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This article describes a ground-up initiative for a volunteer-run digital literacy program in Singapore targeting vulnerable older adults, focusing on the barriers faced in running this program and training these beneficiaries. It further offers possible solutions to overcome these hurdles, providing insight for individuals or organizations seeking to start similar ground-up initiatives., (©Haikel A Lim, Joanne Sze Win Lee, Meng Han Lim, Lynn Pei Zhen Teo, Natalene Siew Wen Sin, Rou Wei Lim, Si Min Chua, Jia Qi Yeo, Nerice Heng Wen Ngiam, Angeline Jie-Yin Tey, Celine Yi Xin Tham, Kennedy Yao Yi Ng, Lian Leng Low, Kai Wen Aaron Tang. Originally published in JMIR Aging (https://aging.jmir.org), 03.05.2022.)

Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ -coactivator 1 α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase- α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: To examine the prevalence and risk factors for hypercalcemia among non-dialysis chronic kidney disease (CKD) patients with mineral and bone disorder (MBD)., Methods: A retrospective cohort study was conducted in Singapore General Hospital, involving all CKD stage 4 and 5 pre-dialysis patients who were on treatment for MBD in June 2016. Each patient was followed up for 1 year and screened for hypercalcemia episodes. Mild, moderate and severe hypercalcemia were defined as corrected calcium of 2.47-3.00, 3.01-3.50 and ≥ 3.51 mmol/l respectively. Patients who were on dialysis, post-renal transplant, post-parathyroidectomy or had no calcium levels taken during the study period were excluded. Details related to patients' clinical information and hypercalcemia episodes were collected. Multivariate logistic regression analysis was performed to evaluate risk factors for hypercalcemia., Results: Of 557 patients, 75 (13.4%) patients developed hypercalcemia. There were 120 (97.6%) mild and 3 (2.4%) moderate hypercalcemia episodes. The daily elemental calcium intake from phosphate binders and usage of vitamin D analogues did not differ between patients with and without hypercalcemia (p > 0.05). After adjusting for covariates, lower baseline iPTH level [odds ratio (OR) 0.96, 95% CI 0.93-0.99], history of hypercalcemia in past 1 year (OR 11.11, 95% CI 3.36-36.75) and immobility (OR 3.34, 95% CI 1.34-8.40) were associated with increased hypercalcemia risk., Conclusion: Hypercalcemia affects a significant proportion of pre-dialysis patients with MBD. More studies should be undertaken to evaluate other risk factors associated with hypercalcemia.

Takayasu's arteritis (TA) is a chronic non-specific vasculitis with variable presentation in different ethnicities and countries. Treatment options vary and are dependent on the stage and presentation of the disease. We aimed to review current literature related to TA, focusing on the role of endovascular treatment in revascularisation. The temporal course of the disease and stage at presentation influence the management of TA. Treatment options include medical therapy, endovascular intervention or surgical vascular reconstruction. The decision to intervene is individualised according to vascular anatomy and the presence of haemodynamically significant lesions. There are currently no clear guidelines regarding the choice between the endovascular and open surgical approaches, but studies have shown that endovascular procedures are associated with slightly higher rates of restenosis while surgical procedures have higher rates of thrombosis. Periprocedural immunosuppression is suggested if the disease is active at the point of intervention. This improves outcomes but at the cost of immunosuppression-related side effects. Careful long-term follow-up is essential due to the risk of disease activation or flare-up, requiring appropriate evaluation of the diseased vessels., Competing Interests: K.K. Yeo has received research support from Medtronic, and honoraria from Abbott Vascular and St. Jude Medical. The other authors have no conflicts of interest to declare.

