amino acids capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Using a series of GAL-4-IE fusion constructs, we previously demonstrated that the minimal transactivation domain (TAD) of the IE protein maps within amino acids 3-89. Additional studies revealed that that the carboxyl terminus of the IE protein may be required for full transactivation activity in vitro. Analyses of the minimal TAD revealed the presence of 13 acidic amino acids and six basic residues giving the TAD region a net negative charge of -7. In addition, there are conserved hydrophobic residues (Leu(12) and Phe(15)) that may be critical for transactivation function. To identify residues essential for IE transactivation and to ascertain if the overall net negative charge of the TAD or the position of specific hydrophobic residues within the IE TAD are critical for the transactivation function, plasmids expressing mutant forms of the TAD were generated using specifically designed mutagenic oligonucleotides and PCR mutagenesis. Mutagenized TADs in which the acidic and hydrophobic amino acid residues were replaced, singly and in combination, with polar, uncharged amino acids were cloned into a GAL-4/CAT reporter expression system and assayed in transient transfection assays. To determine if the carboxyl terminus is necessary for full transactivation activity, a series of constructs that express forms of the IE protein-containing deletions within this region were generated and assayed for transactivation function in transient transfection assays. These assays demonstrated that mutation of any acidic residue, either singly or in combination, or deletion of the carboxyl terminus of the IE protein resulted in a severe impairment of transactivation activity. These results show that both acidic and hydrophobic residues within the IE TAD are critical for transactivation function and that the carboxyl terminus of the IE protein is required for full transactivation activity.

Animals↗
PubMed1997

Two thrombotic complications in a patient with active ulcerative colitis.

A 30-year-old woman is described with both a cerebral venous thrombosis and a deep vein thrombosis of her leg in association with an exacerbation of her ulcerative colitis. Laboratory investigations revealed a transient functional APC resistance, without Factor V mutation, and a mild hyperhomocysteinaemia. After colectomy the APC ratio was normal. These findings may form a partial explanation of the thrombophilia seen in some patients with active ulcerative colitis.

Adult↗
PubMed1996

Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts.

PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Ataxia Telangiectasia↗
PubMed2002

Sp1 is essential for estrogen receptor alpha gene transcription.

The exact molecular mechanisms regulating estrogen receptor (ER)alpha expression in breast tumors are unclear, but studies suggest that the regulation is at least partly transcriptional. We therefore undertook a detailed analysis of ERalpha promoter activity in a number of breast cancer cell lines. We find that the majority of ERalpha promoter activity lies within the first 245bp of the 5'-flanking region of the gene. Three elements essential for full ERalpha promoter transcriptional activity were identified within the -245 to -192bp region in transient transactivation assays using linker-scanner mutation analysis. These three elements include two binding sites for the Sp1 family of transcription factors as well as a non-consensus E box. We show that both Sp1 and Sp3 bind to this region using electrophoretic mobility shift assays. Exogenous expression of Sp1 or Sp3 in Sp1/3-negative Drosophila Schneider SL2 cells results in transactivation of the -245 to +212bp fragment of the ERalpha promoter. These data demonstrate that transcription of ERalpha is dependent upon the expression of members of the Sp1 family.

Animals↗
PubMed2002

Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids.

The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.

Amino Acid Sequence↗
PubMed2002

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L.

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.

Benzyl Alcohols↗
PubMed2000

Identification and characterization of cis-acting elements conferring insulin responsiveness on hamster cholesterol 7alpha-hydroxylase gene promoter.

Bile acid biosynthesis occurs primarily through a pathway initiated by the 7alpha-hydroxylation of cholesterol, catalysed by cholesterol 7alpha-hydroxylase (encoded by CYP7A1). Insulin down-regulates CYP7A1 transcription. The aim of our study was to characterize the sequences of hamster CYP7A1 promoter, mediating the response to insulin. We therefore performed transient transfection assays with CYP7A1 promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster CYP7A1 promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster CYP7A1 insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster CYP7A1. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of CYP7A1; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster CYP7A1 proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin.

Animals↗
PubMed2003

Follow-up of newborns with elevated screening T4 concentrations.

