Persistent Room-Temperature Photodarkening in Cu-Doped ß-Ga_{2}O_{3}.

ß-Ga_{2}O_{3} is an ultrawide band gap semiconductor with emerging applications in power electronics. The introduction of acceptor dopants yields semi-insulating substrates necessary for thin-film devices. In the present work, exposure of Cu-doped ß-Ga_{2}O_{3} to UV light >4 eV is shown to cause large, persistent photo-induced darkening at room temperature. Electron paramagnetic resonance spectroscopy indicates that light exposure converts Cu^{2+} to Cu^{3+}, a rare oxidation state that is responsible for the optical absorption. The photodarkening is accompanied by the appearance of O─H vibrational modes in the infrared spectrum. Hybrid function calculations show that Cu acceptors can favorably complex with hydrogen donors incorporated as interstitial (H_{i}) or substitutional (H_{O}) defects. When Cu_{Ga}-H_{O} complexes absorb light, hydrogen is released, contributing to the observed Cu^{3+} species and O─H modes.

Shape memory polymers with visible and near-infrared imaging modalities: Synthesis, characterization and in vitro analysis.

Shape memory polymers (SMPs) are promising for non-invasive medical devices and tissue scaffolds, but are limited by a lack of visibility under clinical imaging. Fluorescent dyes are an alternative to radiocontrast agents in medical applications, they can be utilized in chemical sensors and monitors and may be anti-microbial agents. Thus, a fluorescent SMP could be a highly valuable biomaterial system. Here, we show that four fluorescent dyes (phloxine B (PhB), eosin Y (Eos), indocyanine green(IcG), and calcein (Cal)) can be crosslinked into the polymer backbone to enhance material optical properties without alteration of shape memory and thermomechanical properties. Examinations of the emission wavelengths of the materials compared with the dye solutions showed a slight red shift in the peak emissions, indicative of crosslinking of the material. Quantitative analysis revealed that PhB enabled visibility through 1 cm of blood and through soft tissue. We also demonstrate the utility of these methods in combination with radio-opaque microparticle additives and the use of laser-induced shape recovery to allow for rapid shape recovery below the glass transition temperature. The crosslinking of fluorescent dyes into the SMP enables tuning of physical properties and shape memory and independently of the fluorescence functionality. This fluorescent SMP biomaterial system allows for use of multiple imaging modalities with potential application in minimally invasive medical devices.

Hedgehog signaling is activated in canine transitional cell carcinoma and contributes to cell proliferation and survival.

Transitional cell carcinoma (TCC) is the most commonly diagnosed tumor of the canine urinary system. Hedgehog (HH) signaling represents one possible novel therapeutic target, based on its recently identified central role in human urothelial carcinoma. The purpose of this study was to determine if HH mediators are expressed in canine TCC and the effect of inhibition of this pathway on cell growth and survival. HH pathway mediators were found to be expressed in five canine TCC cell lines. Indian HH was expressed in tumor cells in five canine bladder tumor tissues, but not in normal canine bladder tissue. Inhibition of HH signaling with cyclopamine and GANT61 led to significantly decreased cell proliferation but had a smaller effect on apoptosis. These results support future investigation of inhibitors of HH signaling in the treatment of canine TCC.

The CombiRx trial of combined therapy with interferon and glatiramer cetate in relapsing remitting MS: Design and baseline characteristics.

BACKGROUND: Interferon-ß1a (IFNB) and glatiramer acetate (GA) are distinct therapies which are both partially effective for relapsing MS. It is not known if combining the two treatments would be more effective. OBJECTIVE: To review the rationale, design, and baseline characteristics of the CombiRx study of combined treatment with IFNB and GA. METHODS: The key inclusion criteria included a diagnosis of relapsing MS, at least 2 episodes of MS activity in the previous 3 years, expanded disability status scale of 0 to 5.5, and no prior treatment with either IFNB or GA. Subjects were randomized to IFNB+GA, IFNB monotherapy, or GA monotherapy in a 2:1:1 ratio. RESULTS: From 2005 to 2009, we enrolled 1008 subjects. The participants were 72.4% female and 87.6% Caucasian with a mean age of 37.7 years. The median duration of symptoms was 2 years at entry into the study, and the mean EDSS was 2.1. On the baseline MRI, the mean total lesion load was 12.2 ml, and 40% of the participants had enhancing lesions. CONCLUSION: We have recruited a population of patients with clinical and MRI characteristics typical for early MS. The study results will aid in deciding on the optimum early treatment. This trial should serve as a model for future studies of combination therapy.

