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R L Kincaid

Publications and source records attributed to R L Kincaid.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation.

Alzheimer Disease↗
PubMed1997

Zinc oxide and amino acids as sources of dietary zinc for calves: effects on uptake and immunity.

Calf starter diets were formulated to contain 60 ppm of Zn, 150 or 300 ppm of Zn in the form of Zn-Met and Zn-Lys, or 300 ppm of Zn in the form of ZnO to compare relative bioavailability and effects on immunity. Holstein heifer calves were weaned at wk 5 and fed experimental starter diets from wk 6 to 12. Feed intake, body weight, Zn concentrations in liver and serum fractions, and mineral concentrations in serum were measured to determine the effects of treatment. In addition, peripheral blood lymphocyte blastogenesis, interleukin-2 production, cytotoxic activity, and the ability of blood neutrophils to phagocytose and kill bacteria were assessed at wk 0, 2, 4, and 6 of the trial. Feed intakes and body weight gains were similar among calves. Concentrations of Zn in serum were elevated in calves fed 300 ppm of Zn as Zn-Met and Zn-Lys but not in calves fed ZnO. Concentrations of Zn in liver were significantly elevated by 300 ppm of Zn in the form of Zn-Met and Zn-Lys (360 micrograms/g) but not by the other Zn treatments or by the control (245 micrograms/g). No treatment had an effect on the concentrations of Lys and Met in serum; however, concentrations of Lys did decrease in serum as the age of the calves increased. There was no significant treatment effect on mitogen-induced lymphocyte blastogenesis, interleukin-2 production, lymphocyte cytotoxicity, or phagocytic and intracellular killing ability of blood neutrophils. These data indicated greater absorption and retention of Zn when administered in the form of Zn-Met and Zn-Lys than that when ZnO was administered to young calves. However, there was no advantage to the immune function of extra dietary Zn.

Amino Acids↗
PubMed1996

Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin.

An inhibitory, "dominant-negative," form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits.

Animals↗
PubMed1996

Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation.

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.

Amino Acid Sequence↗
PubMed1995

Effect of whole cottonseeds and calcium salts of long-chain fatty acids on performance of lactating dairy cows.

Holstein cows were assigned to diets containing no supplemental fat, supplemental fat from whole cottonseed, or supplemental fat from whole cottonseed plus Ca salts of fatty acids (Megalac). The TMR contained 46% forage and 54% concentrate or mixtures of concentrate and whole cottonseed on a DM basis and were fed from wk 3 through 44 of lactation. The mean fat content of the three diets was 3.0, 4.7, and 6.4% of DM for control, whole cottonseed, and whole cottonseed plus Ca salts of fatty acids, respectively. Supplemental fat increased NEL intake, percentage of milk fat, milk fat production, and rate of recovery of BW and body condition. Supplemental fat decreased milk protein production in early lactation, but not in late lactation. Addition of supplemental fat had no significant effect on ruminal concentration of VFA, NH3 N, or in situ digestibility of fiber. The proportion of unsaturated fatty acids in milk fat was increased with supplemental whole cottonseed or whole cottonseed plus Ca salts of fatty acids. During the first 3 mo of lactation, the proportion increased of fatty acids C14 or less, C16, and C18:2. Proportion of fatty acids C16:1 and C18:1 correspondingly decreased. The change in composition of milk fatty acids during early lactation is consistent with the use of body fat for milk synthesis.

Animal Feed↗
PubMed1995

Effect of selenium supplementation of cows on maternal transfer of selenium to fetal and newborn calves.

A trial was conducted to determine the effect of maternal supplementation of Se on transfer of Se to the fetus during late gestation. Holstein cows were randomly assigned at dry-off to receive no Se or 3 mg/d of supplemental Se as selenite delivered via an intraruminal bolus. Supplementation significantly increased concentrations of Se in blood of dams at parturition. Similarly, calves of cows that were supplemented with Se had higher Se in blood and liver. Colostral concentrations of Se were increased by maternal Se supplementation; Se increased in the casein fraction. The Se concentrations in blood, plasma, and liver of calves were positively correlated with the Se concentrations in plasma of the dam at parturition. Concentrations of Se in colostrum and in calf liver also were closely correlated. Concentrations of Se in calf liver at d 42 were closely correlated with concentrations of Se in liver at birth. Maternal supplementation of Se increased Se reserves in the liver of the newborn and in colostrum. Carryover effects of Se supplementation of dams were evident in calves at 42 d of age.

