[Comparison of the tuberculin skin test with the interferon-gamma test in the framework of a surroundings investigation in a prison]. / Vergleich von Tuberkulin-Hauttests und Interferon-Gamma-Tests im Rahmen einer Umgebungsuntersuchung in einer JVA.

After the occurrence of a multidrug-resistant tuberculosis in a prison of Lower Saxony (Germany), 223 contact persons (178 inmates, 45 staff members) were identified. To detect latent tuberculosis infections, all contact persons were tested with the tuberculin skin test (TST) and the interferon-gamma release assay (IGRA). An additional standardised questionnaire on anamnestic information and risk factors was completed by the participants. The statistical analysis included 149 male inmates with a mean age of 39 years. 61.1% of the inmates were born in Germany, 14.8% were born in the USSR or, respectively, its follow-up states. With the TST 29.5% of the inmates were tested positive, whereas with the IGRA the positive rate was 18.1%. For 7 inmates there was a positive IGRA despite a negative TST. The positive predictive value of TST with respect to IGRA was 45.5%, agreement was 79.2% (kappa=0.44). The agreement found in this study is comparable to studies within populations with lower incidence. Numerous investigations outside Europe indicate a high risk of tuberculosis infection among inmates.

Sexually abused girls: patterns of psychopathology and exploration of risk factors.

This paper studies the patterns of psychopathology in sexually abused girls. It also explores some environmental risk factors for psychopathology including abuse characteristics and environmental experiences. The data are derived from the baseline assessment of 81 sexually abused girls referred to the London Child Sexual Abuse Psychotherapy Outcome Study (collaborative Tavistock and Maudsley project). Data about abuse were collected from the parent or foster parent using a standardised, semi-structured interview format. The girls' psychopathology was assessed using the Kiddie-SADS schedule. Widespread and serious psychopathology in sexually abused girls attending a psychotherapy clinic previously reported in a small-scale study was confirmed; so, too, was the extent of comorbidity and impairment of psychosocial functioning. Further, a significant association was found between children looked after away from home and high rates of Separation Anxiety Disorder. No such significant associations were found for Major Depressive Disorder nor impairment of functioning. Multivariate prediction analysis revealed that significant predictors of Major Depressive Disorder consisted of seriousness of abuse, the abuser not being a parent figure, and the abuse not being recent; the only significant predictor of Separation Anxiety Disorder was that the abuser was not a parent figure; finally, impairment of general functioning was strongly predicted by the greater seriousness of abuse and also by the abuser/s not being a parent figure. Theoretical explanations advanced for the reported associations have a sense of face validity: that girls abused by strangers will be at risk of developing Separation Anxiety Disorders; that serious sexual abuse is followed by the development of a Major Depressive Disorder and a high level of impairment of social functioning.

Psychopathology in children from families with blood disorders: a cross-national study.

BACKGROUND: This study examines the prevalence of psychiatric disorders in affected and in unaffected siblings from families with haemophilia or beta-thalassaemia. METHOD: Based on data derived from a cross-sectional and multi-centre study into the resilience of 115 families with blood disorders. Sociodemographic and developmental data were collected from the parent using a standardised and semi-structured interview, and medical data were elicited from the attending clinician. The children's psychopathology was assessed with the Schedule for Affective Disorders and Schizophrenia (K-SADS). RESULTS: Children with beta-thalassaemia were twice as likely to receive a diagnosis of psychiatric disorder and more likely to show a higher degree of impairment of general functioning than haemophilic boys or unaffected children from families with blood disorders. Clinical severity of haemophilia or beta-thalassaemia was not associated with significant differences in prevalence of child psychiatric disorders or impairment. Mothers' evaluation of their relationship with their child as 'less than easy' predicted psychopathology. CONCLUSIONS: The high prevalence of psychopathology in children with beta-thalassaemia reported in this study suggests that specific blood disorders have differential impact on affected children. This difference may be related to medical therapy advances in haemophilia so that haemophilic boys can lead an almost normal life.

Psychotherapy for sexually abused girls: psychopathological outcome findings and patterns of change.

