Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.     

Evidence for a highly nuclease resistant RNA fragment in prosomes

Prosomes, small cytoplasmic particles of mouse erythroblasts were found to contain low molecular weight RNA molecules in the range of 80 nucleotides. Nuclease digestion of prosomes suggests that prosomal proteins cover and protect almost the whole length of their RNA(s). Our results demonstrate clearly that RNA is an intrinsic component of prosomes.     

Design and Concept of Polyzwitterionic Copolymer Microgel Drug Delivery Systems <Emphasis Type="Italic">In Situ</Emphasis> Loaded with Non-steroidal Anti-inflammatory Ibuprofen

Nowadays, the modern pharmaceutical investigations are directed toward obtaining of new polymer micro- and nano-sized drug delivery carriers. In this respect, the use of hydrogel carriers based on polyzwitterions (PZIs) is an opportunity in the preparation of polymer drug delivery systems with desired characteristics. This paper describes the synthesis and characterization of micro-structured p(VA-co-DMAPS) systems with different compositions in situ loaded with Ibuprofen by emulsifier-free emulsion copolymerization (EEC) in water. The mean size of the prepared microparticles was measured by SEM and particles have been visualized by AFM. The inclusion of Ibuprofen in the polyzwitterionic copolymer microgel systems was established by using DSC. In vitro drug release experiments were carried out in order to estimate the ability of the obtained microgels to modify the release of water-insoluble Ibuprofen.     

Evaluation of recombinant caspase specificity by competitive substrates

The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.     

Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T ₂ and T ₂*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as “probeless” magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T ₂- and T ₂*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin’s stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T ₂ relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T ₂ relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.     

Impaired mitochondrial Ca2+ homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca(2+) homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A>G and 3302A>G in tRNA(Leu(UUR)), as well as Rho(0) cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP+ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.     

Tightly bound nonprotamine proteins from ram sperm nuclei studied by one- and two-dimensional peptide mapping

The tightly bound proteins of ram sperm nuclei (TBSP) have been recovered as a fraction co-sedimenting with DNA after high salt-urea deprotamination of the nuclei. TBSP were studied by two-dimensional (2D)tryptic peptide mapping and by one-dimensional (1D)-partial proteolysis mapping. The 2D maps revealed a strong homology among the proteins, irrespective of substantial differences in their molecular masses. This homology was supported also by the 1D-mapping data.The 2D-tryptic maps of TBSP were compared to those of lamb liver lamins but no apparent similarity was detected. TBSP were found to react positively to a test for the presence of carbohydrate residues, suggesting that these proteins are glycoproteins as established earlier for the lamins.The 2D maps of several proteins of seminal plasma origin, used as a control, displayed completely different peptide profiles.     

Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide

Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5-25 microM for homocysteine and glutathione, and in the range of 0.5-200 microM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 microl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35-4.38% and 0.89-4.13%, respectively.