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)

Using the thymidine kinase gene-containing plasmid pTK-1 and the mouse fibroblast thymidine kinase-deficient LTK- cell line, we obtained results indicating that the plasmid molecules suspended in the external medium were introduced into the nuclei by pricking cells on the nuclei with a microneedle. Approx. 25% of the cells pricked in the presence of the plasmid pTK-1 showed TK gene expression, and 2% of the cells became stable TK+ transformants. This 'pricking' method is applicable for introducing DNA into cell nuclei and is more efficient than the conventional calcium phosphate transformation method.
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PMID:The 'pricking' method. A new efficient technique for mechanically introducing foreign DNA into the nuclei of culture cells. 695 71

The growth of tick cells in Leibovitz's L--15 medium supplemented with various concentrations of fetal bovine serum (FBS), tryptose phosphate broth (TPB), and tick egg extract (TEE) was evaluated using a protein assay. A continuous cell line from Rhipicephalus appendiculatus (RA 243) was compared with young lines of cells isolated from embryos of R. appendiculatus (RAE 25) and Rhipicephalus sanguineus (RSE 8). We found fetal bovine serum and tryptose phosphate broth both to be essential supplements. The addition of tick egg extract further stimulated growth. The yield of cellular protein in both young and continuous lines of tick cells increased as a function of the concentration of tryptose phosphate broth from 0 to 10%, and fetal bovine serum from 2.5 to 20%. The growth of the RA 243 line correlated negatively with the size of the inoculum and positively with the concentration of fetal bovine serum, as the greatest increase in cell protein was obtained when cells were seeded at a low density into a medium containing 20% fetal bovine serum. The addition of an extract prepared from eggs of R. sanguineus or Hyalomma excavatum improved yields of cultures and promoted cell growth at low population densities. The protein yield increased as a function of tick egg extract concentration, but 0.8% inhibited growth of the RA 243 line. The RA 243 line could be propagated in a medium supplemented with 10% tryptose phosphate broth, 5% fetal bovine serum, and 0.5% tick egg extract.
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PMID:Effect of medium supplements on tick cells in culture. 713 Nov 99

The bone mineral content in the forearm of 99 male and 62 female patients who had never been treated with vitamin D on hemodialysis was measured to elucidate the relationship between bone mineral content and duration of hemodialysis. We found that reduction in bone mineral content related to the duration of hemodialysis in both sexes. 1--2 microgram/day of 1 alpha-OH-D3 was administered to 35 hemodialysis patients for 2 years. Bone mineral content in the forearm of both patient groups treated with and without 1 alpha-OH-D3 was measured every 3 months. Although bone mineral content in the forearm of the hemodialysis patients decreased, serum Ca, serum-ionized Ca, and serum phosphate levels increased and serum ALK-Pase and serum immunoreactive PTH decreased significantly after administration of 1 alpha-OH-D3. When bone mineral content in the forearm of patients treated with 1 alpha-OH-D3 was compared with that of untreated patients, the loss of bone mineral content in patients treated with 1 alpha-OH-D3 was less than that of the untreated group. In addition to uremic metabolic derangements, intermittently repeated humoral derangement on hemodialysis seems to cause loss of bone mineral content.
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PMID:Effects of 1 alpha-OH-D3 on bone mineral content in the forearm of chronic hemodialyzed patients. 739 38

The efflux of radiolabelled organic cations from the tubular lumen into proximal tubular cells was investigated by using the stop-flow microperfusion method. The efflux rate increased in the sequence: N1-methylnicotinamide (NMeN+) < cimetidine < tetraethylammonium (TEA+) < N-methyl-4-phenylpyridinium (MPP+). Preloading the animals by i.v. infusion or pre perfusion of the peritubular capillaries with NMeN+ increased the efflux rate of MPP+. Luminal efflux was also augmented when the tubular solution was made alkaline with HCO3- or phosphate, whereby HCO3- is more effective than phosphate. Replacement of Na+ by Cs+ showed no effect. With i.v. preloading the animals with NMeN+ and with 25 mM HCO3- in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant Km = 0.21 mmol/l and maximal flux Jmax = 0.42 pmol.cm-1.s-1 and a permeability term with P = 37.7 microns2.s-1. Comparing the apparent luminal inhibitory constant values for MPP+ (Kil,MPP+) with the apparent contraluminal Kicl,NMeN+ values of substrates of homologous series, it was found that (1) limitation by molecular size occurs at the contraluminal cell side earlier than at the luminal cell side; (2) affinity increases with hydrophobicity of the substrates at the luminal cell side, with a steeper or equal slope than at the contraluminal cell side; (3) affinity increases with basicity (i.e. pKa values) at the luminal cell side with a steeper slope than at the contraluminal cell side. Taken together, substrates with low hydrophobicity and low basicity interact at the luminal cell side more weakly than at the contraluminal cell side. On the other hand large, hydrophobic substrates have, at the luminal cell side, a higher affinity than at the contraluminal cell side. Many substrates, however, have equal affinity at the luminal and contraluminal cell sides.
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PMID:Luminal transport system for H+/organic cations in the rat proximal tubule. Kinetics, dependence on pH; specificity as compared with the contraluminal organic cation-transport system. 749 Dec 74

