Biomedical subjects

R B McNeill

Publications and source records attributed to R B McNeill.

6 recordsLinked to original sources

PubMed2000

Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor.

Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689-20692). Here, we show that residues 1290-1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys(1292), Leu(1298), Arg(1299), Arg(1300), Gln(1301), and Ser(1303)). Phosphorylation of NR2B at Ser(1303) by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII.NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260-1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets.

Amino Acid Sequence↗

PubMed1999

CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias.

Early afterdepolarizations (EAD) caused by L-type Ca2+ current (ICa, L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in ICa,L during repolarization. ICa,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. ICa,L augmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

Animals↗

PubMed1999

Medicinal yeast extracts.

Alcoholic extracts of bakers' yeast (Saccharomyces cerevisiae) have been used for over 60 years in over-the-counter medications for the treatment of hemorrhoids, burns, and wounds. Although previous studies suggested that small peptides were responsible for the medical observations, the peptides were never resolved into separate fractions and identified. In the present report, a protein fraction was prepared by RPC18 chromatography of the extract which enhances wound closure in both diabetic and non-diabetic littermates. The peptides are active in nanomolar amounts and are 600 times more active than the initial extract. SDS-PAGE and N-terminal amino acid sequencing identified 4 polypeptides in the extract. Three of the proteins were small molecular weight stress-associated proteins: copper, zinc superoxide-dismutase, ubiquitin, and glucose lipid regulated protein (HSP 12). The fourth protein, acyl-CoA binding protein II, has not been previously associated with stress proteins.

Amino Acid Sequence↗

PubMed1997

Association of brain protein phosphatase 1 with cytoskeletal targeting/regulatory subunits.

Protein phosphatase 1 catalytic subunit (PP1C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)-labeled PP1C revealed four major rat brain PP1C-binding proteins (PP1bps) with molecular masses of approximately 216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size-exclusion chromatography of solubilized extracts separated two main PP1 activities (approximately 600 and approximately 100 kDa). PP1bps and PP1C gamma1 were enriched in the approximately 600-kDa peak, but PP1C beta was enriched in the approximately 100-kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG-PP1C beta than recombinant DIG-PP1C gamma1 or DIG-PP1C alpha. Solubilized PP1bp175 and PP1bp134 interact with PP1C under native conditions, because they both (1) coeluted from size-exclusion and ion-exchange columns; (2) bound to microcystin-LR-Sepharose; and (3) coprecipitated using PP1C antibodies. Trypsinolysis of the approximately 600-kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform-selective binding of PP1C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.

Animals↗

PubMed1995

Interaction of autophosphorylated Ca2+/calmodulin-dependent protein kinase II with neuronal cytoskeletal proteins. Characterization of binding to a 190-kDa postsynaptic density protein.

Subcellular localization of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by interaction with specific anchoring proteins may be an important mechanism contributing to the regulation of CaMKII. Proteins capable of binding CaMKII were identified by the use of a gel overlay assay with recombinant mouse CaMKII alpha (mCaMKII alpha) or Xenopus CaMKII beta (xCaMKII beta) 32P-autophosphorylated at Thr286/287 as a probe. Numerous [32P]CaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation of forebrain extracts localized p190 and p140 to a crude particulate/cytoskeletal fraction and isolated postsynaptic densities. [32P]m-CaMKII alpha-bound to p190 with an apparent Kd of 609 nM (subunit concentration) and a Bmax of 7.0 pmol of mCaMKII alpha subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM [32P]m-CaMKII alpha to p190 was competed by nonradioactive mCaMKII alpha autophosphorylated on Thr286 (EC50% = 200 nM), but to a much lesser extent by nonradioactive mCaMKII alpha autophosphorylated on Thr306 (EC50% > 2000 nM). In addition, nonphosphorylated mCaMKII alpha was a poor competitor for [32P]mCaMKII alpha binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286 promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr306 does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+ concentration.

Animals↗

PubMedDate not supplied

Topical application of yeast extract accelerates the wound healing of diabetic mice.

Alcoholic extracts of yeast have been used as the active ingredient in medications under names such as "tissue or skin respiratory factor," Biodyne (Sperti Drug Co, Cincinnati, Ohio--now defunct), and live yeast cell derivative (LYCD). Beneficial clinical results from the use of LYCD have been reported for the treatment of burns, wounds, and hemorrhoids. The medicinal effects of LYCD have recently been localized to a protein fraction containing a mixture of several peptides. The effects of topical application of the peptide mixture on wounds were examined in diabetic mice, an animal model in which the healing process is disrupted and delayed. Full-thickness wounds were created on the backs of diabetic (DB) and nondiabetic (non-DB) mice. Half of the DB and non-DB mice were treated with 0.05 mL of LYCD after wounding and for 4 successive days. All other mice received vehicle. Wound areas were measured at Day 0 and at 2-day intervals. Mice were sacrificed at 3, 7, 10, 21, and 28 days postinjury. Differences in the extent and quality of healing appeared between DB mice receiving LYCD and DB mice receiving vehicle by day 10 (P < .0001). By 24 days postinjury, DB mice receiving LYCD had achieved 100% wound closure, whereas DB mice receiving vehicle had achieved only 31.4% wound closure. Histologic examination of wounds reflected improved wound healing in DB mice receiving LYCD as compared with those receiving vehicle. A topically applied yeast extract peptide mixture significantly attenuates wound closure and the degree of cellular reorganization of full-thickness excisional wounds of DB mice.

Administration, Topical↗