Biosynthesis and action of nitric oxide in mammalian cells

Nitric oxide (NO) can act as a vasorelaxant, a modulator of neurotransmission and a defence against pathogens. However, under certain conditions, NO can also have damaging effects to cells. Whether NO is useful or harmful depends on its chemical fate, and on the rate and location of its production. Here, we discuss progress in NO chemistry and the enzymology of NO synthases, and we will also attempt to explai its actions in the cardiovascular, nervous and immune systems.     

DNA sequence analysis of mitochondrial Cyt-b and the species status ofLaniarius dubiosus (Rchw. 1899)

In 1899Reichenow described an african bushshrike in juvenile plumage as a new species. He named itLaniarius dubiosus. DNA from type material (feathers and skin) was extracted and DNA sequences from the mitochondrial Cyt-b gene were analysed. Comparisons of DNA-sequences from other bushshrikes (including the type-specimen from Luehder's bushshrikeLaniarius lühderi) support the judgement that dubiosus does not represent a full species rather than a representative of the western subspecies of the Luehder's bushshrikeLaniarius luehderi.

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Zusammenfassung Der vonReichenow (1899) als neue Art beschriebene, sich im Jugendkleid befindliche BuschwürgerLaniarius dubiosus ist nach DNA-Sequenzanalysen des mitochondrialen Cyt-b Genes mit hoher Wahrscheinlichkeit ein Jungvogel der westlichen Rasse des BraunscheitelwürgersLaniarius lühderi. Auf der Basis eines DNA-Stammbaumes von sieben verschiedenen Buschwürgern ist der 1991 neu beschriebeneLaniarius liberatus am wenigsten eng mitL. dubiosus verwandt.

     

Interleukin 1β-converting Enzyme Related Proteases/Caspases Are Involved in TRAIL-induced Apoptosis of Myeloma and Leukemia Cells

The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNFfamily and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1β-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor AcYVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO.

These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

     

Thermophilic Bacilli growing with carbon monoxide

Four strains of obligately thermophilic Bacilli capable of growing with carbon monoxide as a sole carbon and energy source were isolated from settling ponds of a sugar factory. Most of them could be identified as strains of Bacillus schlegelii on the basis of cell wall composition, DNA homology menaquinone and DNA base content. Growth with CO was very fast (t _d =3 h) and was optimal at 65°C. No growth occurred below 50°C. As with the mesophilic carboxydotrophs, hydrogen plus carbon dioxide could also serve as autotrophic substrates. Growth of the isolates with CO depended on the presence of molybdenum in the growth medium. This suggested CO oxidase in the newly isolated Bacilli being a molybdenum hydroxylase similar to the enzymes from the mesophilic carboxydotrophs. Some data characterizing the CO-oxidizing activity in extracts of the thermophilic isolates are also provided.This paper is respectively dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β-adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

New species of Roccellinastrum with an emendation of the genus

The genus Roccellinastrum Follm. emend. Henssen & Vobis is placed in the Lecideaceae. Two new species are described, R. epiphyllum Henssen & Vobis, from Chile and R. neglectum Henssen & Vobis, from New Zealand and Tasmania. The new combination R. candidum (Mull. Arg.) Henssen is proposed. Roccellinastrum is characterized by apothecia of diverse shape without a proper margin, and a developmental morphology corresponding to that of the Lecanorales, by small amyloid asci, branched paraphyses and excipular hyphae, a byssoid–spon–giose thallus composed of thick–walled hyphae, and members of the Chlorococcales as phycobionts. The principal chemical constituent is protocetraric acid. The species have different spore septation and chemistry. Summary in Spanish.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Dermorphins, Opioid Peptides from Amphibian Skin, Act on Opioid Receptors of Mouse Neuroblastoma x Rat Glioma Hybrid Cells

Dermorphin and its Hyp6 analogue are opiate-like heptapeptides originally discovered in frog skin and characterized by the presence of a D-Ala2 residue in their sequence. They were assayed for their capacity to compete with [3H]Leu-enkephalin for binding to opioid receptors in membranes of neuroblastoma x glioma hybrid cells. In the presence of 7 nM-[3H]Leu-enkephalin, the concentrations at which they caused 50% inhibition of [3H]enkephalin binding (IC50 values) are 0.1 micro M and 0.3 micro M, respectively. In contrast, the synthetic L-Ala2-dermorphin shows very low affinity for the opioid receptors. In addition, like other opioid peptides, dermorphin and hyp6-dermorphin inhibit the elevation by prostaglandin E1 (PGE1) of the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) (IC50 values 0.2 micro M and 0.4 micro M, respectively). The inhibition is prevented by the opiate antagonist naloxone, L-Ala2-dermorphin is at least three orders of magnitude less potent in inhibiting the PGE1-evoked increase in the level of cyclic AMP. The results show that peptides with an amino acid sequence quite different from that of the enkephalins can bind to opioid receptors of the hybrid cells.