Hyperactivation of mitogen-activated protein kinase increases phospho-tau immunoreactivity within human neuroblastoma: additive and synergistic influence of alteration of additional kinase activities.

Mitogen-activated protein (MAP) kinase phosphorylates tau in cell-free analyses, but whether or not it does so within intact cells remains controversial. In the present study, microinjection of MAP kinase into SH-SY-5Y human neuroblastoma cells increased tau immunoreactivity toward the phosphodependent antibodies PHF-1 and AT-8. In contrast, treatment with a specific inhibitor of MAP kinase (PD98059) did not diminish "basal" levels of these immunoreactivities in otherwise untreated cells.

The order of exposure of tau to signal transduction kinases alters the generation of "AD-like" phosphoepitopes.

1. The individual and sequential influence of protein kinase C (PKC), protein kinase A (PKA) and mitogen-activated protein kinase (MAP kinase) on human brain tau was examined. 2.

Extensive phosphorylation and axonal transport of triton-soluble neurofilament subunits.

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100-insoluble cytoskeleton. Here we present evidence for multiple populations of NFs and NF subunits, distinguished by differential solubility in Triton, within perikarya and axons of neurons in situ and in culture. We further demonstrate, using microinjection of "tagged" NF subunits and by pulse-chase radiolabeling of endogenous NF subunits, that these soluble NF populations represent precursors for incorporation into the axonal cytoskeleton.

A 26-30 kDa developmentally-regulated tau isoform localized within nuclei of mitotic human neuroblastoma cells.

Tau isoforms migrating at 46-68 and 97-115 kDa were prominent within heat-stable Triton-soluble material, and were present in lesser concentration with Triton-insoluble cytoskeletons, derived from undifferentiated SH-SY-5Y human neuroblastoma cells. Conversely, a 26-30 kDa tau isoform was enriched in the cytoskeleton and detected at relatively minor levels within cytosolic fractions. Pulse labeling with 35S-methionine indicated that this 26-30 kDa "small tau" did not represent a breakdown product of larger isoforms.

Selective activation by bryostatin-1 demonstrates unique roles for PKC epsilon in neurite extension and tau phosphorylation.

Phorbol esters such as 12-O-tetradeonyl phorbol-13 acetate (TPA) induce a time-dependent biphasic effect on protein kinase C (PKC)-mediated events by fostering translocation of cytosolic (latent) PKC to the plasma membrane (where it is activated). Continued treatment, however, depletes the cell's entire PKC complement and induces a functional stake of PKC inhibition. Previous studies from several laboratories have demonstrated that long-term TPA treatment, like treatment with PKC inhibitors, induces neuronal differentiation.

Relationship between selected discourse strategies and student critical thinking.

This study investigated the relationship between use of selected discourse strategies and level of student critical thinking in nursing clinical post-conferences. Selected discourse strategies included: (a) teacher high-level questions, (b) teacher elaboration of student ideas, (c) teacher probing questions, (d) student participation, and (e) student-to-student participation. The level of student critical thinking was defined as the quartile ranking of students (N = 57) on the Watson-Glaser Critical Thinking Appraisal summative measure.

Alterations in cell cholesterol content modulate Ca(2+)-induced tight junction assembly by MDCK cells.

Transepithelial electrical resistance (TER), a measure of tight junction (TJ) barrier function, develops more rapidly and reaches higher values after preincubation of MDCK cells for 24 h with 2 microM Lovastatin (lova), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. While this effect was attributed to a 30% fall in cholesterol (CH), possible effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase the flux of intermediates through the CH biosynthetic pathway.

Refined fibril structures: the hydrophobic core in Alzheimer's amyloid beta-protein and prion as revealed by X-ray diffraction.

From the wide-angle, equatorial X-ray data of a beta-amyloid analogue, we previously calculated the electron density of the constituent beta-crystallite, which assembles as multimers (four to six crystallites) in building the amyloid fibre. In the scattering region where the spacing d < approximately 10 A, the observed reflections were indexed by an orthogonal lattice with a unit cell having a = 9.44 A, b = 6.

Inhibition of proteolysis enhances aluminum-induced perikaryal neurofilament accumulation but does not enhance tau accumulation.

As observed for neurons in situ, phosphorylated neurofilament (NF) epitopes are normally segregated within the axonal cytoskeleton of NB2a/d1 cells. However, accumulations of phosphorylated NFs develop in NB2a/d1 perikarya following exposure to aluminum salts and following inhibition of proteolysis. In the present study, we observed that perikarya of cells exposed to both aluminum and the protease inhibitor C1 (also known as "AllNal") were more intensely labeled by monoclonal antibodies directed against both nonphosphorylated and phosphorylated epitopes than were cells treated with either aluminum or protease inhibitor alone.

Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells.

Aluminum treatment of intact neuroblastoma cells alters neurofilament subunit phosphorylation, solubility, and proteolysis.

Addition of 400 microM AlCl3 to the culture medium for 72 h has been previously shown to induce perikaryal whorls of intermediate-sized filaments in intact mouse NB2a/d1 neuroblastoma cells. Immunoblot analyses demonstrated that in vivo treatment of cells with aluminum induced the de novo appearance of extensively phosphorylated NF-H isoforms in cytoskeletons of undifferentiated cells and increased levels of these isoforms in differentiated cells. Neurofilament subunits isolated from intact cells treated with aluminum were resistant to dephosphorylation in vitro by alkaline phosphatase and to in vitro degradation by endogenous calcium-dependent protease(s).

Coffee oil consumption does not affect serum cholesterol in rhesus and cebus monkeys.

Oil from coffee beans contains the diterpenes cafestol and kahweol, which greatly elevate cholesterol in humans. Consumption of 0.03 g coffee oil (0.

Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C.

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ("C1") and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera.

Calcium influx recruits an additional class of kinases to hyperphosphorylate tau.

SH-SY-5Y human neuroblastoma cells were treated with combinations of the kinase inhibitors HA-1004, W-7 and H-7 and calcium ionophore A23187. Microdensitometric analyses revealed that, in the absence of ionophore-mediated calcium influx, PHF-1 levels were reduced by approximately half in cultures treated with HA-1004 or W-7, but were not reduced by H-7. By contrast, the doubling in PHF-1 immunoreactivity that resulted following ionophore treatment was prevented by all three inhibitors.

Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth.

Intracellular delivery of anti-GAP IgG inhibits the elaboration of neurites by NB2a/d1 cells. However, recent studies indicate that the extent of inhibition is minimized when cells were cultured on poly-L-lysine-coated or laminin-coated versus uncoated plates, suggesting that the role of GAP-43 in neuritogenesis may be specifically related to membrane adhesiveness. We therefore examined the influence of inhibition of thrombin, the neuronal surface protease that restricts neurite outgrowth, on GAP-43-dependent neurite outgrowth.

Calcium modulates aluminum neurotoxicity and interaction with neurofilaments.

We examined the influence of calcium on neurotoxicity of AlCl3 and Al-lactate toward differentiated NB2a/d1 cells. Apart from induction of perikaryal neurofibrillary inclusions, AlCl3 at 1 mM induced no obvious additional signs of toxicity when added to culture medium in the presence of the normal medium CaCl2 content of 1.8 mM, nor when extracellular calcium was decreased by the addition to the medium of 0.

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence.

A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.

Steady state fluorescence energy transfer measurements of human alpha apohemoglobin structure.

A nonfluorescent reagent, 4-phenylazophenylmaleimide [4-PAPM], was attached to the sole cysteine residue [104(G11)] of alpha apohemoglobin (alpha degree) and served as an energy acceptor for the single intrinsic tryptophanyl [14(A12)] donor. This novel fluorescence system provided a transmolecular vehicle by which the overall structure of alpha degree could be monitored in 0.05 M potassium phosphate buffer at 5(0) C.

Kinetics of human apohemoglobin dimer dissociation.

Heme chain exchange was employed to investigate the dimer dissociation reaction of human apohemoglobin in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees C.

Interrelationship between plasma potassium concentration, pulmonary ventilation and electrocardiographic change during and after highly intense exercise.

Objective: The purpose of this investigation was to induce elevated plasma concentrations of potassium (K+) efflux from active muscle cells during intense muscular exercise. The relationship between K+, pulmonary ventilation (VE) and EKG changes, specifically T-wave amplitude, is presently controversial.

Design: Repeated measures design.

Response of drug resistant isolates of Schistosoma mansoni to antischistosomal agents.

The susceptibility of four isolates of Schistosoma mansoni (BH, MAP, MPR-1 and K) to four multiple doses of anti-schistosomal agents (hycanthone, niridazole, oxamniquine, and praziquantel) were evaluated in infected female Swiss albino mice. These schistosomal isolates had been maintained in the laboratory without further drug pressure for 20 to 30 generations. Multiple dosage regimens were used for each drug against each isolate of S.