BACKGROUND: Controversy exists about the efficacy of psychotherapy for the mental health problems of sexually abused children. AIMS: To compare the relative efficacy of focused individual or group therapy in symptomatic sexually abused girls, and to monitor psychiatric symptoms for persistence or change. METHOD: A multi-centre psychotherapy outcome study recruited 71 sexually abused girls aged 6-14 years who were randomly assigned to focused individual psychotherapy (up to 30 sessions) or psychoeducational group therapy (up to 18 sessions). Changes over the course of the study were monitored. RESULTS: Both treatment groups showed a substantial reduction in psychopathological symptoms and an improvement in functioning, but with no evident difference between individual and group therapy. However, individual therapy led to a greater improvement in manifestations of post-traumatic stress disorder (PTSD). CONCLUSIONS: The beneficial effects on PTSD support the use of individual therapy. However, the small sample size and lack of a control group limit conclusions about changes attributable to treatment.

GH regulation of IGF-I and suppressor of cytokine signaling gene expression in C2C12 skeletal muscle cells.

GH is required for normal postnatal growth and metabolism. GH stimulates postnatal growth through induction of IGF-I gene expression. Although the liver is the major site of GH-regulated IGF-I, recent evidence indicates that GH-regulated IGF-I expression in nonhepatic tissues is sufficient for normal postnatal growth. One potentially important nonhepatic site of GH-stimulated IGF-I expression is skeletal muscle, as injection of GH into animals leads to increased IGF-I mRNA in this tissue. Nevertheless, direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore tested the C2C12 myogenic cell line for its response to GH and demonstrate that C2C12 skeletal muscle cells rapidly respond to physiological levels of GH with increased tyrosine phosphorylation of the GH receptor, Janus kinase 2, signal transducer and activator of transcription-5a and -5b, insulin receptor substrate-1, and activation of MAPKs/ERKs and protein kinase B/Akt. In these cells, GH stimulates the expression of IGF-I and two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein and suppressor of cytokine signaling-2. Treatment of C2C12 myoblasts with either the MAPK kinase inhibitor PD98059 or the PI3K inhibitor wortmannin results in higher levels of GH-induced IGF-I and suppressor of cytokine signaling-2 mRNA expression, suggesting that activation of MAPK and PI3K pathways has an inhibitory role in IGF-I and suppressor of cytokine signaling-2 gene regulation. Therefore, C2C12 cells provide the first in vitro model system to study various aspects of GH action in skeletal muscle.

Stat5 phosphorylation status and DNA-binding activity in the bovine and murine mammary glands.

The transcription factors Stat5a and Stat5b are mediators of prolactin signalling in mammary epithelial cells, and are thought to play a role in lactogenesis. In cultured cells, activation of Stat5 activity through phosphorylation results in Stat5 binding to the promoters of at least some of the milk protein genes, thereby stimulating their transcription. However, the mammary biology of Stat5 differs between species, and the role of Stat5 in the bovine mammary gland is not fully understood. We have generated an antibody that specifically recognises the phosphorylated forms of Stat5a and Stat5b and used it to compare the levels of phosphorylated Stat5 with Stat5 DNA-binding activity in bovine and murine mammary tissue. Both Stat5 DNA-binding activity and phosphorylation status in the bovine mammary gland were at near-maximal levels at late pregnancy (27-35 days prior to calving), when at least three of the major milk proteins are not highly expressed. In addition, these studies revealed significant animal-to-animal variation in the level of Stat5 activity in both species. The results are consistent with a role in terminal differentiation of mammary epithelial cells. They also suggest that the stimulation of high-level expression of milk protein genes in the bovine mammary gland is not through activation of the prolactin receptor-Jak2-Stat5 pathway.

Insulin Induction of SOCS-2 and SOCS-3 mRNA expression in C2C12 Skeletal Muscle Cells Is Mediated by Stat5*.

