Liver Nuclear Proteins Research Articles

Effects of Aging on Rat Liver Nuclear Protein Kinase Activity Stimulated by the Thioacetamide Administration

Effects of Aging on Rat Liver Nuclear Protein Kinase Activity Stimulated by the Thioacetamide Administration

Transcriptional regulation of rat alpha 1-acid glycoprotein gene by phenobarbital.

Phenobarbital induces gene transcription of both cytochrome P450IIB (the barbiturate-inducible cytochrome P450 in mammals) and alpha 1-acid glycoprotein, one of the major acute-phase proteins in rats and humans. Analysis of the 5'-regulatory sequences of cytochrome P450IIB and alpha 1-acid glycoprotein genes in rats revealed the presence of a consensus sequence of 10 base pairs, termed the phenobarbital-responsive element or Barbie box, located in a region extending from positions -136 to -127 from the transcription start site of the alpha 1-acid glycoprotein gene. A 17-base pair oligonucleotide probe specific for alpha 1-acid glycoprotein and including the consensus sequence showed, in mobility shift assays, slight binding to liver nuclear protein from untreated animals. This binding was strongly and specifically increased with protein extracts from phenobarbital-treated rats. Transfection of rat primary hepatocytes with the pAGPcat construct induced basal expression of chloramphenicol acetyltransferase activity, which was increased by phenobarbital and dexamethasone treatment of cells. Induction of chloramphenicol acetyltransferase activity by phenobarbital was abolished when hepatocytes were transfected by constructs with a mutation or deletion of the Barbie box sequence. These results strongly suggest that the Barbie box sequence is involved in alpha 1-acid glycoprotein gene regulation by phenobarbital.

In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein.

Previous studies of rat ucp (uncoupling protein) gene organization carried out in this laboratory identified regulatory sequences located in the 5'-flanking region. In this work, DNase I footprint analysis of the enhancer revealed two domains at base pairs (bp) -2444 to -2423 and bp -2352 to -2319. The former domain can bind in vitro, in a cooperative manner, factors related to nuclear factor 1 and Ets1; the latter domain contains a type 3 directly repeated sequence that was shown to be able to bind the retinoid X and triiodothyronine receptors. Moreover, a positive effect of retinoic acid on ucp mRNA levels in immortalized brown adipocytes was observed. DNase I footprint analysis identified two hypersensitive regions, A and B, at bp -509 to -472 and bp -403 to -350, respectively; region A contains a repeated CACCC box, and region B can bind protein related to Ets1. The A box differentially binds liver and brown adipose tissue nuclear proteins and could be involved in uncoupling protein induction. Further analysis showed three foot-printed boxes, C-E, at bp -182 to -159, -147 to -120, and -111 to -85, able to bind in vitro proteins related to nuclear factor 1, cAMP response element-binding protein, and Sp1, respectively.

The Use of Biotinylated Poly(ADP-Ribose) for Studies on Poly(ADP-Ribose)-Protein Interaction

Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([32P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.

Interaction of Proteins with a Cytochrome P450 2B2 Gene Promoter: Identification of Two DNA Sequences That Bind Proteins That Are Enriched or Activated in Response to Phenobarbital

Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.

Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter

Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.

Analysis of the mechanism of glucocorticoid-mediated down regulation of the mouse α-fetoprotein gene

Regulation of α-fetoprotein gene expression by dexamethasone was examined in vivo and in vitro using primary mouse fetal liver cell cultures. Dexamethasone accelerates the developmental down regulation of AFP mRNA pools. However, treatment of primary fetal liver cells in culture does not reduce the AFP mRNA pool and may stabilize both AFP and albumin gene expression. These results indicate that in vivo the effect of dexamethasone may require interaction with another tissue or cell type. The mechanism of the dexamethasone mediated inhibition of AFP was examined by DNase I footprinting and transient expression assays. Two protein-binding regions of the proximal promoter (III and IV) show significant homology to the GRE consensus sequence. DNase I footprinting shows that only region IV can bind purified GR and competition with GRE oligonucleotides indicate that, using adult liver nuclear proteins, no GR is bound in either region. Nuclear protein from adrenalectomized mice show the same protection as controls. These results indicate that GR may not bind to the AFP proximal promoter in the adult. AFP promoter-CAT expression vectors were used to further examine the effect of dexamethasone on AFP expression. AFP promoter-CAT constructs were inhibited by 10 −6 M dexamethasone; while linking of an AFP enhancer to the promoter abolished the effect. We conclude that the in vitro effects on transiently expressed AFP directed expression vectors may be a function of vector structure and/or characteristics of the cells used whereas the in vivo effect may reflect normal regulatory mechanisms.