Background: We examined the association between unwanted sexual experiences and cervical cancer, cervical intraepithelial neoplasia 3, adenocarcinoma in situ, diagnosed ≤25 years of age., Methods: A case-control study of women ≤55 years who attended gynecological hospitals in Australia between 1983 and 2007. Cases were ≤25 years when diagnosed with disease, control group 1 were "older women" >25 years at diagnosis; control group 2 were "well women" ≤25 years attending preventive health clinics. A self-administered postal survey was utilized. The main outcome measures were prevalence of childhood sexual abuse (<16 years) and unwanted adolescent sexual experiences (between 16 and 18 years) in cases compared to controls., Results: Of 400 contactable subjects, 251 participated (62.8%). Prevalence of childhood sexual abuse in cases (26.6% [25/94]) was similar to other groups. Prevalence of childhood genital contact abuse in cases with cervical cancer was 45.5% [5/11], compared to older women (20% [10/50], p = 0.08) and well women (13.8% [8/58], p = 0.01), and was marginally more common compared to well women when adjusted for other lifestyle factors (odds ratio [OR]: 4.7 [1.0-22.6], p = 0.05). Prevalence of unwanted adolescent sexual experiences in cases was 28.9% [33/114]. Prevalence of adolescent penile-genital contact experiences in cervical cancer cases was 46.7% [7/15], compared to older women (9.4%, [6/64], p < 0.001) and well women (13.7%, [10/73], p = 0.003), and was more common compared to well women when adjusted for lifestyle (OR: 5.9 [1.4-24.9], p = 0.02) and sexual health risk factors (OR: 5.6 [1.4-22.1] p = 0.01)., Conclusions: Unwanted sexual experiences with genital contact were a risk factor for invasive cervical cancer ≤25 years, likely due to a complex interplay of biological and environmental factors.

The human PNPLA3 (patatin-like phospholipase domain-containing 3) gene codes for a protein which is highly expressed in adipose tissue and liver, and is implicated in lipid homeostasis. While PNPLA3 protein contains regions homologous to functional lipolytic proteins, the regulation of its tissue expression is reflective of lipogenic genes. A naturally occurring genetic variant of PNPLA3 in humans has been linked to increased susceptibility to alcoholic liver disease. We have examined the modulatory effect of alcohol on PNPLA3 protein and mRNA expression as well as the association of its gene promoter with acetylated histone H3K9 by chromatin immunoprecipitation (ChIP) assay in rat hepatocytes in vitro, and in vivo in mouse and rat models of acute binge, chronic, and chronic followed by acute binge ethanol administration. Protein expression of PNPLA3 was significantly increased by alcohol in all three models used. PNPLA3 mRNA also increased, albeit to a varying degree. ChIP assay using H3AcK9 antibody showed increased association with the promoter of PNPLA3 in hepatocytes and in mouse liver. This was less evident in rat livers in vivo except under chronic treatment. It is concluded for the first time that histone acetylation plays a role in the modulation of PNPLA3 levels in the liver exposed to binge ethanol both in vitro and in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ethanol and its major metabolite acetate both induced histone H3 acetylation in primary culture of rat hepatocytes. The acetylation by ethanol was dependent on the reactive oxygen species and mitogen-activated protein kinase pathway, whereas that by acetate was independent of both pathways. Ethanol increased CYP2E1 protein expression but acetate had negligible effect. The level of phospho-H2AX, an indicator of DNA breaks, was elevated by ethanol but not by acetate. Ethanol and acetate differentially activated mRNA expression for different genes, e.g., IL-6, PPARγ, c-Fos, Egr-1, and PNPLA3 in hepatocytes. The most striking increase (3-fold) was in PNPLA3 mRNA by ethanol with little change by acetate. It was further shown that acetate inhibited histone deacetylase activity. Taken together, these data establish for the first time that ethanol and acetate exhibit differences in their effects on hepatocytes in gene expression, P-H2AX levels, and the mechanism of histone H3 acetylation. The implications of these differences in the actions of ethanol in liver are discussed., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The need to produce more efficient and less polluting vehicles has encouraged mass production of alternative energy vehicles, such as hybrid and electric cars. Many of these vehicles are capable of very quiet operation. While reducing noise pollution is desirable, quieter vehicles could negatively affect pedestrian safety because of reduced sound cues compared to louder internal combustion engines. Three studies were performed to investigate people's concern about this issue. In Study 1, a questionnaire completed by 378 people showed substantial positive interest in quiet hybrid and electric cars. However, they also indicated concern about the reduced auditory cues of quiet vehicles. In Study 2, 316 participants rated 14 sounds that could be potentially added to quiet alternative-energy vehicles. The data showed that participants did not want annoying sounds, but preferred adding "engine" and "hum" sounds relative to other types of sounds. In Study 3, 24 persons heard and rated 18 actual sounds within 6 categories that were added to a video of a hybrid vehicle driving by. The sounds most preferred were "engine" followed by "white noise" and "hum". Implications for adding sounds to facilitate pedestrians' detection of moving vehicles and for aiding drivers' awareness of speed are discussed., (Copyright © 2013 Elsevier Ltd and The Ergonomics Society. All rights reserved.)