The CombiRx trial of combined therapy with interferon and glatiramer acetate in relapsing remitting MS: Design and baseline characteristics.

BACKGROUND: Interferon-ß1a (IFNB) and glatiramer acetate (GA) are distinct therapies which are both partially effective for relapsing MS. It is not known if combining the two treatments would be more effective. OBJECTIVE: To review the rationale, design, and baseline characteristics of the CombiRx study of combined treatment with IFNB and GA. METHODS: The key inclusion criteria included a diagnosis of relapsing MS, at least 2 episodes of MS activity in the previous 3 years, expanded disability status scale of 0-5.5, and no prior treatment with either IFNB or GA. Subjects were randomized to IFNB+GA, IFNB monotherapy, or GA monotherapy in a 2:1:1 ratio. RESULTS: From 2005 to 2009, we enrolled 1008 subjects. The participants were 72.4% female and 87.6% Caucasian with a mean age of 37.7 years. The median duration of symptoms was 2 years at entry into the study, and the mean EDSS was 2.1. On the baseline MRI, the mean total lesion load was 12.2ml, and 40% of the participants had enhancing lesions. CONCLUSION: We have recruited a population of patients with clinical and MRI characteristics typical for early MS. The study results will aid in deciding on the optimum early treatment. This trial should serve as a model for future studies of combination therapy.

Ha-Ras transformation of MCF10A cells leads to repression of Singleminded-2s through NOTCH and C/EBPbeta.

We have previously shown that Singleminded-2s (SIM2s), a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of transcription factors, is downregulated in breast cancer samples and has tumor suppressor activity. However, the mechanism by which SIM2s is repressed in breast cancer cells has not been determined. In this study, we show that transformation of MCF10A cells by Harvey-Ras (Ha-Ras) induces CCAAT/enhance binding protein beta (C/EBPbeta) and activates the NOTCH signaling pathway to block SIM2s gene expression. NOTCH-mediated repression acts through a C-repeat binding factor 1 (CBF1)-independent mechanism, as introduction of CBF1 had no effect on SIM2s expression. Consistent with C/ebpbeta-dependent inhibition of SIM2s, C/ebpbeta(-/-) mouse mammary glands express high levels of SIM2s and reestablishment of C/ebpbeta isoforms decreased SIM2s mRNA levels in C/ebpbeta immortalized mammary epithelial cell lines. These studies illustrate a novel pathway of tumor suppressor gene silencing in Ha-Ras-transformed breast epithelial cells and identify SIM2s as a target of C/EBPbeta and NOTCH signaling.

The remarkable influence of M2delta to thienyl pi conjugation in oligothiophenes incorporating MM quadruple bonds.

