Biomedical subjects

E R Butch

Publications and source records attributed to E R Butch.

5 recordsLinked to original sources

PubMed2001

Radiolabeled cholinesterase substrates: in vitro methods for determining structure-activity relationships and identification of a positron emission tomography radiopharmaceutical for in vivo measurement of butyrylcholinesterase activity.

There is currently great interest in developing radiolabeled substrates for acetylcholinesterase and butyrylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. Using a simple in vitro spectrophotometric assay for determination of enzymatic cleavage rates, the structure-activity relationship for a short series of 1-methyl-4-piperidinyl esters was investigated. Relative enzymatic hydrolysis rates for the well-characterized 1-methyl-4-piperidinyl acetate, propionate, and i-butyrate esters were in agreement with literature values. The 4 and 5 carbon esters of 1-methyl-4-piperidinol were specific for butyrylcholinesterase and cleaved in the rank order n-valerate > n-butyrate >> 2-methylbutyrate, iso-valerate. These spectrophotometric results were also in agreement with in vitro hydrolysis rates in mouse blood and with in vivo regional retention of radioactivity in mouse brain of 11C-labeled analogs. Brain uptake and apparent enzymatic rate constants for 1-[11C]methyl-4-piperidinyl n-butyrate and n-valerate were calculated from in vivo measurements in M. nemistrina using positron emission tomography. Based on higher brain uptake of radioactivity and superior pharmacokinetics, 1-[11C]methyl-4-piperidinyl n-butyrate was identified as a new radiopharmaceutical for the in vivo measurement of butyrylcholinesterase activity.

Acetylcholinesterase↗

PubMed1997

Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice.

Stimulation of the ERK family of protein kinases ('extracellular signal regulated kinases', also known as MAP kinases) plays an important role in the activation of many cell types, including T lymphocytes. ERKs are activated when they are phosphorylated by an upstream activator, the dual-specific protein kinase MEK. To see if aging leads to an impairment of MEK activation in mouse T cells, we used a mobility shift assay in which activation of MEK leads to phosphorylation and altered mobility of ERK-2 kinase. Similarly, we monitored mobility of pp90rsk, a known ERK substrate, as an indication of ERK function. We found an age-related decline in the ability of mouse T cells to activate both MEK and ERK function in response to stimulation by antibodies to the CD3 chain of the T cell receptor. Aging did not alter the kinetics of enzyme activation, but did diminish (by about 2-fold) the maximal level of substrate converted into the slower migrating form. Naive and memory CD4 T cells from young mice were equally able to convert ERK2 to its slower migrating form, suggesting that the decline in MEK function is not likely to be attributable to the shift, with age, from naive to memory T cell predominance. Our data suggest that age-dependent declines in gene activation, including genes for key cytokines like IL-2, may be due to declines in the upstream signals that lead to activation of the MEK/ERK protein kinase cascade.

Aging↗

PubMed1997

Signaling molecules involved in coupling growth hormone receptor to mitogen-activated protein kinase activation.

We have shown previously that GH stimulates the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal-regulated kinases) 1 and 2. To examine pathways coupling GH receptor (GHR) to MAP kinase activation, we have determined the effects of GH on SHC-growth factor receptor bound 2-son of Sevenless (SHC-Grb2-SOS) association and activation of Ras, Raf, and MAP-ERK kinase (MEK). GH promoted the rapid, transient association of SHC with the Grb2-SOS complex, which correlated with the time course of Ras, Raf, and MEK activation. Despite the continuous presence of GH, these activation events were transient with Ras, Raf, and MEK returning to near basal activity by 15 or 30 min. The inactivation of Ras, Raf, and MEK directly correlated with the serine/threonine phosphorylation of SOS and dissociation of SOS from Grb2 but not Grb2 from tyrosine-phosphorylated SHC. Phosphorylation was blocked by the MEK inhibitor, PD98059. Based upon the established functions of the MAP kinase pathway, these data indicate that GH stimulation results in the assembly of a SHC-Grb2-SOS complex that serves to activate Ras and thereby engage the Raf-MEK-ERK pathway. Activation of this pathway generates a feedback kinase cascade that phosphorylates SOS resulting in the dissociation of SHC-Grb2 complexes from SOS, thereby causing a more rapid termination of the signaling pathway than would result from SHC dephosphorylation.

Animals↗

PubMed1996

Characterization of ERK1 activation site mutants and the effect on recognition by MEK1 and MEK2.

To discern MEK1 and MEK2 specificity for their substrate, extracellular signal-regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants could be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 had greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg-208 or Thr-207 to an alanine residue significantly altered the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increased the phosphorylation of Tyr-204 approximately 4-fold yet almost completely eliminated the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine resulted in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK.

Amino Acid Sequence↗

PubMed1992

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