Heat flux estimates of power balance on Proto-MPEX with IR imaging.

The Prototype Material Plasma Exposure eXperiment (Proto-MPEX) at Oak Ridge National Laboratory (ORNL) is a precursor linear plasma device to the Material Plasma Exposure eXperiment (MPEX), which will study plasma material interactions (PMIs) for future fusion reactors. This paper will discuss the initial steps performed towards completing a power balance on Proto-MPEX to quantify where energy is lost from the plasma, including the relevant diagnostic package implemented. Machine operating parameters that will improve Proto-MPEX's performance may be identified, increasing its PMI research capabilities.

Erythropoietin structure-function relationships. Identification of functionally important domains.

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacements at 51 conserved sites predicted to be on the surface of the molecule. Alanine replacements permitted preservation of alpha-helical structure. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type and mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell line, and the human UT7-EPO leukemia cell line. When Arg14 on predicted Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specific bioactivity. In a similar manner, the mutein Arg103-->Ala in Helix C was completely lacking in biological activity, whereas both Ser104-->Ala and Leu108-->Ala had decreased bioactivity. In Helix D, the mutein Gly151-->Ala had markedly decreased bioactivity, whereas that of the adjacent Lys152-->Ala mutein was moderately impaired. In contrast, Ala replacements at three nearby sites on Helix D (147, 146, and 143) resulted in muteins with increased bioactivity. In conclusion, our mutagenesis experiments have identified functionally important domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.

A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.

Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line.

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-M1) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.

Activation of the erythropoietin receptor by the Friend spleen focus-forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation.

The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine-phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.

Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene.

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Densitometric analysis of cytochrome oxidase in ischemic rat brain.

Elevated brain lactate during incomplete ischemia is thought to contribute to the irreversibility of cell damage by interference with mitochondrial respiratory function, that should be evident in reduced cytochrome oxidase (CO) activity. In this study changes in the density of CO staining in a stroke model in the rat were assessed. Brains were analyzed subsequent to 30 min of ischemia followed by 30 min of reperfusion. The effects of postischemic treatment with sodium dichloroacetate (DCA)--a compound used to decrease lactate, were also evaluated. Examination of lateral cortex, hippocampus, and corpus striatum showed different intensities of CO in a distribution consistent with known regional variations in metabolic activity of the forebrain. Known laminar staining patterns in lateral cortex and areal patterns in the hippocampus were also confirmed. Comparable regions in ischemic forebrain were stained less densely for CO than controls. Image analysis demonstrated that the density of CO: (a) was greater in lateral cortex than hippocampus in control; (b) in ischemics was reduced by an equal degree in cortex and hippocampus; (c) lacked regional uniformity in ischemic rats; and (d) was not changed by DCA treatment in the majority of cases of ischemia. Our results suggest that lactate may not be the major determinant of 'selective vulnerability'. Despite elevated lactate levels in lateral cortex when compared to hippocampus in a previous study, the proportionate decrease in CO activity in lateral cortex and hippocampus was equal. However, there was a considerable decrease in CO activity subsequent to high brain lactate and some ischemic hemispheres appeared to respond to DCA treatment. Therefore, the role of excessive lactate in the exacerbation of 'selective vulnerability' warrants further evaluation. CO histochemistry can be used successfully to determine the distribution of pathology and the quality of fixation of ischemic forebrain. Densitometric measurements allowed comparative assessment of degrees of injury and the effects of treatment in discrete anatomical regions. This kind of analysis may allow localization of pathology within specific cellular circuits.

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit.

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.

A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase.

While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.

Photoaffinity labeling of glucocorticoid receptors.

The cross-reactivity of progestins for glucocorticoid receptors was exploited to photoaffinity label glucocorticoid receptors from cultured rat hepatoma (HTC) and mouse lymphoma (S49) cell cytosol. The synthetic progestin, 17 alpha, 21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020), rapidly forms covalent bonds with protein upon irradiation of either cytosol with 350 nm light. Polyacrylamide gel electrophoresis under denaturing conditions reveals a single band photolabeled by R5020 that is not observed when excess nonradioactive dexamethasone is included in the incubation. This protein band corresponds to a molecular weight of about 87,000 in both HTC and S49 cell cytosol; it is entirely absent in cytosol from glucocorticoid-resistant S49(r-) cells which lack receptor-binding activity. Another steroid-resistant mutant, S49 (nti), which exhibits normal levels of steroid-binding activity but increased binding of receptor-steroid complexes by the nucleus, yields a receptor which, when photolabeled, has an apparent molecular weight of only 39,000. These results demonstrate that glucocorticoid receptors can be photoaffinity-labeled; the data are consistent with the notion that the binding form of the receptor consists of a single polypeptide chain, Mr = 87,000, in two different species, rat and mouse, and in cells of either hepatic or lymphoid origin. The data also suggest that the lesion in the steroid-resistant S49 (nti) lymphoma cell line is a mutation of the structural gene for the glucocorticoid receptor which results in the synthesis of a truncated protein.

Effect of 2-deoxyglucose on [32P] phosphate and insulin release from perifused rat pancreatic islets.

The effect of 2-deoxyglucose on glucose mediated insulin and [32P]phosphate release was studied by perifusion of isolated rat pancreatic islets. When islets were perifused with media containing 2.8 mmol/l glucose and 20 mmol/l 2-deoxyglucose for 60 minutes and then exposed to media containing 8.3 or 16.7 mmol/l glucose and 20 mmol/l 2-deoxyglucose for the next 15 minutes, insulin release at either glucose concentration was prompt but blunted. Similarly, islets preincubated (90 min) with [32P] orthophosphate, then perifused with 20 mmol/l 2-deoxyglucose for 75 min and stimulated by either 8.3 or 16.7 mmol/l glucose for the final 15 minutes or 2-deoxyglucose exposure demonstrated obtundation of [32P]phosphate release. Perifusion of islets with 20 mmol/l 2-deoxyglucose alone induced no heightened 32P efflux. These studies suggest that 2-deoxyglucose affects initial events in stimulus-secretion coupling of glucose mediated insulin release.