Oligothiophenes incorporating MM quadruple bonds have been prepared from the reactions between Mo(2)(TiPB)(4) (TiPB = 2,4,6-triisopropyl benzoate) and 3',4'-dihexyl-2,2'-:5',2''-terthiophene-5,5''-dicarboxylic acid. The oligomers of empirical formula Mo(2)(TiPB)(2)(O(2)C(Th)-C(4)(n-hexyl)(2)S-(Th)CO(2)) are soluble in THF and form thin films with spin-coating (Th = thiophene). The reactions between Mo(2)(TiPB)(4) and 2-thienylcarboxylic acid (Th-H), 2,2'-bithiophene-5-carboxylic acid (BTh-H), and (2,2':5',2''-terthiophene)-5-carboxylic acid (TTh-H) yield compounds of formula trans-Mo(2)(TiPB)(2)L(2), where L = Th, BTh, and TTh (the corresponding thienylcarboxylate), and these compounds are considered as models for the aforementioned oligomers. In all cases, the thienyl groups are substituted or coupled at the 2,5 positions. Based on the x-ray analysis, the molecular structure of trans-Mo(2)(TiPB)(2)(BTh)(2) reveals an extended Lpi-M(2)delta-Lpi conjugation. Calculations of the electronic structures on model compounds, in which the TiPB are substituted by formate ligands, reveal that the HOMO is mainly attributed to the M(2)delta orbital, which is stabilized by back-bonding to one of the thienylcarboxylate pi* combinations, and the LUMO is an in-phase combination of the thienylcarboxylate pi* orbitals. The compounds and the oligomers are intensely colored due to M(2)delta-thienyl carboxylate pi* charge transfer transitions that fall in the visible region of the spectrum. For the molybdenum complexes and their oligomers, the photophysical properties have been studied by steady-state absorption spectroscopy and emission spectroscopy, together with time-resolved emission and transient absorption for the determination of relaxation dynamics. Remarkably, THF solutions the molybdenum complexes show room-temperature dual emission, fluorescence and phosphorescence, originating mainly from (1)MLCT and (3)MM(deltadelta*) states, respectively. With increasing number of thienyl rings from 1 to 3, the observed lifetimes of the (1)MLCT state increase from 4 to 12 ps, while the phosphorescence lifetimes are approximately 80 micros. The oligomers show similar photophysical properties as the corresponding monomers in THF but have notably longer-lived triplet states, approximately 200 micros in thin films. These results, when compared with metallated oligothiophenes of the later transition elements, reveal that M(2)delta-thienyl pi conjugation leads to a very small energy gap between the (1)MLCT and (3)MLCT states of <0.6 eV.

Overconsumption and obesity: peptides and susceptibility to weight gain.

Physiological control of feeding is mediated by tonic and episodic signalling systems. These are sometimes thought of as long-term and short-term control. Tonic signals arise from tissue stores whereas episodic signals oscillate periodically with the consumption of food. These physiological controls are paralleled in the motivation to eat by long-acting enduring traits (such as disinhibition) and by short-acting states (such as hunger). Peptides are usually envisaged to exert an action on appetite control through the modulation of states such as hunger and satiety (fullness). Here we provide evidence that peptides involved in tonic regulation--such as leptin--may express a control over appetite motivation through an effect on traits that confer a constant readiness to eat, whereas episodic peptides such as GLP-1 influence appetite motivation through a state such as hunger. The distinction between tonic and episodic regulation, and between traits and states has implications for understanding overconsumption and the susceptibility to weight gain.

Childhood sexual abuse and obesity.

The causes of the current obesity epidemic are multifactorial and include genetic, environmental, and individual factors. One potential risk factor may be the experience of childhood sexual abuse. Childhood sexual abuse is remarkably common and is thought to affect up to one-third of women and one-eighth of men. A history of childhood sexual abuse is associated with numerous psychological sequelae including depression, anxiety, substance abuse, somatization, and eating disorders. Relatively few studies have examined the relationship between childhood sexual abuse and adult obesity. These studies suggest at least a modest relationship between the two. Potential explanations for the relationship have focused on the role of disordered eating, particularly binge eating, as well as the possible "adaptive function" of obesity in childhood sexual abuse survivors. Nevertheless, additional research on the relationship between childhood sexual abuse and obesity is clearly needed, not only to address the outstanding empirical issues but also to guide clinical care.

Post-herniographic abdominal pain syndrome.

PURPOSE: To disclose the frequency of abdominal pain that led to post-procedure hospitalization and the outcome of this major complication. MATERIAL AND METHODS: 576 patients who had undergone herniography during a 13-year period were retrospectively analysed. RESULTS: Nine out of 576 patients (1.6%) undergoing herniography with an iodine contrast medium developed abdominal pain. The pain resolved within 24 h in 6 patients while 3 patients had pain for up to 3 days. CONCLUSION: Patients who present with this pain syndrome thus only need careful clinical observation until asymptomatic, with no need for laparotomy or X-ray examination. Prior to herniography, the patients should be informed about this potential complication.

Synthesis of an organoinsulin molecule that can be activated by antibody catalysis.