Animals↗
PubMed1995

Lipid metabolism in adipose tissue of cows fed high fat diets during lactation.

The adaptations of fat synthesis in adipose tissue to lactational state, rate of milk production, and dietary fat intake were determined for dairy cows. Lipogenesis and esterification were determined in cows of average or high genetic merit for milk production and fed either a control TMR of corn silage, alfalfa, and concentrate (2.5% fat; 1.47 Mcal of NEL/kg); a TMR with whole cottonseeds replacing 12% of the concentrate (4.4% fat; 1.49 Mcal NEL/kg); or a TMR with 12% cottonseeds and 2.7% of Ca salts of fatty acids (6.0% fat; 1.53 Mcal of NEL/kg). Dietary treatments began on d 17 of lactation and continued for 288 d. Lipogenesis and esterification decreased equally from 15 d prepartum to 15 d postpartum in all groups. Cows of high merit had lower rates of lipogenesis and esterification at d 60 than did low merit cows but had higher rates of lipogenesis at d 120. Rates of lipogenesis were decreased by dietary fat treatments. Esterification rates were lowest on the intermediate fat TMR and highest on the highest fat TMR. Lipogenesis was decreased logarithmically by dietary fat intake; this effect was greater as lactation progressed. Adipocyte size and body fat mass decreased during early lactation and then increased for all treatment groups. Supplemental dietary fat reduces de novo synthesis of fatty acid, and this effect increases as lactation progresses.

Adipocytes↗
PubMed1994

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.

Amino Acid Sequence↗
PubMed1994

Regulation of calcineurin phosphatase activity and interaction with the FK-506.FK-506 binding protein complex.

The immunosuppressant FK-506 (tacrolimus) forms a complex with a ubiquitous intracellular receptor, FK-506 binding protein (FKBP12), and this complex inhibits the heterodimeric Ca2+/calmodulin-dependent phosphatase, calcineurin, an essential component of the T-cell receptor signal transduction pathway. Using a series of truncated calcineurin catalytic subunits, we show here that a region within the catalytic subunit that regulates phosphatase activity, the autoinhibitory domain, also regulates the Ca(2+)-dependent interaction of calcineurin with the FK-506.FKBP12 complex. Deletion of this domain produces constitutive activation of the phosphatase as demonstrated by transient transfection experiments in which expression of the truncated protein permitted Ca(2+)-independent induction of interleukin-2 transcription. Thus, deletion of the autoinhibitory domain is necessary and sufficient to constitutively activate calcineurin (CaN). Furthermore, CaN A467-492, an inhibitory peptide based on the autoinhibitory domain from calcineurin (ITSFEEAKGLDRINERMPPRRDAMP), inhibited dephosphorylation of the RII peptide substrate competitively with a Ki = 4 microM, consistent with binding of the autoinhibitory domain at the active site of the enzyme. To assess the role of the autoinhibitory domain in regulating the interaction of CaN with the FK-506.FKBP12 complex, we reconstituted wild type and mutant phosphatase heterodimers using in vitro transcribed and translated subunits. Association of the reconstituted calcineurin heterodimers with FKBP12 was dependent on FK-506. In the case of the wild type heterodimer, association with the FK-506.FKBP12 complex was also dependent upon Ca2+; however, mutant catalytic subunits, in which the autoinhibitory domains were deleted, associated with the drug-binding protein complex in the presence of 10 mM EGTA. These results indicate that the conserved autoinhibitory domain regulates both Ca(2+)-dependent phosphatase activity and association with the FK-506.FKBP12 complex.

Amino Acid Sequence↗
PubMed1994

Activation of the interleukin-4 gene is controlled by the unique calcineurin-dependent transcriptional factor NF(P).