The widening web of epigenetic regulatory mechanisms also encompasses ethanol-induced changes in the gastrointestinal (GI)-hepatic system. In the past few years, increasing evidence has firmly established that alcohol modifies several epigenetic parameters in the GI tract and liver. The major pathways affected include DNA methylation, different site-specific modifications in histone proteins, and microRNAs. Ethanol metabolism, cell-signaling cascades, and oxidative stress have been implicated in these responses. Furthermore, ethanol-induced fatty liver (i.e., steatohepatitis) and progression of liver cancer (i.e., hepatic carcinoma) may be consequences of the altered epigenetics. Modification of gene and/or protein expression via epigenetic changes also may contribute to the cross-talk among the GI tract and the liver as well as to systemic changes involving other organs. Thus, epigenetic effects of ethanol may have a central role in the various pathophysiological responses induced by ethanol in multiple organs and mediated via the liver-GI axis.

Aims: The aim of this study was to determine the effect of chronic ethanol feeding on acetylation of histone H3 at lysine 9 (H3-Lys9) at promoter and coding regions of genes for class I alcohol dehydrogenase (ADH I), inducible nitric oxide synthase (iNOS), Bax, p21, c-met and hepatocyte growth factor in the rat liver., Methods: Rats were fed ethanol-containing liquid diet (5%, w/v) for 1-4 weeks. The global level of acetylation of H3-Lys9 in the liver was examined by western blot analysis. The levels of mRNA for various genes were measured by real-time reverse transcriptase-polymerase chain reaction. The association of acetylated histone H3-Lys9 with the different regions of genes was monitored by chromatin immunoprecipitation assay., Results: Chronic ethanol treatment increased mRNA expression of genes for iNOS, c-jun and ADH 1. Chronic ethanol treatment did not cause increase in global acetylation of H3-Lys9, but significantly increased the association of acetylated histone H3-Lys9 in the ADH I gene, both in promoter and in coding regions. In contrast, chronic ethanol treatment did not significantly increase the association of acetylated histone H3-Lys9 with iNOS and c-jun genes., Conclusion: Chronic ethanol exposure increased the gene-selective association of acetylated H3-Lys9 in the absence of global histone acetylation. Thus, not all genes expressed by ethanol are linked to transcription via histone H3 acetylation at Lys9.

The epigenetic histone modification by ethanol is emerging as one of the mechanisms for its deleterious effects in the liver. In this context, we have investigated the role of histone H3 phosphorylation at Ser10 (P-H3-Ser10), and Ser28 (P-H3-Ser28) in liver after acute ethanol treatment in vivo. Ethanol was administered intraperitoneally in male Sprague-Dawley rats. Ethanol dose-response (1-5 g/kg body weight) and time-course (1-4 h) experiments were conducted, and various parameters were monitored. Steatosis and necrosis (serum alanine aminotransferase) of the liver increased in 4 h, suggesting liver injury. There were differences between P-H3-Ser10 and P-H3-Ser28 at 1 h, with the latter being more sensitive to lower ethanol doses. It was noteworthy that phosphorylation of both serines disappeared at the highest dose used (5 g/kg). We also examined phosphoacetylation of histone H3 at K9S10 and observed a dramatic increase. The changes in histone H3 phosphorylation and phosphoacetylation were also accompanied with expression of early response genes (c-fos, c-jun, mitogen-activated protein kinase phosphatase-1). Chromatin immunoprecipitation assays in samples from 1.5 and 4 h of ethanol administration indicated that increased histone H3 phosphorylation at Ser28 was associated with the promoters of c-jun and plasminogen activator inhibitor-1. In conclusion, this study demonstrates for the first time that in vivo exposure of liver to acute ethanol induced phosphorylation and phosphoacetylation of histone H3, and these modifications are differentially involved in the mRNA expression of genes.

We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The transcription factor antagonist inhibitor of differentiation 3 (Id3) has been implicated in many diverse developmental, physiological and pathophysiological processes. Its expression and function is subjected to many levels of complex regulation. This review summarizes the current understanding of these mechanisms and describes how they might be related to the diverse functions that have been attributed to the Id3 protein. Detailed understanding of these mechanisms should provide insights towards the development of therapeutic approaches to various diseases, including cancer and atherogenesis.