We have developed a methodology of prodrug delivery by using a modified insulin species whose biological activity potentially can be regulated in vivo. Native insulin was derivatized with aldol-terminated chemical modifications that can be selectively removed by the catalytic aldolase antibody 38C2 under physiologic conditions. The derivatized organoinsulin (insulin(D)) was defective with respect to receptor binding and stimulation of glucose transport. The affinity of insulin(D) for the insulin receptor was reduced by 90% in binding studies using intact cells. The ability of insulin(D) to stimulate glucose transport was reduced by 96% in 3T3-L1 adipocytes and by 55% in conscious rats. Incubation of insulin(D) with the catalytic aldolase antibody 38C2 cleaved all of the aldol-terminated modifications, restoring native insulin. Treatment of insulin(D) with 38C2 also restored insulin(D)'s receptor binding and glucose transport-stimulating activities in vitro, as well as its ability to lower glucose levels in animals in vivo. We propose that these results are the foundation for an in vivo regulated system of insulin activation using the prohormone insulin(D) and catalytic antibody 38C2 with potential therapeutic application.

Photosystem II peripheral accessory chlorophyll mutants in Chlamydomonas reinhardtii. Biochemical characterization and sensitivity to photo-inhibition.

In addition to the four chlorophylls (Chls) involved in primary charge separation, the photosystem II (PSII) reaction center polypeptides, D1 and D2, coordinate a pair of symmetry-related, peripheral accessory Chls. These Chls are axially coordinated by the D1-H118 and D2-H117 residues and are in close association with the proximal Chl antennae proteins, CP43 and CP47. To gain insight into the function(s) of each of the peripheral Chls, we generated site-specific mutations of the amino acid residues that coordinate these Chls and characterized their energy and electron transfer properties. Our results demonstrate that D1-H118 and D2-H117 mutants differ with respect to: (a) their relative numbers of functional PSII complexes, (b) their relative ability to stabilize charge-separated states, (c) light-harvesting efficiency, and (d) their sensitivity to photo-inhibition. The D2-H117N and D2-H117Q mutants had reduced levels of functional PSII complexes and oxygen evolution capacity as well as reduced light-harvesting efficiencies relative to wild-type cells. In contrast, the D1-H118Q mutant was capable of near wild-type rates of oxygen evolution at saturating light intensities. The D1-H118Q mutant also was substantially more resistant to photo-inhibition than wild type. This reduced sensitivity to photo-inhibition is presumably associated with a reduced light-harvesting efficiency in this mutant. Finally, it is noted that the PSII peripheral accessory Chls have similarities to a to a pair of Chls also present in the PSI reaction center complex.

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors.

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.

Practical risk-adjusted quality control charts for infection control.

BACKGROUND: Control chart methodology has been widely touted for monitoring and improving quality in the health care setting. P charts and U charts are frequently recommended for rate and ratio statistics, but their practical value in infection control may be limited because they (1) are not risk-adjusted, and (2) perform poorly with small denominators. The Standardized Infection Ratio is a statistic that overcomes both these obstacles. It is risk-adjusted, and it effectively increases denominators by combining data from multiple risk strata into a single value. SETTING: The AICE National Database Initiative is a voluntary consortium of US hospitals ranging in size from 50 to 900 beds. The infection control professional submits monthly risk-stratified data for surgical site infections, ventilator-associated pneumonia, and central line-associated bacteremia. METHODS: Run charts were constructed for 51 hospitals submitting data between 1996 and 1998. Traditional hypothesis tests (P values <.05) flagged 128 suspicious points, and participating infection control professionals investigated and categorized each flag as a "real problem" or "background variation." This gold standard was used to compare the performance of 5 unadjusted and 11 risk-adjusted control charts. RESULTS: Unadjusted control charts (C, P, and U charts) performed poorly. Flags based on traditional 3-sigma limits suffered from sensitivity <50%, whereas 2-sigma limits suffered from specificity <50%. Risk-adjusted charts based on the Standardized Infection Ratio performed much better. The most consistent and useful control chart was the mXmR chart. Under optimal conditions, this chart achieved a sensitivity and specificity >80%, and a receiver operating characteristic area of 0. 84 (P <.00001). CONCLUSIONS: These findings suggest a specific statistic (the Standardized Infection Ratio) and specific techniques that could make control charts valuable and practical tools for infection control.

Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells.

Skeletal myoblasts are inherently programmed to leave the cell cycle and begin the differentiation process following removal of exogenous growth factors. Serum withdrawal results in a marked induction of IGF production which is essential for skeletal muscle differentiation in vitro. However, the potential role of the tyrosine kinase IGF-I receptor (thought to be the principal mediator of both IGF-I and II signaling in skeletal muscle) in the decision of myoblasts to begin differentiation following serum withdrawal is unknown. To explore the role of the IGF-I receptor in this decision by skeletal myoblasts, we functionally inactivated endogenous IGF-I receptors in mouse C2C12 cells using a dominant negative, kinase-inactive IGF-I receptor in which the ATP-binding site lysine (K) at residue 1003 has been mutated to alanine (A). Cell lines with the greatest degree of mutant IGF-I receptor expression (A/K cells) demonstrated functional inactivation of endogenous IGF-I receptors as determined by their impaired ability to phosphorylate the principal substrate of the IGF-I receptor, IRS-1, in response to treatment with IGF-I. In addition, the proliferative response of myoblasts to IGF-I was completely abolished in A/K cells. Following withdrawal of exogenous growth factors, A/K cells demonstrated a marked delay in the induction of the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for differentiation, and a subsequent delay in the induction of muscle creatine kinase activity. Delayed differentiation in A/K cells was associated with prolonged phosphorylation of the cell cycle regulatory retinoblastoma (Rb) protein; it is the un- (or hypo-) phosphorylated form of Rb which is known to promote differentiation in skeletal myoblasts. Thus, the IGF-I receptor regulates the timing of myoblast differentiation induced by serum withdrawal. The delayed differentiation of skeletal myoblasts with functionally inactive IGF-I receptors may result, at least in part, from delayed induction of myogenin gene expression and prolonged phosphorylation of the Rb protein.

Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin.

The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway.

Alternative splicing, gene localization, and binding of SH2-B to the insulin receptor kinase domain.

The SH2-B protein is an SH2-domain-containing molecule that interacts with a number of phosphorylated kinase and receptor molecules including the insulin receptor. Two isoforms of the SH2-B have been identified and have been proposed to arise through alternate splicing. Here we have identified a third isoform of the SH2-B protein, SH2-Bgamma, that interacts specifically with the insulin receptor. This interaction required phosphorylation of residue Y1146 in the triple tyrosine motif within the activation loop of the IR kinase and is one of only two signaling molecules shown to interact directly with this residue of the insulin receptor kinase domain. The intron/exon structure of the SH2-B gene was determined. Alternate splice sites utilized to generate the different isoforms of the SH2-B protein were identified in the 3' end of the SH2-B gene immediately downstream of the exon encoding the core of the SH2 domain. Additionally, the chromosomal location of the SH2-B gene was determined to be the distal arm of mouse Chromosome (Chr) 7 in a region linked to obesity in mice.

Studies on the cellular basis of morphogenesis in the sea urchin embryo. Directed movements of primary mesenchyme cells in normal and vegetalized larvae.

A time-lapse study has been made of the movements of the primary mesenchyme cells in the developing sea urchin larva. It shows that these cells move by pseudopod formation and contraction, and that a transition takes place--within a few hours--from a more or less random cluster, in the early mesenchyme blastula, to a well-organized, coherent pattern on the ectoderm of the gastrula. This organization is achieved by a striking random exploration of the wall of the larva by the pseudopods, followed by their contraction. The final pattern of the mesenchyme reflects those regions of the wall where the contacts between pseudopods and wall are most stable. The mechanism is thus one of selective fixation rather than of selective conduction. The pseudopodal contacts are seen to be continually made and broken, even when the final pattern is formed. The pseudopods of several cells may fuse to form a common pseudopod, these cells then migrating together. This is particularly evident in vegetalized larvae, but is also typical of the ventral side. Despite considerable variations in the way in which the final pattern is achieved, several main phases can be distinguished. The first is a radial displacement of the cells from the vegetal plate onto the presumptive ectoderm, followed by a phase of dispersion. The cells then gradually accumulate at a characteristic level, and form a ring. During this process, and when the ring is formed, the cells tend to accumulate in two clusters along the ring. The pseudopods of the cells in these clusters join into a cable, the end of which is highly branched; it explores the ectoderm, and extends the cell clusters to form branches from the ring. In vegetalized larvae, the pattern of distribution is simplified, but the same principles apply. It is suggested that the variations in the way in which the pattern is achieved are, in all probability, merely a reflexion of the lack of precision in the time sequence of changes in adhesive properties of the primary mesenchyme and blastocoel wall.

