Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Activation ofClostridium perfringens cytotoxic enterotoxin(s) in vivo and in vitro: Role in triggers for sudden infant death

The action ofClostridium perfringens cytotoxic enterotoxins may be activated/exacerbated both in vivo and in vitro by the addition of an activator molecule present in a brush border membrane fraction isolated from young rabbits. Increased concentrations of the activator could be induced by immunologically stimulating rabbits with Ribi adjuvant. Comparative studies suggested that the activator was interferon-gamma (IFN-). In vitro IFN- sensitized cell lines apparently by enhancement of cell permeability, which allowed a more rapid uptake of the toxins, resulting in cell death at lower toxin concentrations. Viral and/or bacterial infections are inducers of IFNs. We propose that some immunologically immature infants are predisposed to infection. In the weeks prior to death, these infants may suffer from an infection that induces the synthesis of IFNs, sensitizing the infant to a more virulent infection and possible sudden death.Florida Agricultural Experiment Station Journal Series No. R-02380     

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_P photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

The effect of pulsed microwaves on passive electrical properties and interspike intervals of snail neurons

The effects of pulsed microwaves (2.45 GHz, 10 μs, 100 pps, SAR: 81.5 kW/kg peak, 81.5 W/kg average) on membrane input resistance and action potential (AP) interval statistics were studied in spontaneously active ganglion neurons of land snails (Helix aspersa), at strictly constant temperature (20.8±.07°C worst case). Statistical comparison with sham-irradiated neurons revealed a significant increase in the mean input resistance of neurons exposed to pulsed microwaves (P ? .05 ). Pulsed microwaves had no visible effect on mean AP firing rate; this observation was confirmed by analysis of interspike intervals (ISIs). Using an integrator model for spontaneously active neurons, we found the net input current to be more variable in neurons exposed to pulsed microwaves. The mean input current was not affected. The standard deviation of ISIs and the autocorrelation of the input current were marginally affected, but these changes were not consistent across neurons. Although the observed effects were less obvious than those reported in other studies, they represent evidence of a direct interaction between neurons and pulsed microwaves, in the absence of macroscopic temperature changes. The data do not suggest a single, specific mechanism for such interaction. © 1993 Wiley-Liss, Inc.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Simple electromyographic biofeedback treatment for chronic pediatric constipation/encopresis: Preliminary report

Pediatric constipation/encopresis is thought to be due, in part, to paradoxical constriction of the external anal sphincter (EAS) muscle during attempted defecation. This inappropriate contraction can lead to delayed, impacted, painful, and infrequent bowel movements. Standard Medical Care (SMC) involves disimpaction with enemas, followed by laxative therapy and diet modification, to maintain frequent soft stools. Using the case control method, the efficacy of SMC alone was compared with SMC plus EAS electromyographic biofeedback aimed at eliminating paradoxical contraction. Thirteen consecutive chronically constipated children received SMC plus biofeedback, and were compared with 13 age- and sex-matched children who received only SMC. Biofeedback subjects demonstrated post-treatment elimination of EAS paradoxical constriction. At 16 months follow-up parents of biofeedback children reported significantly greater improvement in constipation, encopresis, laxative use, and painful bowel movements compared to SMC.This research report was supported by the NIH under grant No. RO1 HD 28160.