Specific Intracellular Proteins Research Articles

Engagement of CD45 during in vitro priming enhances antigen-specific Th cell frequencies.

CD45 is a transmembrane protein tyrosine phosphatase expressed by all lymphoid cells including T cells. Substantial experimental data has shown that CD45 maintains a permissive state for TCR signaling. The highly glycosylated extracellular domain of CD45 may be the site of interaction with regulatory lectin-like counter-receptors on antigen-presenting cells. The mAb NDA5, recognizing a unique but broadly distributed epitope of CD45, was used to study the possible immunoregulatory role of CD45 during anti-CD3 and antigen-specific CD4+ T cell activation. In vitro priming of peripheral blood mononuclear cells with peptide antigens in the presence of mAb NDA5 results in a higher frequency of antigen-specific T cells. The responses of both naive and memory T cells to peptide antigens were sensitive to mAb NDA5-enhanced priming. Anti-CD3 activation of normal resting T cells, in the presence of mAb NDA5, resulted in enhancement of tyrosine phosphorylation of specific intracellular proteins associated with TCR signal transduction. In cultures without antigen, mAb NDA5 down-regulated the cell surface expression of both CD3 and CD4, yet did not stimulate proliferation of resting T cells. Together these results suggest that engagement of CD45 during in vitro priming has a significant effect on the development of antigen-specific T cell populations.

7B2 is a specific intracellular binding protein of the prohormone convertase PC2.

Biosynthetic pulse-chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro-PC2 and that zymogen activation to PC2 occurs following the slow exit of pro-PC2 from the endoplasmic reticulum (ER) and its concentration within the trans-Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro-7B2), which is cleaved within the TGN by a furin-like ubiquitous convertase at the RRKRR155S site to generate 7B2. In this report, we demonstrate that within the ER, pro-7B2 binds pro-PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca2+ dependent and does not require an N-glycosylated pro-PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR152 and greatly diminished by mutations of either the R151 or S156 residues of pro-7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin-like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca(2+)-dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro-PC2, did not eliminate the processing of pro-PC2 to PC2.(ABSTRACT TRUNCATED AT 250 WORDS)

A calorimetric study of the thermal stability of barstar and its interaction with barnase.

The temperature-induced unfolding of single, double, and triple mutants of barstar, the specific intracellular protein inhibitor of barnase from Bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH. The unfolding enthalpy and heat capacity changes are slightly lower than those accepted for highly compact, small, globular proteins. We have found that at pH 2.5, where barstar seems to be in a molten globule state, the protein has a heat capacity between that of the native and the unfolded states and shows some tendency for association. Scanning calorimetry experiments were also extended to the barstar--barnase complex in the neutral and alkaline pH range. The binding constants obtained from DSC studies are similar to those already obtained from other (kinetic) studies. The interaction of barstar and barnase was also investigated by isothermal calorimetry in various buffers within the pH range 6.0-10.0 and a temperature range of 15-35 degrees C. The favorable enthalpy contribution to the binding is about 4 times higher than the entropic one at 25 degrees C. The overall data analysis of the combined calorimetric results has led to the thermodynamic characterization of barstar unfolding and the interaction of barstar and barnase over a wide range of temperatures.

The Differentiation-Related Upregulation of Aryl Hydrocarbon Receptor Transcript Levels Is Suppressed by Retinoic Acid

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binds to a specific intracellular receptor protein -the Ah-receptor - which acts as a transcription factor. Herein we report on the relative levels of Ah-receptor-mRNA and TCDD-induced cytochrome P450 1A1 (Cyp1A1)-mRNA and their modulation by retinoic acid in the human keratinocyte cell line HaCaT. Ah-receptor-mRNA was already present in proliferating keratinocytes and increased 8-fold in the course of differentiation. Addition of 10 nM TCDD did not alter the level of Ah-receptor transcripts. In contrast, addition of 1 μM RA maintained the amount of Ah-receptor-mRNA at the basal level as observed only in proliferating keratinocytes. The transcription of Cyp1A1 was dependent on TCDD-treatment and increased fivefold in more differentiated cells as compared to proliferating cells. Simultaneous addition of retinoic acid revealed an only twofold increase. These results indicate that the expression of the AhR is dependent on the state of differentiation of keratinocytes and seems to be affected by retinoic acid.