The importance of PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors. However, the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception. It is also clear that cell proliferation signaling does not always require PLC, as indicated by the fact that growth factors such as insulin and CSF-1 do not appear to elicit the hydrolysis of PtdIns(4,5)P2, even though the intracellular domains of their receptors carry a PTK domain and the receptors show topologies very similar to those of the PLC-activating growth factors PDGF, EGF, and FGF. The growth factor-dependent activation of PLC is initiated by the formation of a complex between the receptor PTK and PLC-gamma; the formation of this complex is mediated by a specific interaction between a tyrosine phosphate residue on the intracellular domain of PTK and the SH2 domain of PLC-gamma. The receptor PTK subsequently phosphorylates PLC-gamma, of which two distinct isozymes, PLC-gamma 1 and PLC-gamma 2, have been identified. Proliferation of T cells and B cells in response to the aggregation of their respective cell surface receptors is also accompanied by the activation of PLC-gamma isozymes at an early stage. Unlike growth factor receptors, the T cell and B cell receptors lack intrinsic PTK activity but associate with several non-receptor PTKs of the Src and Syk families. Although the specific kinases are not known, one or more of these enzymes phosphorylate and activate PLC-gamma 1 and PLC-gamma 2. Transduction of growth signals by G protein-coupled receptors such as those for thrombin or bombesin also requires PtdIns(4,5)P2 hydrolysis, which, in this instance, is mediated by PLC-beta isozymes. The PLC-beta subfamily consists of four distinct members: PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-beta 4. Agonist interaction with specific G protein-coupled receptors causes the dissociation of Gq proteins into G alpha and G beta gamma subunits and the exchange of GDP bound to G alpha for GTP. The resulting GTP-bound G alpha subunit then activates PLC-beta isoforms by binding to the carboxyl-terminal region of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositide-specific phospholipase C and mitogenic signaling. 749 69

Adipocytic-cytosolic non-receptor protein tyrosine kinase (CytPTK) when activated can substitute for the insulin receptor tyrosine kinase (InsRTK), in manifesting several insulin effects in insulin-receptor independent fashion. Our aims here were to utilize PolyGlu4Tyr, a good experimental exogenous substrate for protein tyrosine kinases (PTKs) in general, for studying qualitative and quantitative parameters of CytPTKs extracted from different tissue cytosols. At the same time, we would search for a unique specific marker specifically characterizing CytPTKs. High speed supernatants of spleen, thymus, smooth muscle, lung and kidney were found to be rich in CytPTK activities. Their specific activities being 6- to 13-fold that of liver or adipose cytosols. Brain, testis and adrenal cytosols were an intermediate source of CytPTK activity, whereas CytPTK activity of heart and skeletal muscle was low. It was also evaluated that the capacity of the cytosol to phosphorylate PolyGlu4Tyr is 15-50% that of the non-stimulated Triton X-100 extractable plasma membrane PTKs. Fractionation of the cytosols on superose 12 column revealed several CytPTKs within the same tissue, their peaks ranging between 30 and 450 kDa. Immunoblotting analysis showed Fyn and Lyn were present in most tissue cytosols. Upon immunoprecipitation, however, with anti-Fyn or anti-Lyn, negligible amounts (< 2%) of the total cellular CytPTK were precipitated. Thus, these general markers of CytPTKs comprise only a minor proportion of the total intracellular PolyGlu4Tyr phosphorylating capacity. To see whether a specific marker for CytPTK could be detected, we also examined the requirement of CytPTKs for divalent ions, their preferred phosphate donor and their sensitivity to inhibition by known PTK inhibitors. We found that the order of reactivity with divalent cations was Co2+ > Mn2+ > Mg2+, while Zn2+ and Ca2+ did not support CytPTK activity. The best phosphate donor was ATP (ED50 = 5 microM), but other nucleoside 3-phosphates could substitute for ATP at high concentrations. With respect to these parameters, no basic difference exists between cytosolic and plasma-membrane PTKs. The PTK inhibitors, genestein and quercetin, inhibited both cytosolic and membranal PTKs at micromolar concentrations. In contrast, staurosporine was a potent inhibitor of CytPTKs (IC50 5-20 nM) and a poor inhibitor of membranal PTKs (IC50 10-40 microM). One of the conclusions we can draw from this study is that tissue cytosols contain PolyGlu4Tyr phosphorylating capacity in quantities greater than previously assumed and that the low level of phosphotyrosine found in cells is not the result of limited intracellular levels of CytPTKs.
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PMID:Non-receptor cytosolic protein tyrosine kinases from various rat tissues. 749 84

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.
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PMID:Structural determinants of substrate selection by the human insulin-receptor protein-tyrosine kinase. 752 41

The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved.
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PMID:How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase. 755 15

Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.
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PMID:Biological activity of prostate-specific antigen isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution. 758 64

The beta gamma subunits of heterotrimeric G proteins (G beta gamma) play a variety of roles in cellular signaling, one of which is membrane targeting of the beta-adrenergic receptor kinase (beta ARK). This is accomplished via a physical interaction of G beta gamma and a domain within the carboxyl terminus of beta ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of beta ARK not only binds G beta gamma but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the G beta gamma binding region of beta ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of beta ARK in order to determine important residues involved in G beta gamma and PIP2 binding. To examine the effects of mutations on G beta gamma binding, we employed three different methodologies: direct G beta gamma binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G beta gamma-mediated beta ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G beta gamma-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of beta ARK to bind G beta gamma, suggesting that a proper alpha-helical conformation is necessary for the G beta gamma.beta ARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both G beta gamma binding and PIP2 binding were abolished following Ala replacement of Trp643, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the beta ARK carboxyl terminus with G beta gamma and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of beta ARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for G beta gamma binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.
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PMID:Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding. 762 21


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