Ethanol treatment increases gene expression in the liver through mechanisms that are not clearly understood. Histone acetylation has been shown to induce transcriptional activation. We have investigated the characteristics and mechanisms of ethanol-induced histone H3 acetylation in rat hepatocytes. Immunocytochemical and immunoblot analysis revealed that ethanol treatment significantly increased H3 acetylation at Lys9 with negligible effects at Lys14, -18, and -23. Acute in vivo administration of alcohol in rats produced the same results as in vitro observations. Nuclear extracts from ethanol-treated hepatocytes increased acetylation in H3 peptide to a greater extent than extracts from untreated cells, suggesting that ethanol either increased the expression level or the specific activity of histone acetyltransferases (HAT). Use of different H3 peptides indicated that ethanol selectively modulated HAT(s) targeting H3-Lys9. Treatment with acetate, an ethanol metabolite, also increased acetylation of H3-Lys9 and modulated HAT(s) in the same manner as ethanol, suggesting that acetate mediates the ethanol-induced effect on HAT. Inhibitors of MEK (U0126) and JNK (SP600125), but not p38 MAPK inhibitor (SB203580), suppressed ethanol-induced H3 acetylation. However, U0126 and SP600125 did not significantly affect ethanol-induced effect on HAT, suggesting that ERK and JNK regulate histone acetylation through a separate pathway(s) that does not involve modulation of HAT. Chromatin immunoprecipitation assay demonstrated that ethanol treatment increased the association of the class I alcohol dehydrogenase (ADH I) gene with acetylated H3-Lys9. These data provide first evidence that ethanol increases acetylation of H3-Lys9 through modulation of HAT(s) and that histone acetylation may underlie the mechanism for ethanol-induced ADH I gene expression.

Id3 is a member of the Id family of transcriptional regulators that have been implicated in the development of multiple tissues. Altered expression of the Id genes and proteins contribute to carcinogenesis and atherosclerosis. Id3 is highly expressed in proliferating skeletal muscle cells but becomes downregulated upon terminal differentiation. We have identified several DNase I protected footprints within a proximal region of the mouse Id3 promoter that has been shown previously to support high levels of transcriptional activity in proliferating skeletal muscle cells. Two of these sites interacted, respectively, in vitro with Sp2 and Egr-1 proteins present in muscle cell nuclear extracts. Mutation analysis revealed that the Sp2 site accounted for a major part of the Id3 promoter activity in proliferating muscle cells whereas the Egr-1 site was dispensable. Consistent with the previously observed downregulation of the endogenous Id3 gene, protein binding to the Sp2 site was substantially reduced with extracts from differentiated muscle cells. Our results reveal Id3 as a potential target for Sp2 and raise the possibility that acute activation and the chronic and maintained expression of Id3 gene might be regulated by different mechanisms.

Poor awareness of psychiatric symptoms is associated with schizophrenia. It is unclear whether this lack of insight extends to the cognitive impairment that affects at least 85% of people with schizophrenia. Given the increasing efforts to develop treatments for cognitive impairment, and the link between awareness of disability and treatment compliance, it is important to understand whether people with schizophrenia have awareness of their cognitive deficits. Ratings of cognitive functioning from 185 outpatients diagnosed with schizophrenia, their clinicians' ratings and objective neuropsychological test results were compared in the following cognitive domains: attention, nonverbal memory, and verbal memory. The results indicated that there was poor concordance among the three assessments. Patients did not agree with their clinician's assessments and did a very poor job of accurately classifying their cognitive status. However, clinicians were also poor at classifying cognitive status consistent with neuropsychological test results. The implications of these findings for understanding insight in schizophrenia are discussed.

The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme. Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein. The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5. Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0. RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment. Part of this was removed in the deletion construct, RNase I Delta(26-38). We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes. The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases., (Copyright 2001 Academic Press.)

Overexpression of the helix-loop-helix motif-containing transcription inhibitor Id3 has been shown to repress muscle-specific gene expression. Consistent with its putative negative regulatory role in the myogenic process, Id3 is highly expressed in proliferating myoblasts but down regulated when myoblasts are induced to differentiate. To investigate how Id3 expression may be transcriptionally regulated, we isolated a mouse Id3 genomic DNA fragment and characterized its organization and promoter activity. Comparison of the Id3 gene from human and mouse demonstrated a conserved exon-intron organization in which the first intron interrupts the C-terminal protein coding region and the second intron interrupts the 3' untranslated region at analogous positions in the two species. Sequence analysis of the 5'-flanking region revealed an unexpected mouse strain-specific genetic polymorphism due to a single base substitution. Deletion analysis revealed that as little as 180 base pairs of the mouse Id3 promoter upstream of the transcription start site is sufficient for a high level of gene expression in proliferating C2C12 myoblasts. In particular, the region between the nucleotide position -180 and -34 appeared to be crucial for maximal reporter gene activity and interacted specifically with C2C12 nuclear proteins. Finally, we showed that, despite the creation of a putative transcription factor-binding site, the genetic polymorphism observed did not affect Id3 promoter activity in proliferating C2C12 cells.