OBJECTIVE: To determine the type and incidence of hyperthyroxinemic disorders detected by follow-up of infants with elevated screening total T4 (TT4) values. STUDY DESIGN: Infants born in Oregon with a screening TT4 measurement >3 SD above the mean were offered enrollment. Serum TT4, free T4, total T3, free T3, and thyroid-stimulating hormone concentrations were measured in study infants and their mothers. RESULTS: Over a 20-month period, 101 infants (51 boys) and their mothers enrolled in the study (of 241 eligible infants), from a total screening population of 80,884; 17 infants were identified with persistent hyperthyroxinemia (TT4 >16 microg/dL). Ten had thyroxine-binding globulin excess (1:8088), 5 had evidence for increased T4 binding but not thyroxine-binding globulin excess (1:16,177), and 2 had findings compatible with thyroid hormone resistance (1:40,442); the other 84 infants had transient hyperthyroxinemia. Sequence analysis revealed a point mutation in the thyroid hormone receptor-beta gene in one infant with thyroid hormone resistance; no mutation was identified in the other infant. CONCLUSIONS: Although neonatal Graves' disease occurs in approximately 1 in 25,000 newborn infants, we did not detect any case among 80,884 infants, most likely because their mothers were receiving antithyroid drugs. Although the other hyperthyroxinemic disorders in the aggregate occur frequently (1:4758) and may benefit from detection, in general they do not require treatment.

Female↗
PubMed2003

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation.

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH*) induces an electron transfer over approximately 15 A from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* <-- W382 <-- W359 <-- W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant tau approximately 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (tau approximately 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield ( approximately 0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (tau approximately 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

Base Sequence↗
PubMed1986

Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene.

Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously.

Bacterial Proteins↗
PubMed1995

The conserved box 1 motif of cytokine receptors is required for association with JAK kinases.

The JAK2 tyrosine kinase is known to associate with the receptors for growth hormone (GH) and erythropoietin (EPO) and with the interleukin-6 receptor signal transducing protein, gp130. Here we demonstrate that chimeric cytokine receptors which contain the cytoplasmic domain of the receptors for GH and EPO or for gp130 can form complexes with JAK2 when transiently co-expressed in HeLa cells. Mutational analyses of chimeras for the the GH and EPO receptors and gp130 demonstrated that box 1, a motif critical for cytokine receptor signal transduction, was required for the association of JAK2. Although JAK2 was capable of associating with all three of the chimeras, JAK1 co-precipitated only with the gp130 chimera. Association of JAK1 and JAK2 with cytokine receptor proteins, therefore, requires the highly conserved box 1 domain, but other sequences within the receptor proteins may influence the specificity of JAK binding. Mutational analysis of JAK2 revealed that multiple or complex protein sequences within JAK2 are required for association with cytokine receptors.

Animals↗
PubMed1995

Angiotensin II receptor endocytosis involves two distinct regions of the cytoplasmic tail. A role for residues on the hydrophobic face of a putative amphipathic helix.

Following agonist stimulation, many receptors are rapidly internalized from the plasma membrane via a mechanism which presumably involves recognition motifs within the cytoplasmic domains of the receptor. We have previously demonstrated (Thomas, W. G., Thekkumkara, T. J., Motel, T. J., and Baker, K. M. (1995) J. Biol. Chem. 270, 207-213) that truncation of the angiotensin II (AT1A) receptor, to remove 45 amino acids from the cytoplasmic tail, markedly reduced agonist stimulated receptor endocytosis. In the present study, we have stably and transiently expressed wild type and carboxyl terminus mutated AT1A receptors in Chinese hamster ovary cells to identify regions and specific amino acids important for this process. Wild type AT1A receptors rapidly internalized (t1/2 = 2.5 min; Ymax = 76.4%) after AII stimulation. Using AT1A receptor mutants, truncated and deleted at the carboxyl terminus, two distinct regions important for internalization were identified: one membrane proximal site between residues 315-329 and another distal to Lys333, within the terminal 26 amino acids. Point mutations (Y302A, Y312A, L316F, Y319A, and K325A) were performed to identify residues contributing to the membrane proximal site. Mutation of Y302A, Y312A, and K325A had little effect on the rate (t1/2 = 4.3, 2.8, and 2.8 min) and maximal amount (Ymax = 81.7, 67.8, and 73.5%) of AII induced internalization. In contrast, L316F and Y319A mutations displayed an approximately 2.5-fold reduction in rate (t1/2 = 6.1 and 6.2 min) and L316F a decreased maximal level (Ymax = 38.1 and 71.4%, respectively) compared to wild type. Interestingly, Leu316 and Tyr319 are closely aligned within the hydrophobic aspect of a putative amphipathic helix, possibly representing an internalization motif for the AT1A receptor. We conclude that the AT1A receptor does not use NPXXY (NPLFY302) motif, first described for the beta 2-adrenergic receptor, to mediate agonist stimulated endocytosis. Rather, two distinct regions of the carboxyl terminus are utilized: one involving hydrophobic and aromatic residues on a putative alpha-helix and another serine/threonine-rich domain.