Cyclosporin A-sensitive promoter activity that is induced by the T cell antigen receptor was identified in the region between base pairs -147 and -17 of the mouse interleukin (IL)-4 gene. Here, we identify a transcriptional factor, NF(P), that binds a sequence from -75 to -69 (ATTTTCC) of the mouse IL-4 gene which corresponds to the human IL-4 gene P sequence. Expression of NF(P) was associated with positive regulation of the IL-4 gene and cyclosporin A sensitivity. The P sequence is homologous to the binding site for the positive transcriptional regulator of the IL-2 gene nuclear factor of activated T cells (NFAT). Binding of NF(P) to the P sequence was completely inhibited by an oligonucleotide corresponding to the NFAT binding site. Like NFAT, NF(P) was also found in the cytosol of resting T cells but translocated to the nucleus via a cyclosporin A-sensitive mechanism after T cell activation. Overexpression of a constitutively active calcineurin caused translocation of NF(P) from the cytosol to the nucleus. NF(P) purified on an oligonucleotide affinity column was 90 kDa, which is smaller than the 120-kDa reported for cNFAT. Purified NF(P) did not bind the NFAT consensus sequence, even in the presence of AP-1 protein. Our results suggest that the NF(P) molecule is not identical to the cNFAT molecule.

Animals↗
PubMed1994

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.

Amino Acid Sequence↗
PubMed1994

Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B.

The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA-sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.

Base Sequence↗
PubMed1994

Calcineurin immunoreactivity in Alzheimer's disease.

Aberrant phosphorylation of tau is linked to formation of the paired helical filaments (PHF) seen in Alzheimer's disease. Protein kinases such as mitogen-activated protein kinase, and calcium-regulated protein kinases may, in part, be responsible for addition of phosphate groups to serine residues of PHFtau; however, less is known concerning the phosphatases which regulate tau. In this report, we used several well-characterized antibodies to document calcineurin immunoreactivity in brain tissue from patients with Alzheimer's disease. We now report that levels of immunoreactive calcineurin are not significantly altered in neocortex and cerebellum of Alzheimer's patients relative to similar regions of age-matched controls. Immunocytochemical studies indicated that calcineurin immunoreactivity was present in dendrites and perikarya of many different neuronal populations in both control and Alzheimer brain. When specific antibodies against PHFtau were used in double-labeling experiments with anti-calcineurin antibodies, calcineurin immunoreactivity was seen in association with neurofibrillary tangles. However, calcineurin was not seen in all tangle bearing neurons. These data suggest that calcineurin levels per se are not significantly altered in Alzheimer's disease, but that calcineurin is distributed around some neurofibrillary tangles and may play a role in regulation of tau phosphorylation.

Aged↗
PubMed1994

The Ca2+/calmodulin-activated, phosphoprotein phosphatase calcineurin is sufficient for positive transcriptional regulation of the mouse IL-4 gene.

We have studied the TCR mediated signal transduction pathways involved in transcriptional regulation of the mouse IL-4 gene. The sequences extending from base pair -766 to +63 of the IL-4 gene were inserted upstream of a luciferase indicator gene. Transcriptional activity was observed when the construct, [pIL-4(-766)], was transfected into either the IL-4 producing cell line, EL-4, or the IL-4 non-producing T cell hybridoma, 68-41, but not in the L929 fibroblast cell line. By analysis of deletion mutants of pIL-4(-766), we identified a transcriptional regulatory element that is tightly associated with a signal coming from the TCR and which controls inducible activation of the IL-4 promoter. By analysis of deletion mutants of pIL-4(-766), this latter element was found between base pairs -147 to -17. Electrophoretic mobility shift assays indicated that expression of a nuclear binding protein with binding sites between base pairs -84 and -55 could be induced. By competition and mutation analysis, the binding motif of this protein was determined to be AAAATTTTCC. Stimulation with ionomycin alone was sufficient to induce activity in pIL-4(-766). Cyclosporin A inhibited both the IL-4 promoter activity and activation of the inducible nuclear protein. Transient over-expression of a constitutively active form of the Ca2+/calmodulin-regulated protein phosphatase, calcineurin was sufficient to cause activation of pIL-4(-766) without any additional stimulus. These results indicate that the signaling requirements for activation of upstream positive regulatory elements of the IL-4 gene are distinct from those of the IL-2 gene. Ca2+ mobilization is sufficient to activate the IL-4 promoter, whereas IL-2 gene transcription requires both Ca2+ mobilization and protein kinase C activation.