Studies with 1,1′-Ethylidenebis(tryptophan), a Contaminant Associated with L-Tryptophan Implicated in the Eosinophilia-Myalgia Syndrome

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [ 3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [ 3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1′-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [H-3]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [ 3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [ 14C]leucine incorporation into proteins. [ 14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

Decreased incorporation of 14C-leucine in different liver nuclear protein fractions at early stages of carbon tetrachloride poisoning in the rat.

Administration of CCl4 to 12-14 h starved Sprague Dawley male rats (220-240 g) at a dose of 5 ml of a 20% (v/v) olive oil solution/kg i.p. decreased the incorporation of 14C-leucine into microsomal and nuclear proteins at 1 or 2 h after the hepatotoxin. Decreased amino acid incorporation capacity in nuclear but not in microsomal proteins returned to control values at 4 h after poisoning. Cycloheximide given i.p. at a dose of 1 mg/kg in saline either alone or before CCl4 also decreased to a similar or greater extent the incorporation of 14C-leucine into both, microsomal and nuclear proteins at 1 h. The CCl4-induced decrease in amino acid incorporation was observable and significant even 1 h after CCl4 and involved histone and different non-histone nuclear protein fractions. Results suggest that decreased protein synthesis at the endoplasmic reticulum occurring in the very early stages of CCl4 poisoning might provoke temporary deficiencies in protein supplies to nuclei with as yet unknown consequences. However, some of the proteins involved have major regulatory properties in DNA expression.

Identification of a liver preference enhancer element of the rat hemopexin gene and its interaction with nuclear factors.

Transcription of hemopexin (Hx) occurs predominantly in the liver. To investigate the contribution of the cis-acting enhancer element to the hepatocyte preferential expression, we performed chloramphenicol acetyl-transferase (CAT) assays in HepG2 cells. A strong enhancer element was identified by successive truncation to reside in the region -157/-104 from the cap site. The Hx region -145/-125 interacts with rat liver nuclear proteins, as shown by standard DNA-protein binding assays. The nucleotide sequence -141AGACTTTGACCT-130 includes, in reverse orientation, a direct repeat of the imperfect AGGTCA sequence, one of the recognition motifs of the steroid-thyroid hormone receptor superfamily. That this AGGTCA repeat is an enhancer core of the Hx element was affirmed by mutational analyses. In electrophoretic mobility shift assays, oligonucleotides, corresponding to binding regions of chicken ovalbumin upstream promoter transcription factor (COUP-TF) and apolipoprotein AI regulatory protein 1 (ARP-1) but not of hepatocyte nuclear factor-4 (HNF-4), competed with the Hx element for binding sites. Co-transfection analyses indicated that HNF-4 does not affect CAT expression whereas ARP-1 and COUP-TF repress it. Antibody supershift analyses suggested that HNF-4 and COUP-TF may not be major factors binding to the Hx element.

Binding of nuclear proteins to transcription regulatory elements in selenium deficiency

Dietary selenium deficiency in rats increased binding of liver nuclear proteins to DNA regulatory sequences which activate transcription in response to oxidative stress. In contrast, binding to the activator protein-1 control sequence, which is inhibited by oxidation, was decreased in selenium deficiency.

Cloning and tissue-specific functional characterization of the promoter of the rat diazepam binding inhibitor, a peptide with multiple biological actions.

Diazepam binding inhibitor (DBI) is a 10-kDa polypeptide that regulates mitochondrial steroidogenesis, glucose-induced insulin secretion, metabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABAA receptors. To investigate the regulation of DBI gene expression, three positive clones were isolated from a rat genomic library. One of them contained a DBI genomic DNA fragment encompassing 4 kb of the 5' untranslated region, the first two exons, and part of the second intron of the DBI gene. Two other overlapping clones contained a processed DBI pseudogene. Several transcription initiation sites were detected by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription startpoint usage. Transient expression experiments using DNA fragments of different length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequence, whereas full activation was achieved with 423 bp of the 5' untranslated region. DNase I protection experiments with liver nuclear proteins demonstrated three protected regions at nt -387 to -333, -295 to -271, and -176 to -139 relative to the ATG initiation codon; in other tissues the pattern of protection was different. In gel shift assays the most proximal region (-176 to -139) was found to bind several general transcription factors as well as cell type-restricted nuclear proteins which may be related to specific regulatory patterns in different tissues. Thus, the DBI gene possesses some features of a housekeeping gene but also includes a variable regulation which appears to change with the function that it subserves in different cell types.