Amino Acid Sequence↗
PubMed1996

Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone. Possible allosteric mechanism of channel blockade.

Mechanisms of ion channel blockade by noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) have been particularly difficult to elucidate. We have combined here transient expression of embryonic, adult, and a mutated adult muscle AChR associated with a slow channel syndrome (Ohno, K., Hutchinson, D. O., Milone, M., Brengman, J. M., Bouzat, C., Sine, S., and Engel, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 758-762) with single channel recordings to determine subunit specificity and mechanisms of action of the prototype glucocorticoid hydrocortisone (HC). HC affected in a similar manner the gating kinetics of all types of muscle AChR, producing briefer openings with normal amplitudes. We postulate that this steroid acts as a noncompetitive inhibitor of the AChR and that its mechanism of action can be interpreted in terms of blocking models. The forward rate constant for the blocking process was also similar for all channel types, indicating that the structural differences between them are not responsible for the effect. The reduction in the channel open time was not dependent on agonist concentration; it was slightly voltage dependent, suggesting that HC binds to a site located inside the membrane that senses the electric field. Recordings at high acetylcholine concentration in the presence of HC showed a reduced number of openings per activation period and the long closed times typically observed in the desensitization phenomenon. In competition studies with the classical open channel blocker QX-222, HC induced an early termination of the burst, suggesting that the two act at different sites. Taken together the results support the existence of specific sites sensed by the membrane field, different from those of open channel blockers and probably located at the lipid-protein interface. From this site(s), glucocorticoids and other hydrophobic noncompetitive inhibitors could allosterically mediate channel blockade.

Adult↗
PubMed2000

Regulation of the human O(6)-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300.

O(6)-Methylguanine-DNA methyltransferase (MGMT)(1), a ubiquitous DNA repair protein, removes O(6)-alkylguanine from DNA, including cytotoxic O(6)-chloroethylguanine induced by chemotherapeutic N-alkyl N-nitrosourea-type drugs, e.g. 1,3-bis(2-chloroethyl)-1-nitrosourea. Treating the pancreatic carcinoma cell line MIA PaCa-2 with trichostatin A (TSA), a specific inhibitor of histone deacetylase, increased MGMT mRNA and protein levels by 2-3-fold. Surprisingly, TSA treatment increased MGMT promoter-dependent luciferase activity by some 40-fold in a transient reporter expression assay. Deletion and point mutation analysis showed that two AP-1 binding sites in the MGMT promoter are involved in activation by TSA. Ectopic expression of the transcriptional coactivators cAMP response element-binding protein-binding protein (CBP) and p300, which have intrinsic histone acetyltransferase activity, enhanced luciferase expression. Overexpression of adenovirus E1A, which binds CBP/p300, strongly inhibited both basal and TSA-inducible MGMT promoter activity, while a mutant E1A, defective in binding CBP/p300, did not. Chromatin immunoprecipitation assays revealed that TSA treatment increased histone acetylation in the endogenous MGMT promoter region, which also showed association with CBP/p300. Taken together, our results indicate that targeted histone acetylation results in the remodeling of chromatin by recruitment of the coactivator CBP/p300, and constitutes an important step in regulating MGMT expression.

Acetylation↗