Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.
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PMID:Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family. 186 33

The distribution of neutral endopeptidase (NEP; EC 3.4.24.11) was examined in the alimentary tract of the rat. Immunoreactive NEP and NEP mRNA were localized to epithelial cells of the small intestine and to muscle cells in the stomach, small intestine, and colon by immunohistochemistry and in situ hybridization histochemistry. NEP antisera recognized a protein on Western blots of membranes from gastric, jejunal, and colonic mucosa and gastric muscle with an electrophoretic mobility identical to that of recombinant human NEP (approximately 95 kDa). An antisense cRNA probe to NEP hybridized to RNA of approximately 3.5 kb and approximately 6.5 kb, corresponding to the primary transcripts of rat NEP, on Northern blots of total RNA from the jejunal mucosa. NEP message was detected in mRNA from jejunal and colonic mucosa and gastric, jejunal, and colonic muscle using a ribonuclease protection assay. NEP enzymatic activity, assessed by DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was highest in homogenates of jejunal mucosa (868 +/- 98 pmol.h-1 x micrograms protein-1) and was between 49- and 413-fold lower in other gastrointestinal tissues. The cellular origin of NEP in the gastric and colonic mucosa could not be determined.
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PMID:Distribution and abundance of neutral endopeptidase (EC 3.4.24.11) in the alimentary tract of the rat. 846 Jul 3

Mammalian pancreatic ribonuclease (RNase) was conjugated chemically via a disulfide bond to human or murine epidermal growth factor (EGF). The conjugation between EGF and RNase was ascertained by SDS-PAGE using reduced and nonreduced conjugates. The EGF-RNase conjugate retained potent RNase activity and competed with 125I-EGF for binding to EGFR to the same extent as unconjugated EGF. Both the human and murine EGF-RNase conjugates showed dose-dependent cytotoxicity against EGFR-overexpressing A431 human squamous carcinoma cells with IC50 values of 3 x 10(-7) M and 6 x 10(-7) M, respectively, whereas free RNase had an IC50 of 10(-4) M. Against the EGFR-deficient small-cell lung cancer cell line H69, the EGF-RNase conjugate had no cytotoxic effect. The Human EGF-RNase conjugate showed dose-dependent cytotoxicity against other squamous carcinoma cell lines (TE-5, TE-1) and breast cancer cell lines (BT-20, SK-BR-3, MCF-7) and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell line. Excess free EGF blocked EGF-RNase conjugate cytotoxicity against A431 cells. These results suggest that the EGF-RNase conjugate may be a more effective anticancer agent with less immunogenicity than coventional chimeric toxins.
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PMID:Epidermal growth factor receptor-dependent cytotoxic effect by an EGF-ribonuclease conjugate on human cancer cell lines--a trial for less immunogenic chimeric toxin. 867 51

Hypothalamic norepinephrine (NE) plays an important role in the control of sexual behavior and in the secretion of gonadotropin. Our previous study showed that coitus induced simultaneous increases in hypothalamic NE and GnRH releases in female but not in male rabbits. To investigate the activities in noradrenergic neurons during the coitus-induced process of an LH surge, we measured tyrosine hydroxylase (TH, the rate-limiting enzyme in NE synthesis) and NE transporter (NET, a key protein for NE cellular reuptake) mRNA levels in locus coeruleus (LC) noradrenergic cells in female New Zealand White rabbits. Changes in LC-TH and LC-NET mRNA levels were also measured in males as controls. Female rabbits were killed before coitus and at 15, 30, 60, 120, and 240 min after coitus (n = 6-7/time point); males were killed before and at 30, 60, and 120 min after coitus (n = 3/time). Individual brainstems were sectioned, the LC neurons punched, and TH and NET mRNAs were quantified by ribonuclease protection assay (RPA). Rabbit-specific TH (330 bp) and NET (503 bp) cDNAs were used as probes in the RPA for gene-specific signals. A rabbit 'house-keeping' cDNA (cyclophilin, 158 bp) was also cloned and used as an internal marker for tissue RNA content. Trunk blood was collected to determine serum LH levels. In female rabbits, serum LH levels rose by 15 min after coitus, reached peak concentrations at 1-2 h, and declined thereafter. The time interval for changes in TH and NET mRNA levels in females was similar to that in serum LH levels. Both TH and NET mRNAs increased significantly by 15 min (73% and 85% respectively) and were elevated for 2 h (87% and 111% respectively). TH mRNA levels returned to basal levels by 4 h after coitus, whereas NET mRNA values were elevated throughout the 4 h of observation. In contrast, LH, TH and NET mRNA levels did not change after coitus in males. The enhanced gene expression of both TH and NET in the LC in females, in accord with our previous demonstration of increased hypothalamic NE release, suggests that regulation of NE synthesis and reuptake is an integral part of the coitus-induced NE/GnRH/LH surge process that includes the initiation, sustenance or recovery of the release and/or storage of these neurochemicals.
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PMID:Tyrosine hydroxylase and norepinephrine transporter mRNA levels increase in locus coeruleus after coitus in rabbits. 946 Jun 52

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the alpha-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an approximately 500 bp sequence upstream of the mouse Pde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putative cis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed footprints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.
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PMID:Structure and upstream region characterization of the human gene encoding rod photoreceptor cGMP phosphodiesterase alpha-subunit. 977 Jun 45

It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reverse-transcription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and self-renewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1beta, IL-3, leukemia inhibitory factor, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor-enriched fetal liver hematopoietic cells resulted in a 40-fold to 80-fold expansion of total hematopoietic cells and in a 2.5-fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic-derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self-renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.
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PMID:Hematopoietic support and cytokine expression of murine-stable hepatocyte cell lines (MMH). 982 30