Terminal myogenic differentiation involves an irreversible transition from a proliferative state to a post-mitotic quiescent state. We showed here that in addition to the previously reported down regulation of G(1)-related cyclin-associated kinase activities, this transition was also accompanied by an extensive reorganization of the cyclin-cdk complexes, including a dramatic shift of cdk2 from cyclin A to cyclin D3. Moreover, the inhibition of cdk activity also correlated with an increase in the expression of the p27(kip1) cdk inhibitor and in its association with the cyclin-cdk2 complexes. Since depletion of p27 substantially reduced the cdk inhibitor activity present in differentiated muscle cells, we believe that the increase in p27 expression along with the reorganization of the cyclin-cdk2 complexes may play an important role in the inhibition of cdk2 activity during the differentiation process.

Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domain-binding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogen-activated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.

We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 +/- 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.

This study identified the unique and primary contributions of several concurrent risk factors for poor adherence to treatment recommendations in a clinic population of individuals with chronic psychotic disorders, i.e. 48% had DSM-IV diagnoses of schizoaffective disorder, 38% had schizophrenia, paranoid type, 12% had schizophrenia, undifferentiated type, and 2% had affective disorder with psychotic features. The target cohort consisted of 87 consecutive admissions to a continuing day treatment program. As part of a services-oriented quality assurance program, clinical staff completed rating scales for all patients. These included the BASIS-32 rating scale, which consisted of the following five subscales: psychosis; depression/anxiety; impulsive/addictive behavior; relation to self and others; and daily living and role functioning, and the Working Alliance Inventory-short form (therapist version), which consisted of the following three subscales: goal; task; and bond. These data were used to identify risk factors that weaken a patient's adherence to medication and non-medication treatment during the first 2 weeks of treatment in the clinic. Medication treatment consisted of both typical and atypical neuroleptic medications, with most patients being on multiple medications. Correlational analyses suggested that many of the risk factor variables were significantly associated with poor treatment adherence. Regression analyses suggested that the degree of psychoticism was most strongly associated with poor adherence to medication treatment and that difficulties relating to self and others were the strongest predictor of poor adherence to non-medication treatment. A large-sample services research design such as this can begin to determine patterns of associations between previous identified risk factors and poor treatment adherence in individuals with chronic psychotic disorders.

Intramolecular interaction within the Ca(2+)-regulated 34 kDa actin-bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin-binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > or = 2:1 stoichiometry, which is 5-fold greater than the intact protein aa1-295 as assessed by cosedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind (45)Ca(2+) on blots and displayed Ca(2+)-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca(2+)-binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kDa proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin-binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments.

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.

We report the molecular cloning of a novel guanylate-binding protein (GBP), termed mouse GBP3 (mGBP3) in Friend virus-induced mouse erythroid progenitor (FVA) cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGK(S/T) and DXXG, but lack the third element, (N/T)KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides (GTP, GDP and GMP). Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K(m) and Vmax values of 77 +/- 4 microM and 21 +/- 0.5 pmol min-1 microgram-1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylation/methylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number U44731.

The Dictyostelium discoideum 34-kDa protein is an F-actin bundling protein that demonstrates diverse distributions in the cell during cell shape changes and cell movement. The protein is expressed at a very low level in the amoeba, just 0.4% of the total cell protein. This presents a challenging problem when purifying sufficient protein for structural and biochemical studies. The purification procedure is lengthy and yields only a few milligrams of protein. An alternative protein expression system, that of the bacterial T7 expression system, was used to produce large quantities of recombinant 34-kDa protein (r34-kDa). The soluble r34-kDa protein constitutes up to a quarter of the total bacterial protein, and was purified to homogeneity by a modification of the purification procedure for the native D. discoideum 34-kDa protein (N34-kDa). The r34-kDa possesses all the same functional characteristics as the N34-kDa protein with respect to its interactions with F-actin in vitro: it bound to and cross-linked F-actin, mediated F-actin bundle formation, directly bound calcium, and demonstrated calcium-sensitive F-actin binding activities.