Previously, by a yeast 2-hybrid screen, we identified signal transducer and activator of transcription 5b (Stat5b) as a substrate of the insulin receptor (IR). We demonstrated that refeeding of fasted mice leads to rapid activation of Stat5 proteins in liver, skeletal muscle, and fat, suggesting that Stat5b is a physiological target of insulin. Here, we show that injection of glucose or insulin into fasted mice leads to robust activation of both Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insulin stimulates tyrosine phosphorylation of Stat5a and Stat5b by 3-5-fold. This degree of Stat5 activation in vitro is significantly lower than what we observe in vivo and inversely correlates with IRS-1/2 levels. We can recapitulate robust insulin activation of Stat5 in C2C12 cells by stable overexpression of the human IR (hIR). To identify insulin-activated genes that are Stat5 targets, we also overexpressed an IR mutant (LA-hIR) that signals normally for mitogen-activated protein kinase- and phosphatidylinositol 3-kinase-dependent pathways but is deficient in Stat5 signaling in response to insulin. We demonstrate that insulin induces the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The induction of SOCS-3 by insulin is reduced but not lost in the LA-hIR cells. Therefore, our results suggest that insulin induction of SOCS-2, and in part SOCS-3 mRNA expression, is mediated by Stat5 and can be independent of mitogen-activated protein kinase and phosphatidylinositol 3-kinase-signaling pathways.

Mechanism of STAT3 activation by insulin-like growth factor I receptor.

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.

Behavioural psychopathology of child sexual abuse in schoolgirls referred to a tertiary centre: a North London study.

The sexually abused girls in this study were a sub-sample of a group of girls referred to a Regional Centre for Psychotherapy for the whole of London, North Thames. An inclusion criterion was that they were psychologically symptomatic and so it is likely that they were more problematic cases causing concern in their locality. The control clinical group consisted of referrals to local Child and Family consultation services, were an opt-in matched sample and not a total clinic referral sample. In addition, the reasons for referral covered both child disorder and family problems. It is, therefore, important to bear in mind the differences between these two groups. Certain clear cut findings have emerged from this study. No disorders specific to child sexual abuse in girls were identified but the extent and severity of the disturbance in the sexually abused sample was most striking. In these girls an event (CSA), together with referral because of emotional symptoms, was associated with enhanced severity of disorder and comorbidity particularly with reference to a cluster of disorders comprising post-traumatic stress disorder, depressive disorder, anxiety disorders (general and separation), social phobias and reactive attachment disorder. In the community clinic sample the identified disorders were mainly those of separation anxiety disorders and adjustment. Wide comorbidity was common in the sexual abuse sample and also severity of impairment was notable when compared to the clinic sample. However, because of the selected nature of the abuse group the findings are not generalisable beyond the population from which they emerged. The view is advanced that there are strong grounds for exploring the utility of psychodynamic psychotherapy in similar samples of sexually abused girls. These findings are discussed in the light of the current literature.

STAT activation by the PDGF receptor requires juxtamembrane phosphorylation sites but not Src tyrosine kinase activation.

Activation of the platelet-derived growth factor (PDGF) receptor tyrosine kinase induces tyrosine phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. Since the PDGF receptor also activates the Src tyrosine kinase, it is possible that Src mediates tyrosine phosphorylation of STATs in PDGF-treated cells. Consistent with a role for Src in STAT activation, we found that a PDGF receptor juxtamembrane tyrosine residue required for Src activation is necessary and sufficient for activation of STATs 1 and 3. To test the Src requirement further, we made other mutations in the PDGF receptor juxtamembrane region that increased or decreased Src binding. In epithelial and fibroblast cells, PDGF activated STAT1, 3 and 6 in the absence of detectable binding and activation of Src. In addition, PDGF induced c-myc RNA expression and DNA synthesis even though Src was not detectably activated. The activation of MAP kinase and the induction of c-fos gene expression both correlated with STAT but not Src activation by the receptor. We conclude that juxtamembrane tyrosine phosphorylation is necessary for both Src tyrosine kinase and STAT activation by the betaPDGF receptor, but that both processes are regulated independently by this region.

Early life family disadvantages and major depression in adulthood.

