Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) < 40 microM), 16 (84%) showed >40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases.

GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

Tobacco use and skin disease.

BACKGROUND: The primary objective of this review is to evaluate the mucocutaneous manifestations of tobacco use. METHODS: Computerized literature searches were conducted for English language articles related to skin/mucous membrane disease and use of tobacco. The primary criterion for assessing data quality and validity was the demonstration of a causal relationship between tobacco use and skin/mucous membrane disease. RESULTS: This review of the literature shows that a number of disorders and diseases of the skin and mucous membranes are related to tobacco use. CONCLUSIONS: Since millions of persons use tobacco despite its well publicized relationship to increased mortality, knowledge of the mucocutaneous morbidity associated with tobacco use may help physicians in counseling their patients.

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity.

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide>cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.

Human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL SCID) models of toxoplasmosis.

Toxoplasmosis is a potentially fatal opportunistic infection of immunocompromised hosts. Improved animal models of toxoplasmosis are needed to more nearly approximate conditions that occur in immunocompromised humans. The development of models of toxoplasmosis using human peripheral blood lymphocytes (hu-PBL) transplanted into severe combined immunodeficiency (SCID) mice is described here. Transplantation of hu-PBL into SCID mice without prior conditioning of the mice resulted in detectable differences in quantitative histological scores of brain inflammation due to Toxoplasma gondii infection, but did not alter mortality when compared to SCID mouse controls. The lack of detectable differences in survival were due to inadequate engraftment of hu-PBL, as assessed by flow cytometry. Unconditioned hu-PBL SCID mice had low titre T. gondii-specific antibody detectable after infection. When pretransplantation conditioning with irradiation and antiasialo GM 1 (n-glucolyl neuraminic acid) antibody was used, prolonged hu-PBL engraftment was observed in SCID mice, which was associated with worsened histopathology and usually impaired survival when compared with SCID mouse controls. When pretransplantation conditioning with irradiation, antiasialo GM antibody and polyethylene glycol-conjugated IL-2 was used, prolonged hu-PBL engraftment was also documented, but this did not affect survival from T. gondii infection when compared with similarly conditioned SCID mouse controls. The latter conditioning protocol resulted in hu-PBL SCID mice producing high titre T. gondii-specific antibody after infection. Conditioned hu-PBL SCID mice had evidence of increased T. gondii-induced inflammatory scores when compared with conditioned SCID mice. These models show promise for the study of the pathogenesis of toxoplasmosis and conditioned hu-PBL SCID mice may have applications for the evaluation of novel therapies for toxoplasmosis in immunocompromised humans.

Distribution of parasite stages in tissues of Toxoplasma gondii infected SCID mice and human peripheral blood lymphocyte-transplanted SCID mice.

The establishment of Toxoplasma gondii infection in the tissues of SCID mice and SCID mice transplanted with human peripheral blood lymphocytes (PBL) was investigated. The presence of bradyzoites and tachyzoites was analysed in hu-PBL SCID mice using Southern blot of reverse transcriptase-polymerase chain reaction products for the expression of B1, BAG1 and SAG1 of T. gondii. BAG1 was present by week 1 in brain, lung, liver and spleen of some animals; by week 3, BAG1 was present in all animals and in all of these tissues. In contrast, SAG1 was rarely detected until week 2 (mainly in the lung and brain) and by week 3, some animals still did not have detectable SAG1 in brain, lung, liver and spleen. SAG1 expression was increased in the lungs of animals transplanted with human PBL compared to nontransplanted SCID mice. Human PBL engraftment was demonstrated, initially in uninfected mice, by the presence of human CD3+ T cells in the spleen (3.1 x 10(5) positive cells) and peritoneal cavity (3.4 x 10(5) cells) 4 weeks after transplantation. The final outcome of infection was not influenced by the presence of human PBL, with similar mortality in human PBL transplanted and nontransplanted mice. These studies provide a detailed analysis of the kinetics and distribution of both the cyst and tachyzoite stage of T. gondii. This system has been established to allow evaluation of therapies against T. gondii immunodeficient mice in the presence of human immune cells.