To study biological character and function of epithelial rests of Malassez (ERM) in human periodontal ligament, we have developed a serum-free culture system of epithelial cells (ME) derived from ERM. The mitogenic effects of fibroblast growth factor (FGF)-1, FGF-2, and FGF-7/keratinocyte growth factor (KGF) on ME, human periodontal ligament-derived fibroblasts (PLF), human oral epithelial cells (OE), and human submandibular gland-derived epithelial cells (SGE) were investigated under a serum-free culture condition. FGF-1 and FGF-7/KGF stimulated the growth of both ME and SGE but FGF-2 had no effect. On the other hand, FGF-1, FGF-2, and FGF-7/KGF increased the OE proliferation. These results suggested that the divergent requirement of FGF ligands among these cells would be attributed to the different expression pattern of FGF receptor (FGFR) messenger ribonucleic acid (mRNA) isotypes. Therefore, we examined the expression of FGFR isotypes in these cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of ME-and SGE-derived mRNAs revealed the presence of mRNA encoding FGFR2-IIIb, a high affinity receptor for FGF-1 and FGF-7/KGF. However, no mRNAs for other FGFR isotypes were detected in both ME and SGE. On the contrary, OE expressed FGFR1-IIIc, FGFR3-IIIb, and FGFR4 mRNAs as well as FGFR2-IIb. These results indicate that FGF binding sites on ME dominantly bind to FGF-1 and FGF-7/KGF, which transduce their signals via FGFR2-IIIb. Immunohistochemical analysis, PCR-Southern, ribonuclease protection assay (RPA), and Western blotting revealed that PLF expressed FGF-7/KGF mRNA and its peptide. These observations suggest that FGF-7/KGF might mediate epithelial-mesenchymal interactions between ME and PLF to maintain normal structure and function of periodontal ligament.
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PMID:Isolation and serum-free culture of epithelial cells derived from epithelial rests of Malassez in human periodontal ligament. 1114 56

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).
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PMID:VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis. 1124 11

The signal transduction pathways that mediate GH-dependent regulation of IGF-I gene expression remain poorly defined. To establish a GH-responsive in vitro model system to study the effect of GH on the expression of the endogenous IGF-I gene, primary hepatocytes from adult male rats were prepared. These cells expressed both the GH receptor and the IGF-I gene, as demonstrated using a ribonuclease protection assay. Western blot analyses using antibodies directed against the phosphorylated forms of the ERKs, signal transducer and activator of transcription-5, and Akt/protein kinase B, a protein kinase that is downstream of PI3K, demonstrated GH-dependent phosphorylation of these signaling molecules. These signaling molecules are components of the major signal transduction pathways that are activated by GH. To determine whether GH-responsive IGF-I gene expression could be demonstrated in these cells, hepatocytes were treated without or with 50 ng/ml GH for 3--48 h. IGF-I mRNA levels increased within 3 h, and a maximal 2.2-fold increase was observed after 24 h of GH treatment. To determine whether ERK activation contributes to GH-induced IGF-I gene expression, hepatocytes were treated for 12 h without or with 50 ng/ml GH and 50 microM PD98059, an inhibitor of MAPK kinase-1 and -2. Treatment with PD98059 did not have a significant effect on GH-induced IGF-I gene expression. Similar studies were performed using 50 microM LY 294002, an inhibitor of PI3K. These studies demonstrated that treatment with LY 294002 completely abrogated GH-induced IGF-I gene expression. In contrast, PI3K-specific doses of another inhibitor of PI3K, wortmannin, failed to inhibit the GH-induced increase in IGF-I mRNA levels. In summary, rat hepatocytes in primary culture provide a good model system to study GH-induced IGF-I gene expression, and the results of these studies suggest that a signaling pathway inhibited by LY 294002, possibly a PI3K-dependent pathway, is important for GH-stimulated IGF-I gene expression in hepatocytes.
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PMID:LY 294002, an inhibitor of phosphatidylinositol 3-kinase, inhibits GH-mediated expression of the IGF-I gene in rat hepatocytes. 1151 77

Mammary gland development is regulated by complex interactions among mammogenic hormones and locally derived paracrine growth factors. In epithelial tissues, keratinocyte growth factor (KGF or FGF-7) originates in the stroma while its receptor (KGFR or FGFR2-IIIb) is present only in the epithelium. Previous work showed that estrogen but not progesterone could stimulate the synthesis of KGF in mammary stroma in vivo. The effects of 17 beta-estradiol and progesterone on KGFR expression in vivo were examined in these studies. Peripubertal and mature virgin mice received subcutaneous injections of hormone in sesame oil after which KGFR mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. Estradiol treatment caused a dose- and time-dependent decrease in KGFR mRNA level in mice from both age groups while stimulating ductal growth after 7 days of treatment. Inhibition of KGFR expression was near maximal at an estradiol dose of 2 microg after 1 day of treatment. Progesterone injection increased KGFR mRNA levels but this effect correlated with the stimulation of ductal growth. However, when progesterone was co-administered with estradiol, KGFR mRNA levels were maintained in the absence of any effect on ductal growth. Thus, estradiol inhibited KGFR mRNA only when elevated unopposed by progesterone. These data show that KGFR expression is determined by the ratio of estradiol and progesterone and suggests a mechanism through which these hormones can co-operate to optimize their growth-promoting effects. Consequences of hormone imbalance are also implicated.
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PMID:In vivo inhibition of keratinocyte growth factor receptor expression by estrogen and antagonism by progesterone in the mouse mammary gland. 1169 52


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