BACKGROUND: There is evidence that exposure to social and family disadvantages in childhood are a risk factor for adult depression. AIMS: To explore the effects of multiple adversity in early childhood on adult depression, and the relative effects of the different adversities. METHOD: This study utilises data from the Newcastle Thousand Family Study. Information on childhood disadvantages was collected when the participants were 5 years old, and information on mental health was gathered when they were 33 years old. Mental health data were scrutinised blind to the evidence of early disadvantage, and best-estimate diagnoses of major depressive disorder were made according to DSM-III-R criteria. RESULTS: Multiple family disadvantages in childhood substantially increase the risk of suffering a major depressive disorder in adulthood. Such disadvantages include family or marital relationship instability, a combination of poor mothering and poor physical care, and a combination of dependence on social welfare and overcrowding. For females major depression was linked in particular to the quality of parenting in early life. CONCLUSIONS: Social and family (especially multiple family) disadvantages during childhood predispose individuals to an increased risk of major depression in adulthood.

Stat3 plays an important role in oncogenic Ros- and insulin-like growth factor I receptor-induced anchorage-independent growth.

The role of signal transducers and activators of transcription (STATs) in receptor protein-tyrosine kinase (PTK)-induced cell growth and transformation was investigated using an inducible epidermal growth factor receptor-Ros chimeric receptor called ER2 and a constitutively activated insulin-like growth factor I receptor called NM1, both of which are able to induce anchorage-independent growth of NIH 3T3 cells. ER2 and NM1 receptor PTKs are able to cause Stat3 activation. Co-expressing the dominant negative Stat3 mutant with ER2 or NM1 in transiently or stable transfected cells resulted in a dramatic inhibition of colonies induced by these receptor PTKs and a moderate inhibition of their mitogenicity in monolayer. Therefore, Stat3 is not only important for initiation of transformation, as demonstrated by inhibition of the epidermal growth factor-inducible colony formation of the ER2 cells by the mutant, but it is also required for the maintenance of transformation, as evidenced by reversion of the NM1 transformed cells. The DNA binding and transcriptional activities of the endogenous Stat3 were greatly inhibited in the ER2 and NM1 cells co-expressing the Stat3 mutants. We conclude that activated function of Stat3 is required for the establishment and maintenance of Ros and insulin-like growth factor I receptor PTK-induced cell transformation.

Stat5 is a physiological substrate of the insulin receptor.

Using the cytoplasmic domain of the insulin receptor (IR) in a yeast two-hybrid screen, we identified a cDNA clone encoding the C-terminal 308 amino acids of human Stat5b (Stat5b-Ct). Stat5b-Ct is tyrosine phosphorylated by purified IR kinase domain in vitro. Insulin stimulates tyrosine phosphorylation of overexpressed Stat5b-Ct and endogenous Stat5 in cells overexpressing IR. Stat5 may be a direct target of the IR and, as a member of the Stat family of transcription factors, may play a role in the regulation of gene transcription by insulin. In support of this hypothesis, perfusion of mouse liver with insulin promotes rapid tyrosine phosphorylation of Stat5 and activation of Stat5 DNA binding. Moreover, refeeding of fasted mice leads to rapid tyrosine phosphorylation and stimulation of enhanced DNA-binding activity of Stat5 extracted from liver, skeletal muscle, and adipose tissues. Taken together, our data strongly suggest that IR interacts with and phosphorylates Stat5 in vitro and in tissues physiologically sensitive to insulin.

Contribution of both STAT and SRF/TCF to c-fos promoter activation by granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that has been shown to support call proliferation in murine fibroblasts engineered to stably express both chains of the human GM-CSF receptor (NIH-GMR). Because the proto-oncogene c-fos is believed to provide a link between short-term signals elicited at the membrane and long-term cellular response, we chose to study the mechanism of GM-CSF-dependent cell regulation using c-fos promoter activity as a molecular marker in both NIH-GMR transfectants and in the CD34+ cell line TF-1. The importance of c-fos and related AP-1 activity in GM-CSF signalling was suggested by a tight correlation between GM-CSF-dependent activation of the c-fos promoter and cell proliferation and by the inhibitory effect of a trans-dominant c-fos mutant on cell growth. To evaluate the contribution of the serum response factor (SRF) associated with the ternary complex factor (TCF) and of STAT proteins to c-fos promoter activation in response to GM-CSF, the SRF binding site (SRE) and/or the STAT binding site (SIE) were inactivated. In serum-free medium, both SRE and SIE are essential to c-fos promoter activation by GM-CSF in NIH-GMR transfectants and in TF-1 cells. No response to GM-CSF was observed when both sites were mutated. The nature of the STAT family member was further investigated by Wester blots and DNA retardation assays using an SIE probe. Our data indicate that GM-CSF induced DNA binding of both STAT1 and STAT3 in NIH-GMR and mainly of STAT3 in TF-1 cells. STAT5 tyrosine phosphorylation was also observed in TF-1 cells. Finally, expression of a dominant negative MAPK mutant, ERK192A, resulted in a decrease of both SRE- and SIE-dependent activation of c-fos promoter by GM-CSF, suggesting that STAT1/3 are regulated not only by tyrosine kinases, but also partially by MAPK.

Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins.

Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs--JAK1, JAK2, and Tyk2--become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-beta receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggests that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.

Glucocorticoid and phorbol ester effects in 3T3-L1 fibroblasts suggest multiple and previously undescribed mechanisms of glucocorticoid receptor-AP-1 interaction.

A number of mechanisms have been proposed to account for glucocorticoid hormone and mitogen effects observed on the transcription of engineered exogenous target genes. However, the effect of the co-addition of these agents on the full range of endogenous responsive genes in a given cell type has not been addressed. We detected 19 metabolically labeled proteins or in vitro translation products whose synthesis was altered in 3T3-L1 fibroblasts in response to glucocorticoid hormone using two-dimensional electrophoresis on large-format gels. Co-addition of the mitogenic phorbol ester, tetradecanoylphorbolacetate (TPA), with glucocorticoid hormone resulted in independent (one species), synergistic (six species), and antagonistic (five species) effects on the glucocorticoid regulation of individual gene products, while seven other glucocorticoid regulated species were not affected. These and additional observations suggest direct and gene-specific effects of glucocorticoid receptor and AP-1 on the transcription of responsive genes, and for some of these genes the pattern of regulation is not accounted for by mechanisms described to date. In addition, the pattern of regulation of five species is consistent with a role in mediating the opposing physiological effects of glucocorticoid hormone and growth factors in these cells.

Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins.

Binding of interferons IFN-alpha and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). Interferon-alpha activates both Stat91 (M(r) 91,000; ref. 1) and Stat113 (M(r) 113,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs 3, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-alpha and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-alpha response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs alpha and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-alpha and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91.

A common nuclear signal transduction pathway activated by growth factor and cytokine receptors.

Growth factors and cytokines act through cell surface receptors with different biochemical properties. Yet each type of receptor can elicit similar as well as distinct biological responses in target cells, suggesting that distinct classes of receptors activate common gene sets. Epidermal growth factor, interferon-gamma, and interleukin-6 all activated, through direct tyrosine phosphorylation, latent cytoplasmic transcription factors that recognized similar DNA elements. However, different ligands activated different patterns of factors with distinct DNA-binding specificities in the same and different cells. Thus, unrelated receptors may activate a common nuclear signal transduction pathway that, through differential use of latent cytoplasmic proteins, permits these receptors to regulate both common and unique sets of genes.

Cell-free activation of a DNA-binding protein by epidermal growth factor.

Growth factors such as platelet-derived growth factor and epidermal growth factor (EGF) bind to and activate cell-surface receptors with intrinsic tyrosine kinase activities. Receptor activation elicits multiple physiological changes in target cells, including alterations in gene expression. Receptor tyrosine kinase signalling involves recruitment of proteins into a signalling complex through interactions between receptor autophosphorylation sites and the src-homology region-2 (SH2) domains on these signalling proteins. Diverse signals can subsequently be generated, depending on the specific receptor and cell type. How such signals are transmitted to the nucleus is poorly understood, but because the transcriptional activation of many genes by growth factors occurs in the absence of new protein synthesis, one or more signals emanating from growth factor receptors must directly affect transcription factors. We report here the activation by EGF of a DNA-binding protein in a cell-free system where activation of DNA binding requires ligand, receptor, ATP and phosphotyrosine-SH2 interactions.

Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.