Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.

By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A+T rich and contained different regulatory elements. By using a two step procedure (Heparin-sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD.     

Mathematical modelling of intra- and extracellular potentials generated by active structures: effects of a step change in structure diameter

A mathematical model developed in our laboratory is used to estimate and analyse extracellular potentials generated in a volume conductor by a geometrically inhomogeneous structure with a step increase or a step decrease in its diameter. The transmembrane potentials were calculated using the model of Hodgkin and Huxley (1952) and the method of Joyner et al. (1978). Variations in waveforms of the transmembrane and extracellular potentials were described and discussed. Differences in waveforms of the extracellular potentials and in declines of their components are due to changes in the source which generates these potentials. In case of a propagation block the peak-to-peak amplitude of the extracellular potentials calculated over the area of the block may be higher than that over the area of propagation of action potentials. The possible applications of the results to the analysis of extracellular potentials recorded around actual motoneurons during their orthodromic or antidromic activation are discussed.     

Computer Simulation of the Beta Structure,with Different Lattice Ions and Reactivity,of Hydroperoxo-Metal Complexes Using an Embedded-Cluster Method

The chemical properties of beta zeolite are interconnected with its structure in a complicated way. In general terms, the strength of the acid sites in the beta phase depends strongly on its structure, topology and composition. The quantum chemical calculations of the beta zeolite cluster model have been carried out in order to predict how the cluster's local structure influences the geometric parameters and thus affects the chemical functionality of the beta zeolite. Computer simulations of a beta zeolite model with one lattice aluminium, or boron, or titanium or vanadium ions and of its acidic centres have been carried out. The acidity of the framework oxygen atoms in the vicinity of the lattice atom centre has been calculated and compared. The reactivity of catalytic species comprisingthe lattice ion and a hydroperoxo ligand has been studied as an oxidising site.     

Limited variability of CTG/CAG repeats in <Emphasis Type="Italic">Lycopersicon</Emphasis> nuclear DNA

Seven clones containing (CTG)_n/(CAG)_n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.     

Threshold stimulation and accommodation of the Hodgkin-Huxley axon.

The charge-duration and strength-duration relations for just threshold rectangular stimuli were numerically investigated for the Hodgkin-Huxley axons of different lengths and different membrane capacitances under normal conditions and blockage of the development of accommodative processes. Two linear portions could be distinguished on the charge-duration curve. One of them followed the Weiss law. The other one represented a portion of a straight line passing through the zero point of the coordinates. The slope of the second portion was determined by the charge for very short stimuli (Q0), the slope of the first portion, and the maximum time to excitation (tau max). The rheobase reflected the slope of the second portion. Upon varying the fibre length the slope of the first and the second linear portions and the rheobase changed. The membrane capacitance substantially affected both the value of Q0 (as in the case of myelinated fibres) and the rheobase. The accommodative processes affected the Q0, the slope of the first line, tau max, and, consequently, the rheobase. The effect of potassium activation was stronger than that of sodium inactivation. The slope of the first line, tau max, and the rheobase might be considered more comprehensive indicators of the accommodative processes than the usually used indicators.     

Chemotaxis of cholera vibrios: a study method and the relation to different substances

The chemotaxis of V. cholerae in response to 56 different substances (amino acids, carbohydrates, salts, etc.) has been studied by the methods of visual observation and quantitative determination. Attractants, neutral substances and one repellent have been revealed. Adler's method (1973) has been modified with regard to the requirements for the working procedures in handling the causative agents of highly dangerous infections.     

New method for the histochemical demonstration of dipeptidyl aminopeptidase I activity using a novel anthraquinoyl hydrazide substrate.

A new method for the histochemical localization of dipeptidyl aminopeptidase I (DPP I, cathepsin C), based on a newly synthesized substrate-Gly-L-Phe-5-chloro-1-anthraquinoyl hydrazide.HCl (Gly-Phe-CAH), is proposed. The enzyme activity liberates 5-chloro-1-anthraquinoyl hydrazine (CAH)--a water-insoluble brown-reddish compound, which precipitates on the enzyme locations. The primary reaction product reacts simultaneously or, otherwise, by post-coupling with p-anisaldehyde (p-AA), thus converting to the reddish-violet amorphous hydrazone--the final reaction product. The validity of enzyme localization is thus assured by the insolubility of the primary reaction product and does not depend on the rate of the second reaction step. The enzyme studied is successfully localized in different rat organs using the newly proposed technique.     

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the β-amyloid precursor protein (βAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with β-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon γ (INFγ). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of β-amyloid, or thiorphan, an inhibitor of neprilysin involved in β-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNγ, which can influence the progression of the AD, significantly reduced the enzyme activity.