Two Polypeptides that are Secreted from Suspension-Cultured Tobacco Cells in the Presence of Brefeldin A have Chitin-Binding Domains

Summary Polypeptides SN9 and SN11 are secreted from suspension-cultured tobacco cells in the presence of Brefeldin A. Both polypeptides have N-terminal chitin-binding domains very similar to those of class I chitinases from tobacco. After SDS-polyacrylamide gel electrophoresis under denaturing conditions, the SNll band exhibited chitinase activity. Antibodies directed against barley chitinase as well as against class I chitinases of tobacco cross-react with SN11. However, no cross-reaction took place with polypeptide SN9, which could also not degrade glycolchitin. Both polypeptides could be localized in vacuoles of tobacco mesophyll protoplasts. Whereas SNll seems to be one of the well characterized class I chitinases (EC 3.2.1.14) of tobacco, SN9 appears to be a lectin-like protein without chitinase activity. Experiments with SN11- and SN9-specific DNA and immunoprobes suggest that the Brefeldin A insensitivity of the secretion of SN9 and SN11 is due to a specific intracellular protein transport mechanism and not to Brefeldin A-induced synthesis of these polypeptides.

The mechanism of dioxin toxicity: relationship to risk assessment.

Risk characterization involves hazard identification, determination of dose-response relationships, and exposure assessment. Improvement of the risk assessment process requires inclusion of the best available science. Recent findings in the area of dioxin toxicity have led to a major effort to reassess its risk. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as "dioxin," is the most toxic member of a class of related chemicals including the polyhalogenated dibenzo-p-dioxins, dibenzofurans, biphenyls, naphthalenes, azo- and azoxy-benzenes, whose toxicities can be expressed as fractional equivalencies of TCDD. These chemicals exert their effects through interaction with a specific intracellular protein, the Ah receptor. While binding to the receptor is necessary, it is not sufficient to bring about a chain of events leading to various responses including enzyme induction, immunotoxicity, reproductive and endocrine effects, developmental toxicity, chloracne, tumor promotion, etc. Some of these responses appear to be linear at low doses. Immunotoxicity and effects on the reproductive system appear to be among the most sensitive responses. The Ah receptor functions as a transcriptional enhancer, interacting with a number of other regulatory proteins (heat shock proteins, kinases, translocases, DNA binding species). Interaction with specific base sequences in the DNA appear to be modulated by the presence of other growth factors, hormones and their receptors as well as other regulatory proteins. Thus, dioxin appears to function as a hormone, initiating a cascade of events that is dependent upon the environment of each cell and tissue. While Ah receptor variants exist, all vertebrates examined have demonstrated such a protein with similar numbers of receptors and binding affinity for TCDD. Most species respond similarly to dioxin and related compounds. While a given species may be an outlier for a given response, it will behave like other animals for other responses. For both in vivo and in vitro end points where animal and human data exist, such as enzyme induction, chloracne, immunotoxicity, developmental toxicity, and cancer, the sensitivity of humans appears similar to that of experimental animals. Current levels of environmental exposure to this class of chemicals may be resulting in subtle responses in populations at special risk such as subsistence fisherman and the developing infant, as well as in the general population. Increased understanding of the mechanism of dioxin's effects as well as elucidation of exposure-dose relationships is leading to the development of a biologically based dose-response model in the ongoing process of incorporating the best science into the risk assessment of TCDD and related compounds.

Mechanistic studies of a signaling pathway activated by the organic dimerizer FK1012.

The T-cell receptor (TCR) signaling pathway is initiated by regulated association of TCR chains, including the zeta chain. A recently reported method for inducing the dimerization or oligomerization of targeted proteins in cells used the TCR pathway as a test system. In cells transfected with cDNA encoding MZF3E, a chimeric receptor comprising the intracellular domain of the zeta chain and three copies of FK506-binding protein (FKBP), low concentrations of a synthetic dimer of the natural product FK506 (FK1012) activated the expression of reporter genes. We set out to examine the signaling pathway initiated by FK1012. We characterized the effect of FK1012 on MZF3E and a second chimeric receptor, MZF1E, which contains the zeta chain and one copy of FKBP. Only MZF3E gave FK1012-activated signaling, as shown by an increase in the kinase activity associating with MZF3E, and the appearance of specific phosphotyrosine-containing proteins. Signaling required localization of MZF3E to the inner plasma membrane, and activation of gene transcription in response to FK1012 was dependent on the protein phosphatase calcineurin and the transcriptional activator NF-AT. Some signaling events in the pathway had different kinetics when activated by MZF3E instead of the TCR, however. An unexpected requirement for the prolonged activation of calcineurin was observed. Synthetic dimerizers can be used to gain control over cellular processes that require the association of specific intracellular proteins. The TCR signaling pathway was selected as an initial test system; we show here that one can indeed activate this signaling pathway by inducing the oligomerization of the cytoplasmic tail of the zeta chain with the cell-permeable reagent FK1012.

Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras.

Integrins are heterodimeric, transmembrane cell adhesion receptors that have recently been shown to function in transmembrane signal transduction. To examine the specific role of integrin intracellular domains in signal transduction, chimeric receptors containing various integrin intracellular domains coupled to a reporter consisting of the transmembrane and extracellular domains of the small, non-signaling subunit of the interleukin-2 receptor were expressed in cultured human fibroblasts and assayed for their ability to trigger tyrosine phosphorylation of the 125-kDa cytoplasmic tyrosine kinase, pp125FAK. Tyrosine phosphorylation of pp125FAK was induced in cultured fibroblasts that transiently expressed chimeric receptors containing either the beta 1, beta 3, or beta 5 integrin intracellular domain and were selected by magnetic bead sorting. However, expression of chimeric receptors containing either the alpha 5 or an alternatively spliced form of the beta 3 intracellular domain (beta 3B), as well as those lacking an intracellular domain, failed to induce tyrosine phosphorylation of pp125FAK. These results indicate that information contained in the beta 1, beta 3, or beta 5 integrin intracellular domain is sufficient to stimulate integrin-mediated tyrosine phosphorylation of specific intracellular proteins and that integrin extracellular and transmembrane domains are not required for inducing tyrosine phosphorylation. Our results also indicate that alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction, and they further suggest that the carboxyl-terminal region of specific beta integrins may play a role in the signal transduction pathway involving extracellular matrix molecules.

The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene.

Modification of specific intracellular proteins by ubiquitin targets them for degradation. We describe a yeast enzyme, Doa4, that is integral to the degradation of ubiquitinated proteins and is required in diverse physiological processes. Doa4 appears to function late in the proteolytic pathway by cleaving ubiquitin from substrate remnants still bound to protease. The human tre-2 oncogene encodes a deubiquitinating enzyme similar to Doa4, indicating a role for the ubiquitin system in mammalian growth control.

Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events.

Ligation of the multimeric T-cell antigen receptor complex (TCR) triggers a pleiotropic cellular activation response that includes lymphokine secretion, cell-cycle progression, and ultimately, T-cell proliferation. The earliest detectable biochemical event triggered by TCR cross-linkage is the tyrosine phosphorylation of specific intracellular proteins, which, in turn, propagate receptor-mediated signals into the cytoplasm and nucleus. In this study, we have examined the effects of pervanadate, a powerful inhibitor of protein tyrosine phosphatases (PTP), on the activation state of the human leukemic T-cell line, Jurkat. Treatment of Jurkat cells with pervanadate rapidly induced a series of proximal T-cell activation events that closely resembled those induced by TCR-dependent stimuli. Moreover, pervanadate treatment, like TCR cross-linkage, stimulated interleukin-2 production in wild-type Jurkat cells, indicating that the biochemical events initiated by this TCR-independent stimulus were sufficient to induce lymphokine gene expression. Exposure of intact cells to pervanadate also stimulated the in vitro catalytic activities of both p59fyn and p56lck, src family kinases strongly implicated in TCR-mediated signaling. The stimulatory effects of pervanadate on protein tyrosine kinase-mediated signaling events were accompanied by a marked inhibition of CD45-associated PTP activity. However, the ability of pervanadate to stimulate tyrosine phosphorylation in CD45-negative Jurkat cells suggests that PTPs other than CD45 are important intracellular targets for pervanadate. These studies demonstrate that inhibition of PTP activities in Jurkat cells leads to a T-cell activation response that is remarkably similar to that induced by TCR crosslinkage.

Ubiquitin as a degradation signal.

For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.

