Reversible beta-pleated sheet formation of a phosphorylated synthetic tau peptide.

Serine416 of human tau protein is believed to be phosphorylated in Alzheimer neurofibrillary tangles. We synthesized a fragment of tau, consisting of amino acids 408-421 in both non-phosphorylated and serine416-phosphorylated forms. Circular dichroism in a trifluoroethanol-water mixture indicated a beta-turn----beta-pleated sheet conformational transition upon phosphorylation. The beta-structure formation is intermolecular and can be inhibited by addition of Ca2+ ions or a phosphorylated tripeptide, but not with its non-phosphorylated analog. The presence of the phosphorylated tau peptide did not facilitate the formation of beta-pleated sheets of a phosphorylated neurofilament fragment. Multivalent cations induced a conformational transition of this phosphorylated neurofilament peptide, but the effect was less specific than the transition induced in the tau fragment, and it could also be reversed with the competing phosphorylated tripeptide.

Solid-phase synthesis of phosphopeptides.

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.

Taurine levels in discrete brain nuclei of rats.

Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-putamen, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.

Concentrations of GABA and glycine in discrete brain nuclei. Stress-induced changes in the levels of inhibitory amino acids.

Concentrations of gamma-aminobutyric acid (GABA), glycine and serine have been measured in 44 microdissected areas of the brain of the rat. All three amino acids were ubiquitously present and distributed unevenly in the brain. Very high levels of GABA were found in the anterior hypothalamic and medial preoptic nuclei and the substantia nigra; high levels were found in the interpeduncular and red nuclei in the mesencephalon and in several hypothalamic nuclei. Glycine was distributed fairly uniformly with large concentrations in certain lower brainstem nuclei. In these areas, the concentrations of glycine exceeded those of serine, while the serine-glycine ratio was 4.5:1 in the caudate nucleus, 4:1 in the cerebellum and 2.5:1 in the cerebral cortical areas. Acute stress induced with formalin (pain) resulted in a significant depletion of levels of GABA in the hypothalamus and the lower brainstem but not in the cortical areas. In the same animals, concentrations of glycine doubled in the cerebral cortex and remained unchanged elsewhere in the brain. Increased motor and behavioral activity after the acute administration of a large dose of amphetamine were associated with a 2-5-fold increase in the levels of glycine in brain, and markedly elevated the concentrations of GABA in the major biogenic amine-containing cell groups only (substantia nigra, locus coeruleus and dorsal raphe).

Distribution and stress-induced increase of glutamate and aspartate levels in discrete brain nuclei of rats.

Concentrations of glutamate and aspartate have been measured in 45 microdissected brain areas and nuclei in rat. Both amino acids are ubiquitously present and distributed unevenly in the central nervous system. Very high glutamate levels were found in the cerebellum and the insular cortex, high levels in neocortical and limbic cortical areas, and in the nuclei of the medial hypothalamus. Aspartate is distributed rather uniformly with the highest concentration in the hypothalamic arcuate nucleus and the lowest in the midbrain central gray matter and the cerebellum. Acute formalin (pain) stress elevated glutamate and aspartate levels in the cortical areas and substantia nigra significantly, but had minor or no effects on other brain nuclei. Increased locomotor and behavioral activities due to a high dose of amphetamine resulted in a 2-5-fold increase of glutamate and aspartate concentrations, particularly in the biogenic amine-containing brain nuclei.

[Effect of 1,2:5,6-dianhydrogalactitol and 1,2:5,6-dianhydro-3,4-diacetylgalactitol on DNA synthesis in the cells of melanoma B16, bone marrow, small intestine epithelium, spleen and liver of mice]. / Vliianie 1,2:5,6-diangidrogalaktitola i 1,2:5,6-diangidro-3,4-diatsetilgalaktitola na sintez DNK v kletkakh melanomy B16, kostnogo mozga, épiteliia tonkogo kishechnika. selezenki i pecheni myshei.