Age-related adipose tissue mRNA expression of ADD1/SREBP1, PPARgamma, lipoprotein lipase, and GLUT4 glucose transporter in rhesus monkeys.

Aging has been shown to have an effect on the capacity to differentiate preadipocytes and on the expression of some genes expressed in adipose tissue. The mRNA levels of adipocyte differentiation-related genes were examined in rhesus monkeys (Macaca Mulatta) ranging in age from 7 to 30 years. The effect of aging on the expression of peroxisome proliferator activated receptor gamma (PPARgamma), adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1 (ADD1/SREBP1), CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL), GLUT4 glucose transporter, and adipsin were examined by slot blot analysis. Significant inverse correlations were observed between age and the mRNA levels of PPARgamma, ADD1/SREBP1, LPL, and GLUT4. The coordinate downregulation of these genes may be linked to the declining fat mass of senescent animals. There was no correlation between age and the mRNA levels of adipsin. The mRNA levels of these genes were not correlated to body weight orfasting plasma insulin. These findings indicate that aging may have an effect on the adipocyte differentiation program and this effect appears to be gene specific.

Insulin receptor substrate-1 enhances growth hormone-induced proliferation.

GH exerts a variety of metabolic and growth-promoting effects. GH induces activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, JAK2, resulting in tyrosine phosphorylation of the GHR and activation of STAT (signal transducer and activator of transcription), Ras-mitogen-activated protein kinase, and phosphoinositol 3-kinase signaling pathways, among others. GH-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins has been demonstrated in vitro and in vivo. IRS-1 is a multiply phosphorylated cytoplasmic docking protein involved in metabolic and proliferative signaling by insulin, IL-4, and other cytokines, but the physiological role of IRS-1 in GH signaling is unknown. In this study, as noted by others, we detected in murine 3T3-F442A pre-adipocytes GH-dependent tyrosine phosphorylation of IRS-1 and specific GH-induced coimmunoprecipitation with JAK2 of a tyrosine phosphoprotein consistent with IRS-1. We further examined this interaction by in vitro affinity precipitation experiments with glutathione-S-transferase fusion proteins incorporating regions of rat IRS-1 and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins containing amino-terminal regions of IRS-1 that include the pleckstrin homology, phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains, but not those containing other IRS-1 regions or glutathione-S-transferase alone, bound JAK2 from cell extracts. Tyrosine-phosphorylated JAK2 resulting from GH stimulation was included in the amino-terminal IRS-1 fusion precipitates; however, neither tyrosine phosphorylation of JAK2 nor treatment of cells with GH before extraction was necessary for the specific JAK2-IRS-1 interaction to be detected. In contrast, in this assay, specific insulin receptor association with the IRS-1 phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains was insulin and phosphotyrosine dependent, as previously shown. To test for significance of IRS-1 with regard to GH signaling, IRS- and GHR-deficient 32D cells were stably reconstituted with the rabbit (r) GHR, either alone (32D-rGHR) or with IRS-1 (32D-rGHR-IRS-1). As assayed by three independent methods, GH induced proliferation in 32D-rGHR cells, even in the absence of transfected IRS-1. Notably, however, GH-induced proliferation was markedly enhanced in cells expressing IRS-1. Similarly, GH-induced mitogen-activated protein kinase activation was significantly augmented in IRS-1-expressing cells relative to that in cells harboring no IRS-1. These results indicate that IRS-1 enhances GH-induced proliferative signaling.