We examined the effect of glucocorticoids on the proliferation and differentiation of skeletal muscle cells using the C2C12 cell line. We found that treatment with glucocorticoids enhanced muscle cell differentiation but had only minor effects on the clonal growth rate of C2C12 cells. The stimulatory effect of glucocorticoids on myogenic differentiation was reflected in the increased expression of muscle-specific genes, creatine kinase (CK) and acetylcholine receptor gamma subunit (AChR). Dexamethasone had no effect on CK and AChR mRNA stability and enhanced transcription from a CAT reporter genes containing the 3.3kb 5' flanking region of the murine CK gene (-3300MCK-CAT). Since dexamethasone did not affect the expression levels of the myogenic regulatory genes such as myoD and myogenin, the enhancement of muscle-specific transcription might reflect an increase in the functional activity of the regulatory proteins. Other possible mechanisms involved in the differentiation-enhancing effect of glucocorticoids are discussed.

We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each other's inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells out that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5' flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Uroguanylin is a small peptide isolated from opossum urine that activates membrane guanylate cyclases. We report the isolation by molecular cloning of cDNAs encoding the 109 amino acid preprouroguanylin containing the active uroguanylin peptide at its C-terminus. Preprouroguanylin mRNAs of 1.2 kb were detected throughout the small and large intestine and in the atria and ventricles of heart, but not in kidney, stomach or liver. Transfection of COS-1 cells with the uroguanylin cDNA resulted in prouroguanylin secretion. Both uroguanylin and prouroguanylin were isolated from opossum plasma. Thus, uroguanylin is made by the intestine and heart and circulates as a bioactive form of uroguanylin and the inactive prouroguanylin.

Induction of many primary-response genes by a variety of stimuli occurs in a transient manner. The precise mechanism responsible for these transient kinetics is not completely understood. We report here that the orphan nuclear receptor, TIS1, which is a potent sequence-specific transcription factor, was transiently induced by the adrenergic agonist isoprenaline in the C2C12 skeletal-muscle cell line. Moreover, we showed that the rapid decline in mRNA level after peak induction was due in part to a specific desensitization of the isoprenaline-mediated induction pathway. Desensitization of the induction response presumably occurred at the level of the receptor, as agents that either bypass the adrenergic receptor or activate alternative signalling pathways were able to induce TIS1 expression in the desensitized cells. However, stimulation by agents that directly activate intracellular enzymes also resulted in the signal-transduction-pathway-specific desensitization of TIS1 inducibility. Our results suggest that the pathway-specific nature of the desensitization process may be important for directing an integrated response to multiple physiological stimuli.

One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

The TIS21 gene is a primary response gene that is induced rapidly and transiently in 3T3 cells by the tumor promoter and mitogen tetradecanoyl phorbol acetate. The predicted open reading frame of the TIS21 cDNA encodes a protein of 158 amino acids with no obvious similarity to any known protein. Antiserum prepared to TIS21 recombinant protein produced in Escherichia coli precipitates a 17-kDa protein from Swiss 3T3 cells. The 2040-nucleotide 3'-untranslated region of the cDNA includes an unusual T18 sequence. The TIS21 gene has a single 1.4-kilobase intron which interrupts the open reading frame and is otherwise identical to the cDNA sequence. The 5'-flanking sequence of the TIS21 gene contains TATA and CAAT box-type sequences, three potential Sp1 sites, two putative cyclic AMP response elements, two potential AP1 binding elements, and an AP2 element. A possible Z-DNA structure of 29 AC repeats is present 660 nucleotides from the start of transcription. Expression from a luciferase reporter construct containing a 460-nucleotide fragment of the TIS21 promoter is induced by tetradecanoyl phorbol acetate, forskolin, epidermal growth factor, and serum, despite the absence of a consensus serum response element.

TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O-Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13-acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene.

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Immunolatex spheres, originally developed as visual markers for scanning electron microscopy, were employed as membrane density perturbation reagents. Methacrylate spheres were bound to antibody molecules and used to label antigens on erythrocytes. Ghosts prepared from labeled cells were subjected to isopycnic centrifugation on continuous sucrose and dextran gradients. It was found that the labeled erythrocyte membranes had a substantially higher density than unlabeled membranes. The extent to which the membrane density was shifted on a given gradient depended on the number, size and density of the latex spheres and could be closely predicted by theory. These results suggest that the reagents and techniques described here have potential application for the isolation of plasma membranes from more complex cell types.