A Fos-Jun element in the first intron of an alpha 2u-globulin gene.

The hepatic expression of the alpha-2u-globulin gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect alpha 2u-globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an alpha 2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M2 peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG3 hepatoma cells treated with TPA. Co-transfection with fos and jun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.

Trans-activation of the alpha 1-acid glycoprotein gene acute phase responsive element by multiple isoforms of C/EBP and glucocorticoid receptor

alpha 1-Acid glycoprotein (AGP) is a major acute phase protein synthesized primarily by the liver. The AGP gene is transcriptionally activated in hepatocytes during the acute phase response to bacterial lipopolysaccharide. In this study, we analyzed an acute phase responsive element (APRE) located between nucleotide residues -127 to -104 relative to the transcription initiation site of the mouse AGP gene. Binding studies show that several trans-acting factors interact with the APRE. Using monospecific antibodies we demonstrate that three isoforms of the CCAAT/enhancer-binding protein (C/EBP) family, namely C/EBP alpha, C/EBP beta, and C/EBP delta, bind to the APRE. Furthermore, with liver nuclear protein from control animals, C/EPB alpha is the predominant form that binds to the APRE, whereas with nuclear proteins from acute phase-induced animals, C/EBP alpha is replaced by C/EBP beta. The mechanism of activation of the AGP gene during the acute phase response appears to involve an exchange of C/EBP alpha by C/EBP beta. C/EBP delta does not play a role in this reaction. Interestingly, the C/EBP binding site of the APRE partially overlaps a functional glucocorticoid responsive element. We present evidence that both purified C/EBP alpha and glucocorticoid receptor bind strongly to the APRE. By site-specific mutation, we have identified the C/EBP and glucocorticoid receptor binding sites in the APRE. These mutants were used in expression vectors to demonstrate that both C/EBP and glucocorticoid receptor are essential for maximal response to interleukin-6 and dexamethasone. These results demonstrate that the APRE is a composite binding site for multiple factors that are responsible for the transcriptional control of the mouse AGP. Finally, functional analyses indicate that C/EBP alpha, C/EBP beta, and C/EBP delta are strong transcriptional trans-activators of the AGP APRE in hepatoma cells. These data suggest that the regulatory activity of the C/EBP with the APRE in the liver may require interactions with adjacent proteins.

CCAAT/enhancer-binding protein-related proteins bind to the unusual promoter of the aspartate aminotransferase housekeeping gene.

The cytosolic aspartate aminotransferase (cAspAT) gene is ubiquitously expressed but it is regulated by hormones in a tissue-specific manner. In vitro DNase I footprinting studies of a 260-base pair fragment carrying the basal promoter activity revealed that three CCAAT sequences bind liver nuclear proteins (protected regions P2, P3, P4). Competition studies, the heat resistance of these proteins, and identical footprints obtained using a recombinant CCAAT/enhancer-binding protein (C/EBP) alpha fragment indicate that they belong to the C/EBP family of transcription factors. A fourth protected region P1, overlapping the P2 region, was observed in the liver in the presence of competing oligonucleotides containing the C/EBP site. In cotransfection experiments, the C/EBP beta protein trans-activated 10-15-fold the cAspAT gene promoter in HepG2 cells. Deletion studies revealed that regions P2 and P4 are critical for promoter activity. In gel retardation experiments, the P4 region bound different C/EBP-related proteins in different tissues: the brain protein is heat sensitive in contrast to the liver protein. The synthetic oligonucleotide OL 1-2, which covers the P1 and P2 regions, binds C/EBP-like proteins as well as NF1 and CP1 (or NFY) transcription factors, but the preferential binding of one of these protein is tissue-specific. In summary, using the cAspAT gene promoter, we have shown that ubiquitously active promoters may be recognized by different proteins in different tissues.

High level activity of the mouse CCAAT/enhancer binding protein (C/EBP alpha) gene promoter involves autoregulation and several ubiquitous transcription factors.