Tyrosine phosphorylation is an obligatory event in IL-2 secretion.

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.

Tumor necrosis factor production by Kupffer cells requires protein kinase C activation

Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock. In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear. Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters. We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production. Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS ( Escherichia coli 0111:B4) or LPS in the presence of H-7, a selective PKC inhibitor. Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-α. We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 μ M, while H-8 (a cyclic nucleotide specific inhibitor) had no effect. The effect of H-7 was dose dependent and present at varying concentrations of LPS. Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation. The inhibitor H-7 (10 μ M) also significantly inhibited LPS signaled prostaglandin E 2 release in Kupffer cells. Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipititation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32 Pi. Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 ± 0.9-fold) and a 33-kDa protein (3.1 ± 1.0-fold) which were inhibited by H-7. We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.

Two-Dimensional Gel Autoradiographic Analyses of the Effects of 1,25-Dihydroxycholecalciferol on Protein Synthesis in Clonal Rat Osteosarcoma Cells*

The steady state synthesis of L-[35S]methionine-radiolabeled cellular proteins by two rat osteogenic sarcoma cell lines (G2 and C12) was examined by two-dimensional polyacrylamide gel electrophoresis under basal conditions and after 72-h treatments with 10 nM 1,25-dihydroxycholecalciferol or triamcinolone acetonide. Computer analysis resolved 681 spots, with mol wt ranging from 10-105K and isoelectric points ranging from 4.0-8.0. Fourteen spots were abundant (greater than or equal to 2000 parts/million), with the remainder occurring in limited abundance (150-2000 parts/million) in both clones. Only 28 proteins were radiolabeled at significantly different rates by G2 and C12 cells under basal conditions. The high degree of similarity in the identity and relative abundance of proteins synthesized by these distinct subclones suggests that minor changes in the levels of specific intracellular proteins may have major effects on the osteoblastic phenotype. 1,25-Dihydroxycholecalciferol [1,25-(OH)2D3] or triamcinolone acetonide treatment induced qualitative and quantitative changes in the synthesis of specific subsets of proteins, including induction of novel proteins, complete repression of proteins synthesized under basal conditions, and significant increases or decreases in the levels of others. 1,25-(OH)2D3 significantly altered the levels of 13 proteins in G2 cells and 28 proteins in C12 cells. 1,25-(OH)2D3 enhanced the synthesis of two proteins (no. 304 and 2506) in both subclones. The remainder of the proteins affected by 1,25-(OH)2D3 were unique to the subclone. With the exception of protein 304, the changes induced by 1,25-(OH)2D3 differed from those induced by triamcinolone acetonide, suggesting that unique proteins modulate the osteoblastic phenotype in response to these steroids.

Deficient Collagen-Induced Activation in the Newborn Platelet

We have investigated the impaired secretion response of neonatal platelets. We compared the response of washed neonatal and adult platelets to thrombin and collagen, and to specific activators of calcium flux (inositol trisphosphate) and protein kinase C activation (oleoyl-acetyl glycerol). Neonatal platelets show no impairment of aggregation, secretion of [14C]serotonin or phosphorylation of specific intracellular proteins in response to thrombin, inositol trisphosphate, or oleoyl-acetyl glycerol. However, neonatal platelets have a markedly decreased response to collagen. To further evaluate this deficient response, we examined specific aspects of the collagen activation pathway. Collagen-platelet interaction as measured by adhesion of platelets to collagen-coated dishes showed no difference in adhesion of neonatal platelets compared to adult controls (20.1 +/- 11.6 versus 18.6 +/- 9.3%). The presence of GPIa/IIa, a Mg2(+)-dependent collagen receptor, was evaluated by flow cytometric analysis of binding of fluorescein-tagged monoclonal antibody, 6F1 (directed against GPIa/IIa). There was no difference either in the percent of platelets that bound antibody (80 versus 81%) or in the mean fluorescence intensity of the adult and neonatal samples. Phosphoinositide hydrolysis was decreased in neonatal platelets in response to collagen but normal in response to thrombin. Neonatal platelets released more arachidonic acid than adult platelets in response to thrombin (29.5 +/- 3.2 versus 19.6 +/- 1.8%) but less than adult platelets in response to 10 micrograms/mL collagen (3.2 +/- 1.1 versus 9.3 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombolamban, the 22-kDa platelet substrate of cyclic AMP-dependent protein kinase, is immunologically homologous with the Ras family of GTP-binding proteins.

Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMP-dependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [gamma-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both one-dimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is specific for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [alpha-32P]GTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTP-binding proteins.

Estrogenic control of spermidine/spermine N1-acetyltransferase activity in rat uterus.

It is widely accepted that specific intracellular receptor proteins are implicated in the estrogen regulation of gene expression, growth and differentiation in target cells. The unoccupied forms of these receptors, as suggested by Jensen and Jacobson1 and Gorski et al.,2 reside primarily in the cytoplasm and translocate to the nucleus after hormone interaction. A revision of this classical “two-step” model has recently indicated that both native and ligand-bound forms of the receptors are localized in the nucleus, as supported by biochemical3 and immunocytochemical4 data. The binding of estrogen with receptor leads to a change in the conformational state of the protein, which increases its affinity for nuclear components and DNA.3, 4 A number of nuclear acceptor sites have been proposed, including specific DNA sequences,5 ribonucleoprotein,6 basic nonhistone proteins, nuclear matrix,8 and acidic nonhistone protein DNA complexes.9 The binding of activated receptor with DNA or other nuclear components triggers the synthesis of specific proteins, growth, and differentiation in responsive tissues.KeywordsEstrous CycleOrnithine DecarboxylaseDiamine OxidasePolyamine MetabolismPolyamine LevelThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ah receptor: relevance of mechanistic studies to human risk assessment.

Studies of the toxic actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in numerous animal models and in human and animal cells in culture have established that the most characteristic pathologic lesions produced by this compound result from events initiated by the interaction of TCDD with a specific intracellular receptor protein, the Ah receptor. Although most research on the interaction of TCDD with the Ah receptor has focused on establishing involvement of this receptor complex in specific toxic responses, recent application of modern cell and molecular biology techniques is yielding new insights into the mechanism(s) of signal transduction. Elucidation of these mechanisms is essential for understanding the molecular basis of the cell and species specificity which is a hallmark of TCDD toxicity. This knowledge should provide the framework for development of a more toxicologically relevant risk assessment model.

Ah Receptor: Relevance of Mechanistic Studies to Human Risk Assessment

Studies of the toxic actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in numerous animal models and in human and animal cells in culture have established that the most characteristic pathologic lesions produced by this compound result from events initiated by the interaction of TCDD with a specific intracellular receptor protein, the Ah receptor. Although most research on the interaction of TCDD with the Ah receptor has focused on establishing involvement of this receptor complex in specific toxic responses, recent application of modern cell and molecular biology techniques is yielding new insights into the mechanism(s) of signal transduction. Elucidation of these mechanisms is essential for understanding the molecular basis of the cell and species specificity which is a hallmark of TCDD toxicity. This knowledge should provide the framework for development of a more toxicologically relevant risk assessment model.

Platelet-derived growth factor-inducible genes respond differentially to at least two distinct intracellular second messengers.

Platelet-derived growth factor (PDGF) stimulates expression of the c-myc, c-fos, JE, and KC genes in BALB/c/3T3 cells. Here we show that these genes respond differentially to at least two distinct intracellular second messengers generated by PDGF. A broad body of data support the view that response of the c-myc and c-fos genes to PDGF is channeled, at least in part, through activation of protein kinase C. Both PDGF and protein kinase C agonists stimulate transcription of c-myc and c-fos. Down regulation of protein kinase C inhibits the transcriptional induction of c-fos and c-myc by PDGF. In contrast, protein kinase C agonists do not stimulate transcription of JE and KC. Down regulation of protein kinase C does not inhibit the ability of PDGF to stimulate transcription of JE and KC. The differential response of these four genes to PDGF and 12-O-tetradecanoylphorbol-13-acetate correlates with differential phosphorylation of specific intracellular proteins. PDGF treatment stimulates phosphorylation of intracellular proteins at 31, 32, and 80 kilodaltons. Down regulation of protein kinase C prevents phosphorylation of the 80-kDa protein in response to PDGF, but phosphorylation of the smaller proteins is not affected. Finally, PDGF which induces all four genes (and stimulates phosphorylation of three proteins) is a much more potent mitogen than protein kinase C agonists which regulate only some of these events.