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Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.

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Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.

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Because the catalytic domain of dual leucine zipper-bearing kinase (DLK) bears sequence similarity to members of the mitogen-activated protein (MAP) kinase kinase kinase subfamily, this protein kinase was investigated for its ability to activate MAP kinase pathways. When transiently transfected and overexpressed in either COS 7 cells or NIH3T3 cells, wild type DLK potently activated p46(SAPK) (SAPK/JNK) but had no detectable effect in activating p42/44(MAPK). DLK also activated p38(mapk) when overexpressed in NIH3T3 cells. A catalytically inactive point mutant of DLK had no effect in these experiments. Consistent with its specificity in activating SAPK, DLK activated Elk-1 but not Sap1a-mediated transcription. In NIH3T3 cells, activation of SAPK by v-Src was markedly attenuated by coexpression of K185A, a catalytically inactive mutant of DLK, suggesting that this mutant could function in a dominant negative fashion in a pathway that leads from v-Src to SAPKs. In a series of co-transfection experiments, activation of p46(SAPK) by DLK was not inhibited by dominant negative mutants of Rac1 and Cdc42Hs, PAK65-R, or PAK65-A, but was attenuated by MEKK1(K432M). DLK(K185A) did not inhibit the ability of constitutively active MEKK1 to activate SAPK. Moreover, K185A significantly inhibited the activation of SAPK by constitutively active V-12 Rac1 and V-12 Cdc42Hs. These results suggest that DLK lies distal to Rac1 and/or Cdc42Hs but proximal to MEKK1 in a pathway leading from v-Src to SAPKs activation.

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The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent Mr >/= 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-Delta520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulin-dependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.

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Molecular cloning using a degenerate oligonucleotide-based polymerase chain reaction was undertaken to test the possibility that novel, developmentally regulated protein kinases are expressed in the embryonic mouse kidney. Several receptor tyrosine kinase and serine/threonine kinase cDNA clones were identified. One of these, designated DLK, represented a novel gene product whose 3.6-kilobase transcript was expressed in a tissue-specific and developmentally regulated fashion. Several clones encoding the entire open reading frame were isolated and sequenced. The identified open reading frame encodes an 888-amino acid polypeptide that defines a new subfamily within the mixed lineage protein kinase family. Sequence analysis revealed: 1) a kinase catalytic domain most characteristic of serine/threonine kinases but hybrid between members of the family of microtubule-associated protein kinase kinase kinases and the fibroblast growth factor receptor family; 2) two putative alpha-helical leucine zipper motifs separated by a 25-amino acid charged intermediate segment but lacking an NH2-terminal basic domain; and 3) COOH-terminal and NH2-terminal proline-rich domains suggestive of src homology 3 (SH3) domain binding regions. Rabbit polyclonal immune sera generated against a carboxyl-terminal bacterial fusion protein recognized a protein with an apparent molecular mass of 130 kDa in COS 7 cells that were transiently transfected with a full-length DLK cDNA expression vector. Moreover, COS 7 cells transiently transfected with an epitope-tagged DLK expression vector expressed protein with an apparent molecular mass of 130 kDa that became autophosphorylated on serine and threonine in an in vitro kinase assay.

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An immunoglobulin G (IgG(2b)) producing hybridoma cell line (S3H5/gamma2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible.

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A human umbilical vein endothelial cell (EC)/monocyte (MC) coculture system was used to dissect cell:cell interactions associated with production of procoagulant activity (PCA) in response to two common stimuli of intravascular coagulation in vivo (LPS and immune complexes). We found that the presence of MC at a ratio of 1 MC:10 EC increased the sensitivity of EC to LPS by 4 logs and the maximal response approximately 20-fold. Aggregated IgG alone did not stimulate the system, but in the presence of small amounts of LPS (1 to 10 ng/ml) aggregated IgG was a powerful stimulus. More than 90% of the PCA was tissue factor as shown by clotting studies and mRNA analysis. PCA was not produced by either cell alone under the conditions of study, but was produced in large amounts when the EC and MC were cocultured. The supernatant from the coculture stimulated virgin EC, but not MC, to synthesize tissue factor. The major factor in the supernatant was IL-1 beta as shown by measuring IL-1 beta, IL-1 alpha, and TNF-alpha in supernatants and by blocking the production of PCA by preincubation of supernatants with anti-cytokine antibodies. Small amounts of TNF-alpha were present in the supernatant but anti-TNF-alpha did not inhibit PCA production. Studies using recombinant cytokines established that IL-1 beta was the most potent of the cytokines tested, that cytokines potentiated each other, and that the results could be explained in quantitative terms by the amounts of IL-1 beta measured. These data emphasize that cell:cell interactions are likely to modulate procoagulant events in vivo in the presence of both LPS and immune complexes, and that IL-1 beta may be an important cytokine in these events.

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Renal cortical and glomerular mRNA for alpha 1 (I) and alpha 1 (IV) collagen were measured by filter hybridization during experimental anti-glomerular basement membrane disease in the rabbit. The abundance of alpha 1 (IV) mRNA was 5 times greater in total RNA isolated from glomeruli as compared with whole renal cortex from normal rabbits. In contrast, there was no difference in the relative amounts of alpha 1 (I) procollagen mRNA in these two fractions. Four days after the administration of anti-glomerular basement membrane antisera, a time histologically characterized by glomerular inflammatory cell infiltration without crescent formation, beta-actin mRNA were increased 17-fold in glomeruli and 4-fold in whole renal cortex. Renal cortical mRNA for alpha 1 (I) and alpha 1 (IV) were increased 7-fold (p = 0.07) and 9-fold (p less than 0.05), respectively compared with normal rabbit kidney cortex. In contrast, there was no significant difference in the abundance of these mRNA in glomeruli at day 4. By day 7, cortical alpha 1 (I) and alpha 1 (IVP mRNA had increased 17- and 10-fold, respectively, and these transcripts had increased 13- and 7-fold in glomeruli. Cortical alpha 1 (I) mRNA remained elevated for 35 days. These data show that large changes in collagen mRNA levels occur early in this model of crescentic nephritis in the rabbit, and that extraglomerular collagen mRNA accumulates very rapidly when glomerular inflammation occurs. Extraglomerular collagen synthesis associated with intraglomerular inflammation may help to explain the common association of interstitial fibrosis with glomerulonephritis, particularly in the periglomerular area.

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