Distribution and possible origin of neuropeptide-containing nerve elements in the mammalian liver

The intrahepatic distribution of nerve fibres is highly species dependent, therefore we searched for a species where the innervation pattern is similar to that of the human liver. Livers of rats, cats, guinea pigs and humans were used. The different nerve elements were identified by ABC immunohistochemistry and analysed semiquantitatively. Large numbers of neuropeptide Y (NPY) and dopamine-β-hydroxylase immunoreactive (IR) nerve fibres were observed in the human and guinea pig liver, and they were in close contact with portal triads, central veins and ran parallel with liver sinuses. A few substance P, somatostatin and vasoactive intestinal polypeptide IR nerve fibres were also detected intralobularly, while galanin nerve fibres were only observed around portal triads. In the rat liver only a few NPY-positive nerve fibres were found, exclusively in portal tracts. Some nerve cell bodies (IR for NPY and somatostatin) were also found in the liver of guinea pigs, young cats and humans, therefore some of the nerve terminals might originate from these intrinsic ganglia. It can be concluded that the innervation pattern of the guinea pig liver shows the highest similarity to that of the human liver.

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein–cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

Protective role of pinacidil against adrenaline-induced myocardium injury in guinea pig liver mitochondria

We investigated the role of the ATP-sensitive potassium channel opener pinacidil and blocker glibenclamide on guinea pig liver mitochondrial function, and a possible significance of pinacidil in the pharmacological treatment during myocardium dystrophy. First, a series of experiments was performed to determine the effect of pinacidil and glibenclamide on mitochondrial oxygen consumption. We found that pinacidil increased the rate of mitochondrial respiration for FAD-generated substrate (succinate oxidation), but was most effective for α-ketoglutarate oxidation with enhancement of respiratory control ratio. Oxidation of FAD-generated substrate inhibited efficiency of phosphorylation for α-ketoglutarate oxidation in pinacidil-treated animals. Glibenclamide decreased the rate of respiration with the lowest value of efficiency of phosphorylation, especially for α-ketoglutarate oxidation. A second series of experiments was performed to determine the effects of pinacidil and glibenclamide on oxidative phosphorylation during adrenaline-induced myocardium dystrophy. The increase in respiratory control ratio and efficiency of phosphorylation for α-ketoglutarate oxidation was greater than for succinate oxidation in mitochondria of pinacidil-pretreated animals during myocardium dystrophy. Inhibitory analysis with malonate suggested that endogenous succinate increased oxidation of NADH-generated substrates in mitochondria. Pinacidil is mainly involved in the adrenaline-induced alterations of mitochondrial function due to elevation of phosphorylation efficiency for α-ketoglutarate oxidation and a decreased level of lipid peroxidation.

A Rapid Transglutaminase Assay for High-Throughput Screening Applications

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca2+-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The Km of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 μM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.