Alterations in DNA synthesis induced by 1,2:5,6-dianhydrogalactitol (DAG) and 1,2:5,6-dianhydro-3,4-diacetyldianhydrocalactitol (Diac-DAG) were studied in host normal cells and tumor cells. After administration of these antitumor drugs to melanoma B16-bearing mice, DNA synthesis in host tissues (bone marrow, gastrointestinal mucosa, spleen, liver) got recovered more rapidly than DNA synthesis in melanoma B16. Diac-DAG differed from DAG from the standpoint of damage to DNA synthesis in normal cells. Only DAG inhibited the DNA synthesis in liver cells. Inhibition of DNA synthesis in the bone marrow and spleen with Diac-DAG was less remarkable than with DAG.

Investigation of metabolites of tofizopam in man and animals.

The biotransformation of the labelled forms of 1-(3,4-dimethoxy-phenyl)-4-methyl-5-ethyl-7,8-dimethyoxy-5H-2,3-be nzodiazepine (Grandaxin, tofizopam) has been investigated after oral administration in animals and man. The major part of urinary metabolites was found to be conjugated with glucuronic acid. The metabolites were separated using TLC technique and analysed by GLC and GLC-MS. The chief way of the metabolic information of tofizopam is demethylation, however, the position of CH3 elimination as well as the rate of it was different in various species.

Damages of DNA synthesis in normal and tumor cells with sugar alcohol derivatives.

The rates of incorporation of 2-14C-thymidine into DNA of melanoma B16, bone marrow, gastrointestinal mucosa, spleen and liver at various time after administration of dianhydrogalactitol (DAG), 3,4-diacetyldianhydrogalactitol (DiacDAG) and 3,4-disuccinyldianhydrogalactitol (DisuDAG) at maxima nonlethal single doses to tumor-bearing mice were studied. The sugar alcohol derivatives induced the stable inhibition in DNA synthesis of tumor cells. DNA synthesis in normal dividing cells was shown to recover more rapidly than in melanoma B16 cells after administration of all drugs. DisuDAG is characterized by stronger inhibitory effect on DNA synthesis in melanoma B16 cells at the half of the single maxima nonlethal dose compared with DAG and DiacDAG. Differing from DAG, DiacDAG and DisuDAG did not effect the incorporation of 2-14C-thymidine into DNA of liver cells. In vivo inhibition of DNA synthesis in melanoma B16 cells with DiacDAG was not due to damage of the TCA soluble fraction.

Investigation of the metabolites of tofizopam in man and animals by gas-liquid chromatography-mass spectrometry.

The metabolites of tofizopam [Grandaxin; 1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine] have been studied in patients and animals. The major pathway of the metabolic transformation of tofizopam was found to be demethylation. The position in which demethylation takes place and the rate of this process in various species were determined. Gas-liquid chromatography-mass spectrometry-mass chromatography was used for the identification of the metabolites.

Specific and sensitive method for the determination of tobanum using gas chromatography with electron-capture and mass fragmentographic detection.

Quantitative gas--liquid chromatographic (GLC) and GLC--mass spectrometric (MS) methods for the determination of Tobanum, a new beta-blocking agent, in human plasma have been developed. After solvent extraction, a bis-trifluoroacetyl derivative is formed which is measured by an electron-capture detector. The quantitation is controlled by using an internal standard, propranolol hydrochloride, which is added to all samples. The electron-capture detector response is linear in the concentration range used, 5--150 ng/ml. The identity and quantity of the GLC peaks is confirmed by GLC--MS. The minimum detectable concentration of Tobanum is 1 ng using 3-ml plasma samples.

Pharmacology of GYKI-41 099 (chlorpropanol, Tobanum) a new potent beta-adrenergic antagonist.

The compound GYKI-41 099, as a beta-adrenergic antagonist, is 3-8 times more potent than propranolol in vitro and in vivo. Its antiarrhythmic effectiveness surpasses that of propranolol and pindolol inhibiting the ouabain arrhythmia in dogs and cats. GYKI-41 900 has a negligible cardiodepressant activity; it is not cardioselective. The compound shows a rapid and long lasting effect. There was a prolonged elimination of the radioactivity after the injection of 14C-41 099 to rats and dogs. The half life of the unlabeled substance in humans was more than 10 hours.