The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBP alpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBP alpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBP alpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region -350/+3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBP alpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers and mutation of this site drastically reduces transcription of C/EBP alpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NF kappa B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBP alpha. Furthermore, transient expression of C/EBP alpha and to a lesser extent C/EBP beta expression vectors, results in transactivation of a cotransfected C/EBP alpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBP alpha gene is regulated by C/EBP alpha but other C/EBP-related proteins may also be involved.

Structure and expression of the genes encoding peroxisomal β-oxidation enzymes

To investigate the mechanism of induction of the peroxisomal β-oxidation enzymes by peroxisome proliferators, genes of these enzymes were cloned from rat liver and their structures analyzed. The acyl-CoA oxidase gene was found to produce two forms of the enzyme differing in their amino acid sequences in a limited region, through alternative splicing of the two copies of the third exon. The amino acid sequence of the bifunctional enzyme suggests, compared with those of its mitochondrial counterparts, that the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities are located on the amino- and the carboxyl sides, respectively. Two copies of the 3-ketoacyl-CoA thiolase gene were identified per haploid genome. One gene named A is constitutive and encodes a thiolase precursor carrying 36 amino acid residues of amino-terminal presequence, whereas the other, termed B, is remarkably induced by peroxisome proliferators and specifies a precursor having a 26-residue presequence. Functional analysis of the upstream sequence of the acyl-CoA oxidase gene revealed three functionally different regions, one of which had the character of ciprofibrate-responsive enhancer. In this region, two sequences were identified as binding-sites of rat liver nuclear proteins. A gene transfection study indicated that these sequence elements (termed A and B) play important roles in the induction of the gene, the former acting positively whereas the latter probably is acting negatively.

Carbon tetrachloride induction of rapid changes in liver nuclear protein factors capable of sequence-specific binding to regulatory elements in the long terminal repeat of polytropic-class endogenous murine leukemia virus-related proviruses.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus-related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCl4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5' of the consensus enhancer sequences) and the 3' terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3-6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCl4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCl4 exposure are discussed.

Competitive studies relating to tryptophan binding to rat hepatic nuclear envelopes as a sensitive assay for unknown compounds

Our laboratory has reported that L-tryptophan binds to a rat liver nuclear envelope protein and this binding is saturable, stereospecific and of high affinity. Utilizing an in vitro [ 3H]tryptophan binding assay to hepatic nuclear envelopes, we have determined the effects of using excess unlabeled L-tryptophan from a number of different suppliers. This study reports that, based on our in vitro binding assay, some significant differences were observed when implicated L-tryptophan in cases of the eosinophilia-myalgia syndrome obtained from a Japanese manufacturer, Showa Denko, was assayed, in contrast to non-implicated L-tryptophan from other suppliers. An isolated impurity of Showa Denko L-tryptophan, 1,1′-ethylidenebis(tryptophan) alone or together with non-implicated L-tryptophan or its breakdown product, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, did not appreciably affect the in vitro [ 3H]tryptophan binding to hepatic nuclear envelopes as did the Showa Denko L-tryptophan. Our data, derived with our in vitro binding assay system, suggests that implicated L-tryptophan from Showa Denko contains a compound/s (unknown at present) other than 1,1′-ethylidenebis(tryptophan), which alters in vitro [ 3H]tryptophan binding. The significance of the impurity/ies involved remains to be determined.

Characterization of rat liver nuclear proteins which recognize the cAMP responsive element

1. 1. The data herein reveal the existence of cAMP-responsive element (CRE)-binding factors (CRF) in the nuclear extracts from cAMP-treated rat liver. 2. 2. DNAase I and DMS footprinting analysis showed that the CRFs protected the CRE (−77 to −92) in the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the TGACGTCA motif in a consensus oligodeoxynucleotide based on the sequence of the CRE's of 6 cAMP-regulated genes (C32mer). 3. 3. Competition assays indicate that the CRF(s) is a CGTCA-specific, ATF/CREB-like factor(s). 4. 4. Southwestern (SW) blot analysis detected 2 apparent CRFs which have molecular weights of about 30 and 32 kDa, respectively. 5. 5. Based on the comparison of the size and binding specificity of the CRFs with the CREBs reported to date, the CRFs appear to be novel CRE-binding nuclear factors.