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.

TIS genes are rapidly and transiently induced by tetradecanoyl phorbol acetate in 3T3 cells. We analyzed the developmental appearance of a number of the TIS genes to determine whether, in a normal physiological context, these genes have common or distinct mechanisms of regulation. Each TIS gene has a distinct tissue specificity and/or developmental profile.

We report here the nucleic acid sequence and deduced amino acid sequence of a cDNA for TIS7, a gene induced by mitogens in 3T3 cells and by nerve growth factor in PC12 pheochromocytoma cells. A fragment of the TIS7 gene has previously been cloned from murine fibroblast cells infected with Newcastle Disease Virus. The cDNA sequence suggests some similarities with murine interferons. TIS7 mRNA can also be transiently induced by double-stranded RNA. We suggest that the TIS7 protein may be an autocrine factor that attenuates or amplifies the initial ligand-induced signal.

Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both potent tumor promoters in a mouse skin carcinogenesis experiment. OA was much more toxic than TPA for murine embryo cell lines such as Swiss 3T3 cells or C3H10T1/2 cells. TPA is a potent mitogen for 3T3 cells; in contrast OA was unable to stimulate DNA synthesis in these cells. TPA induces a family of primary response genes, the TPA induced sequence (TIS) genes, in a wide variety of cells. Although OA induced modest levels of TIS mRNA expression, the time course of the induction of TIS1 and TIS8 mRNA was delayed when compared to induction by TPA or peptide mitogens such as fibroblast growth factor (FGF). In addition TPA-mediated down-regulation of protein kinase C attenuated TIS gene induction by OA, but not by FGF.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.

We have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind 125I-labeled EGF; each lacks a functional EGF receptor. We used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in 125I-labeled cell extracts or in [35S]methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.

Rat PC-12 pheochromocytoma cells, in response to nerve growth factor (NGF), stop proliferating and differentiate into cells resembling sympathetic neurons. This model of cell differentiation was used to investigate the expression of a previously isolated collection of mitogen-induced primary response sequences cloned from murine 3T3 cells; the TIS (tetradecanoyl phorbol acetate-induced sequences) genes (Lim et al., 1987). The TIS cDNAs were used to probe RNA isolated from PC-12 cells stimulated with NGF and other agents. Six of these messages were rapidly and transiently induced by NGF, tetradecanoyl phorbol acetate (TPA), or epidermal growth factor (EGF). Expression of these TIS genes generally resembled the NGF-stimulated induction of c-fos. In contrast, one TIS gene (TIS 10), induced by mitogens in 3T3 cells, was not induced by NGF, TPA, or EGF in PC-12 cells. Like c-fos, these TIS genes induced by NGF could also be superinduced by the combined administration of NGF and benzodiazepine. Elevated potassium ion, which leads to the induction of c-fos in PC-12 cells via activation of a voltage-dependent Ca2+ channel, also induces all TIS genes, with the notable exception of TIS 10. The induction of this family of genes may be involved in the general transduction of extracellular signals into biological responses.

The tumor promoter tetradecanoyl phorbol acetate (TPA) is also a potent mitogen for murine 3T3 cells. We have previously described the isolation of variant Swiss 3T3 cell lines unable to proliferative in response to TPA. In this report we sought to identify genes that are stimulated by TPA as 'primary responses', i.e., without intervening protein synthesis. We constructed a cDNA library in lambda gt10, using RNA from quiescent 3T3 cells treated with TPA in the presence of cycloheximide (CHX). Of 50,000 recombinant phages, we identified 50 isolates that demonstrated preferential hybridization to cDNA probes generated from TPA-stimulated cells. One of the clones contains a fragment of the proto-oncogene c-fos. Twenty-nine of the remaining 49 clones fall into six cross-hybridization families. All the characterized clones detected mRNAs that are also inducible by epidermal growth factor, fibroblast growth factor or elevated serum. TPA induction of all the characterized messages is rapid and transient. All these mRNAs are superinduced by a combination of mitogens and CHX. Induction of these messages following TPA addition also occurs in subconfluent 3T3 cultures; expression of these genes is, therefore, not restricted to the G0/G1 transition. Expression of all six clones was also induced by TPA and other mitogens in 3T3-TNR9 cells, a TPA non-proliferative variant.