Synthesis of N‐Substituted Piperidine Salts as Potential Muscarinic Ligands for Alzheimer's Applications

Several heterocyclic N‐piperidine substituted salts were synthesized that were found to inhibit the specific binding of the antagonist [³H] quinuclidinyl benzilate in radioligand muscarinic binding assays (³H‐QNB) in bioassays. One of the heterocyclic salts, compound 7 , met the significance criteria in these assays (>50% inhibition) at 10 μM of the nonselective muscarinic antagonist (³H‐QNB) in cells of the Wistar rat cerebral cortex. Furthermore, this compound displayed 61% inhibition at 10 μM of the antagonist (³H‐QNB) for the M₅ receptor (IC₅₀ 6.34 μM, K_i 3.93 μM, n_H = 0.996) in human recombinant CHO cell lines. These data obtained from Ricerca Biosciences suggested that compound 7 was selective for the M₅ receptor. Another study from the Czech Academy of Sciences demonstrated that compound 7 was 3 to 8 times more potent at M₂ than other subtypes of muscarinic receptors in competition with antagonist N‐methylscopolamine and selective for the M₁ receptors over M₃ and M₅ in antagonizing accumulation of inositol phosphates induced by muscarinic agonist carbachol.     

Concerning the interconnections of some parameters of self-organizing oxyhydrate gels and their experimental determination

The appearance of a pulsating current in the elongated cell filled with the zirconium oxyhydrate gel under short-circuited electrodes and constant mixing was shown. The pulsating character of the current is expressed in the form of current splashes. The reason for the appearance of current splashes is the self-organization of the gel with time; to be more exact, the reason is the constant conformational reorganization of the oxyhydrate matrix. When magnetic field acts on the zirconium oxyhydrate gel, increases in both the current values and the current splashes are observed. The increase of the gel particle sizes in a magnetic field is detected. Thus, it is of interest to consider the connection between the period of appearance of current splashes, splashes amplitudes and the sizes of gel aggregate regions.

In this regard, it is important to find out the connection between the parameters of the self-organizing gel cell. We suppose that:

1. In the process of self-organizing, the concentration of monomers forming the matrix in a gel area may change with time.

2. Solution of the task is independent of time. Generally speaking, it is possible to find a time-dependant solution of the task, but it is hardly worth doing.

High-performance liquid chromatographic profiling of indolylacryloylglycine and its possible precursors in body fluids

Indolylacryloylglycine (IAcrGly) is one of the physiological components of urine, although its source and its role in the human organism have not yet been unambiguously established. Changes in the IAcrGly excretion level have been found under some physiological (age dependence, seasonal variations) and pathological (photodermatoses, muscle dystrophy, liver cirrhosis) conditions. The proposed method for IAcrGly, indolylacrylic acid and its possible precursors, namely indolyllactic and indolylpropionic acids, involves deproteinization and extraction of urine on a Sep Pak C₁₈ cartridge. HPLC analysis was carried out using a DataApex liquid chromatograph, equipped with an LCD 2082 UV detector, signals being acquired with a CSW workstation. The chromatographic column was Spherisorb ODS, 5 μm (125 × 4 mm I.D.), the mobile phase for isocratic elution was ethanol-1% acetic acid (27:73) and the flow-rate was 0.7 ml/min. The lower response limit is about 1 μmol/l for all metabolites at 280 nm.     

A comparison of sequential extraction procedures for fractionation of arsenic,cadmium, lead,and zinc in soil

Twelve soil samples differing in physicochemical properties and total element contents were extracted by three sequential extraction procedures to determine As, Cd, Pb, and Zn bound to individual soil fractions and are defined by individual operational procedures. In the case of arsenic, two additional sequential extraction schemes were designed entirely for fractionation of soil containing arsenic were tested. The results confirmed that determination of element proportions bound to individual soil fractions is strongly dependent on the extracting agent and/or procedure applied within individual extracting schemes. As expected, absolute values of the elements released among the individual extracting procedures are weakly comparable. More reliable results were determined for the more mobile soil elements i.e. cadmium and zinc, in the fractions characterizing the most mobile proportions of investigated elements where significant correlations with basic soil characteristics were observed. In contrast, ambiguous results were observed for As and Pb, for both the individual extraction procedures and the effect of the soil characteristics. Regardless of the studied element, the poorest results were determined for reducible and oxidizable soil fractions. The application of at least two independent procedures or modification of the extraction scheme according to element investigated and/or particular soil characteristics can also be helpful in definition of element pattern in soils in further research.     

Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation

Carboxyl groups containing magnetic and non-magnetic microspheres were used in solid-phase reversible immobilization (SPRI) of genomic DNA. Magnetic non-porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)--P(HEMA-co-EDMA), poly(glycidyl methacrylate)--PGMA and P(HEMA-co-GMA) microspheres with hydrophilic properties were prepared by dispersion copolymerization of the respective monomers in the presence of colloidal iron oxides. DNA from chicken erythrocytes and DNA isolated from bacterial cells of Bifidobacterium longum was used for testing of adsorption/desorption properties of magnetic microspheres. The occurrence of false negative results in polymerase chain reaction (PCR) caused by the presence of extracellular inhibitors in DNA samples has been solved using SPRI. The P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres were used for isolation of DNA from different dairy products followed by PCR identification of Bifidobacterium strains.     

Chromatographic behaviour and purification of linear lambda phage and plasmid DNA molecules on 2-hydroxyethyl methacrylate-ethylene dimethacrylate-based supports

The HEMA-BIO 1000 support, which is based on a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, was used for separation of lambda DNA and its fragments and plasmid pBR322 DNA. The separation of fragments greater than 6.6 kbp was demonstrated according to the slalom chromatography mechanism on column for size-exclusion chromatography in the case of linear lambda DNA fragments. The influence of particle size of column packing, mobile phase rate, and KCl concentration in mobile phase is discussed. The purification of plasmid DNA pBR322 using size-exclusion chromatography was more rapid compared to gel electrophoresis. The presence of salts in the eluate is not disadvantageous. DNA can be recovered from the eluate by ethanol precipitation. Plasmid DNA pBR322 isolated in this way was suitable for different biological applications (cleavage with restrictases, electrotransformation into bacterial cells).     

The mechanism of η(η5:η5-fulvalene)-di-μ-chloro-bis (η5-cyclopentadienyltitanium) formation in the system Cp2TiCl2 : LiAlH4 : Olefinic solvent

Summary The reduction of Cp₂TiCl₂ with LiAlH₄ in olefinic solvents at temperatures above 100° leads to the formation of µ-( ⁵ : ⁵-fulvalene)-di-µ-chloro-bis( ⁵-cyclopentadienyl-titanium) (1) and finally to µ-( ⁵ : ¹⁵-fulvalene)-di-µ-hydrido-bis ( ⁵-cyclopentadienyltitanium). The first step of the reduction includes mainly the formation of the dimer of bis( ⁵-cyclopentadienyl)titanium(III) chloride, (Cp₂TiCl)₂. The mechanism of its conversion into (1) involves the formation of an intermediate bis( ⁵-cyclopentadienyl)titanium(III) hydride species which reacts immediately with the olefinic solvent to give bis( ⁵-cyclopentadienyl)alkyltitanium(III) compounds. The formation of the fulvalene ligand in (1) is explained by the interaction of the alkyltitanium compound with the cyclopentadienyl ligands of (Cp₂TiCl)₂ resulting in hydrogen transfer to the olefinic solvent.     

The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.     

Zur Frage der „Exoelektronen“-Emission bei Metallen

Ohne Zusammenfassung

---