Applying artificial neural network models to clinical decision making.

Because psychological assessment typically lacks biological gold standards, it traditionally has relied on clinicians' expert knowledge. A more empirically based approach frequently has applied linear models to data to derive meaningful constructs and appropriate measures. Statistical inferences are then used to assess the generality of the findings. This article introduces artificial neural networks (ANNs), flexible nonlinear modeling techniques that test a model's generality by applying its estimates against "future" data. ANNs have potential for overcoming some shortcomings of linear models. The basics of ANNs and their applications to psychological assessment are reviewed. Two examples of clinical decision making are described in which an ANN is compared with linear models, and the complexity of the network performance is examined. Issues salient to psychological assessment are addressed.

An evaluation of the toxicities of 2'-fluorouridine and 2'-fluorocytidine-HCl in F344 rats and woodchucks (Marmota monax).

The toxicities of 2'-fluorouridine (2'-FU) and 2'-fluorocytidine-HCl (2'-FC) were separately evaluated in 2 species, male Fischer 344 (F334) rats and woodchucks. Particular attention was focused on the ability of these nucleosides to induce toxicities similar to those induced by the antiviral drug fialuridine (FIAU). 2'-FU or 2'-FC was administered to F344 male rats by intravenous injection at doses of 5, 50, and 500 mg/kg/day for 90 consecutive days and to male and female woodchucks at doses of 0.75 and 7.5 mg/kg/day for 90 consecutive days. Clinical chemistry, hematology, and urinalysis (woodchuck only) profiles were assessed during and at the termination of the study. At necropsy, organs were weighed and tissues collected for routine histologic analysis. Cytochrome c oxidase activity, citrate synthase activity, and mitochondrial DNA content were measured, and micronucleus formation in the bone marrow (rats only) was evaluated. No adverse clinical effects were observed in either species. Rats treated with high doses of either 2'-FU or 2'-FC had body weights that were 90% of those of controls. 2'-FU and 2'-FC both induced a moderate decrease in the median lymphocyte count, and 2'-FC and 2'-FU induced a mild increase in mean corpuscular hemoglobin and mean corpuscular volume. Both compounds caused slight to moderate, reversible, histologic changes in the spleen and thymus. In the woodchuck, 2'-FC caused a slight increase in mean absolute lymphocytes, and 2'-FC and 2'-FU slightly increased hepatic periportal vacuolation and/or mononuclear cell infiltration. In summary, neither compound showed evidence of the toxicity induced by fialuridine in either species. Although compound effects were observed, none of these effects were considered to be adverse, and the no-observed adverse effect level was determined to be 500 mg/kg/day for both compounds in the male F344 rat and 7.5 mg/kg/day in the woodchuck.

Mapping genotype to phenotype for linkage analysis.

We model functions that use genetic information as input and trait information as output to understand genetic linkage in complex diseases. Using simulated data from GAW11, we have applied categorical classification methods and neural network analysis. We use sharing at selected markers as input, and the classification of the sib pair (for example, affected-affected or affected-unaffected) as output. In addition, our methods include environmental risk factors as predictors of phenotype. Categorical and neural network methods each led to results consistent with findings from other methods such as the logistic regression method of Rice et al. [this issue]. Post-analysis comparison with the GAW11 answers showed that these methods are capable of detecting correct signals in a single replicate. One advantage of our methods is that they allow analysis of the entire genome at once, so that interactions among multiple trait-influencing loci may be detected. Furthermore, these methods can use a variety of sib pairs rather than affected pairs only.

Transcutaneous monitoring of carbon dioxide tension after cardiothoracic surgery in infants and children.