Animals↗
PubMed1994

Expression of a calmodulin-dependent phosphodiesterase isoform (PDE1B1) correlates with brain regions having extensive dopaminergic innervation.

Cyclic nucleotide-dependent protein phosphorylation plays a central role in neuronal signal transduction. Neurotransmitter-elicited increases in cAMP/cGMP brought about by activation of adenylyl and guanylyl cyclases are downregulated by multiple phosphodiesterase (PDE) enzymes. In brain, the calmodulin (CaM)-dependent isozymes are the major degradative activities and represent a unique point of intersection between the cyclic nucleotide- and calcium (Ca2+)-mediated second messenger systems. Here we describe the distribution of the PDE1B1 (63 kDa) CaM-dependent PDE in mouse brain. An anti-peptide antiserum to this isoform immunoprecipitated approximately 30-40% of cytosolic PDE activity, whereas antiserum to PDE1A2 (61 kDa isoform) removed 60-70%, demonstrating that these isoforms are the major CaM-dependent PDEs in brain. Quantification of PDE1B1 immunoreactivity on immunoblots indicated that striatum contains 3-17-fold higher levels of PDE1B1 than other brain regions, with lowest immunoreactivity in cerebellum. In situ hybridization demonstrated high levels of PDE1B1 mRNA in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Moderate mRNA levels were observed in dentate gyrus, cerebral cortex, medial thalamic nuclei, and brainstem, whereas negligible mRNA was detectable in the globus pallidus, islands of Calleja, substantia nigra, and ventral tegmental area. Immunocytochemistry confirmed that the majority of PDE1B1 protein was localized to the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, PDE1B1 immunoreactivity was ubiquitous, while PDE1A2 immunostaining was restricted to a minor subset of striatal neurons. The expression of PDE1B1 protein and mRNA correlate strongly with areas of the brain that are richest in dopaminergic innervation; indeed, there are strikingly similar distributions for PDE1B1 and D1 dopamine receptor mRNAs. Since D1 receptor binding activates adenylyl cyclase, and striatal neurons lack CaM-sensitive forms of cyclase, the high amount of this PDE implies an important physiological role for Ca(2+)-regulated attenuation of cAMP-dependent signaling pathways following dopaminergic stimulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Prostaglandin E2 and other cyclic AMP elevating agents inhibit interleukin 2 gene transcription by counteracting calcineurin-dependent pathways.

We have previously shown that prostaglandin E2 and other cAMP elevating agents inhibit the nuclear transcription of the human IL-2 gene by interfering with a Ca(2+)-sensitive T cell signal transduction pathway. Calcineurin, a Ca2+/calmodulin-dependent 2B protein phosphatase, is an essential component of the T cell receptor signal transduction pathway leading to IL-2 gene expression. We have therefore tested the hypothesis that this phosphatase may be a target for the inhibitory effects of cAMP on IL-2 gene transcription. We report here that PGE2 markedly reduces the IL-2 promoter activity that is induced by a constitutively active form of calcineurin. In contrast to the complete inhibition of promoter activity produced by the immunosuppressants cyclosporin A and FK-506, this partial block suggests that PGE2 modulates downstream events needed for lymphokine gene activation. Overexpression of calcineurin in Jurkat cells decreases their apparent sensitivity to the inhibitory effects of PGE2 consistent with the fact that this enzyme plays a physiological role in dephosphorylating substrates of cAMP-dependent kinases in several tissues. These results provide evidence that cAMP-dependent pathways may antagonize calcineurin-regulated cascades for T cell activation in vivo, and suggest crosstalk between the Ca2+ and the cAMP signaling pathways during T cell activation.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A alpha).

A complementary DNA for human calcineurin A alpha (protein phosphatase-2B), encoding a protein of 521 amino acids, was isolated from a hippocampus library. The deduced human sequence differs from that of mouse in only two amino acids, demonstrating that the structure of this catalytic subunit has been strictly conserved during mammalian evolution. Such high homology is in contrast to that seen for calcineurin A gamma, an isoform that shows only 88% identity between human and mouse (Muramatsu, T. and Kincaid, R.L. (1992) Biochem. Biophys. Res. Commun. 188, 265-271).

Amino Acid Sequence↗