UNLABELLED: In this prospective investigation, we evaluated the efficacy and accuracy of transcutaneous monitoring of CO2 (TC-CO2) in infants and children after cardiothoracic surgery. Cardiothoracic surgery patients whose ETCO2 and arterial CO2 values did not correlate (gradient > or = 5 mm Hg) during the first postoperative hour underwent placement of the TC electrode (30 of 33 patients). If the TC-CO2 to arterial difference was > or = 5 mm Hg, the TC-CO2 electrode was recalibrated and reapplied on another site. If the discrepancy was still > or =5 mm Hg, the case was considered a clinical failure and no further data were collected (3 of 30 patients). If the arterial to TC gradient was <5 mm Hg, the patient was included in the data collection (27 of 30 patients). One to five sample sets (TC and arterial CO2) were collected from these patients. Statistical analysis included linear regression analysis and Bland-Altman analysis. The cohort for the study included 27 patients ranging in age from 2 days to 9 yr and in weight from 3.2 to 25 kg. A total of 101 sample sets were analyzed. The mean +/- SD absolute difference between the TC-CO2 and arterial CO2 was 1.7 +/- 1.4 mm Hg (range 0-9 mm Hg). The TC-CO2 to arterial CO2 difference was 0-2 mm Hg in 82 of 101 values (81%), 35 mm Hg in 18 of 101 values (18%), and >6 mm Hg in 1 of 101 values (1%). Linear regression analysis revealed a slope of 0.90, an r value of 0.9410, and an r2 value of 0.8854 (P < 0.0001). Bland-Altman analysis revealed a bias of 0.58 mm Hg with a precision of +/- 2.1 mm Hg when comparing the TC-CO2 with the arterial CO2. IMPLICATIONS: We conclude that, with certain caveats in mind, including the need to correlate the transcutaneous CO2 with an initial arterial CO2 value, transcutaneous CO2 monitoring can be used to estimate arterial CO2 in most neonates and children after cardiothoracic surgery.

Nucellain, a barley homolog of the dicot vacuolar-processing protease, is localized in nucellar cell walls.

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

Overexpression of superoxide dismutase in Trypanosoma cruzi results in increased sensitivity to the trypanocidal agents gentian violet and benznidazole.

The parasitic protozoan Trypanosoma cruzi is exposed to toxic oxygen metabolites which arise from drug metabolism or immune mechanisms, in addition to those produced by endogenous processes. Identification and functional analysis of parasite enzymes which confer protection against oxidative stress is therefore of importance. To investigate the role of T. cruzi superoxide dismutase (SOD) we transfected epimastigotes with an expression vector containing a putative Fe-SOD gene homologue and achieved overexpression of enzyme activity (5-8 fold). Inhibition studies carried out on the partially purified enzyme revealed azide and H2O2 sensitivity and cyanide insensitivity, the profile expected of an Fe-isoform. Phenotypic analysis of transformed parasites showed that they were more susceptible than control cells to growth inhibition by the trypanocidal drug benznidazole and by gentian violet, an agent which can be used to decontaminate blood supplies in endemic areas. These results may reflect an imbalance in the antioxidant defences of the parasite produced as a result of overexpression of Fe-SOD.

Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi.

Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of l-dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris (TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF) required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and Mr on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43% identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin (I50: >15 microM, >15 microM and 2.6 microM, respectively) than that of human MIF (I50 0.2 microM). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis, the cestode Hymenolepis diminuta, or the trematodes Schistosoma mansoni, S. japonicum and S. haematobium, or the free-living nematode, Caenorhabditis elegans, which does contain an MIF-related gene.

Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase.

The Expressed Sequence Tag database has been screened for cDNA clones encoding prostaglandin D2 synthases (PGDSs) by using a BLAST search with the N-terminal amino acid sequence of rat GSH-dependent PGDS, a class Sigma glutathione S-transferase (GST). This resulted in the identification of a cDNA from chicken spleen containing an insert of approx. 950 bp that encodes a protein of 199 amino acid residues with a predicted molecular mass of 22732 Da. The deduced primary structure of the chicken protein was not only found to possess 70% sequence identity with rat PGDS but it also demonstrated more than 35% identity with class Sigma GSTs from a range of invertebrates. The open reading frame of the chicken cDNA was expressed in Escherichia coli and the purified protein was found to display high PGDS activity. It also catalysed the conjugation of glutathione with a wide range of aryl halides, organic isothiocyanates and alpha,beta-unsaturated carbonyls, and exhibited glutathione peroxidase activity towards cumene hydroperoxide. Like other GSTs, chicken PGDS was found to be inhibited by non-substrate ligands such as Cibacron Blue, haematin and organotin compounds. Western blotting experiments showed that among the organs studied, the expression of PGDS in the female chicken is highest in liver, kidney and intestine, with only small amounts of the enzyme being found in chicken spleen; in contrast, the rat has highest levels of PGDS in the spleen. Collectively, these results show that the structure and function, but not the expression, of the GSH-requiring PGDS is conserved between chicken and rat.

Hepatocellular toxicosis associated with administration of carprofen in 21 dogs.

A diagnosis of hepatocellular toxicosis attributable to carprofen administration was made in 21 dogs on the basis of development of clinical signs and clinicopathologic abnormalities associated with hepatic disease and histopathologic documentation of hepatic necrosis. Clinical signs of toxicosis were anorexia, vomiting, and icterus. Hyperbilirubinemia and high serum activities of alanine transaminase, alkaline phosphatase, and aspartate transaminase were the most notable clinicopathologic abnormalities. In 7 of 9 dogs in which urinalyses were performed, abnormalities suggestive of renal tubular disease were detected. Clinical course of toxicosis was variable; however, most dogs had resolution of clinical signs and improvement or resolution of biochemical abnormalities with discontinuation of the drug and administration of supportive care. As with any medication, clients should be informed of possible adverse effects and reactions associated with administration of carprofen. In the event of those signs, clients should be instructed to immediately discontinue administration of carprofen to their dog and contact their veterinarian.

Purification and characterization of a tumor lipid-mobilizing factor.

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.

Interaction of macrophage-migration-inhibitory factor with haematin.

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione-agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Aman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2, 143-149]. The l-dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC50 100-200 nM). The inhibition was non-competitive and showed positive co-operativity (h=5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.

Regulation of glucose transport and c-fos and egr-1 expression in cells with mutated or endogenous growth hormone receptors.

To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR(GHR(1-638)), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHR(deltaP)) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR(1-454)) were examined. Not only did GHR(1-454) mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also mediated GH-stimulated transcriptional activation via Elk-1, a transcription factor associated with the c-fos Serum Response Element. Thus, the C-terminal half of the cytoplasmic domain of GHR is not required for GH-stimulated c-fos transcription, suggesting that increased calcium is not required for GH-stimulated c-fos expression. In CHO cells lacking all but five N-terminal residues of the cytoplasmic domain (GHR(1-294)), GH did not induce c-fos or egr-1 expression or stimulate 2-deoxyglucose uptake. Further, in 3T3-F442A fibroblasts with endogenous GHR, GH-stimulated c-fos expression and 2-deoxyglucose uptake were reduced by the tyrosine kinase inhibitors herbimycin A, staurosporine, and P11. Herbimycin A and staurosporine inhibit JAK2 and tyrosyl phosphorylation of all proteins stimulated by GH, whereas P11 inhibits the GH-dependent tyrosyl phosphorylation of only some proteins, including extracellular signal regulated kinases ERK1 and -2, but not JAK2. Taken together, these results implicate association of GHR with JAK2 and GH-stimulated tyrosyl phosphorylation of an additional cellular protein in GH-stimulated glucose transport and c-fos and egr-1 expression. These studies also indicate that, in contrast to spi-2.1, the N-terminal half of the cytoplasmic domain of GHR is sufficient to mediate stimulation of c-fos and egr-1 expression and Elk-1 activation, supporting multiple mechanisms for GH signaling to the nucleus.