Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

NOCICEPTIVE RESPONSES IN INTERLEUKIN-6-DEFICIENT MICE TO PERIPHERAL INFLAMMATION AND PERIPHERAL NERVE SECTION

The cutaneous nociceptive response threshold to mechanical and thermal stimulation, the development of hyperalgesia and plasma extravasation after subcutaneous injection of carrageenan and the development of autotomy behaviour after nerve section were assessed in interleukin-6-deficient (IL-6^−/−) and age-matched wild-type (IL-6^+/+) mice. IL-6^−/−mice had significantly lower response threshold to both mechanical and thermal stimulation in comparison to IL-6^+/+controls. Both IL-6^−/−and IL-6^+/+mice developed hyperalgesia to mechanical and thermal stimulation after localized carrageenan injection, but the magnitude of the hyperalgesia was less in the IL-6^−/−than in the IL-6^+/+controls. IL-6^−/−mice also exhibited less plasma extravasation after carrageenan injection. No difference was noted between males and females in basal nociception and inflammatory hyperalgesia. However, female IL-6^−/−mice exhibited autotomy behaviour, a sign of neuropathic pain, significantly more frequently and after a shorter interval following peripheral nerve injury than male IL-6^−/−or male and female IL-6^+/+mice. It is suggested that IL-6^−/−mice exhibited numerous changes in nociceptive responses compared to controls, some of which are sex related. The mechanisms of these changes in relation to null-mutation of the IL-6 gene and the influence of genetic background are discussed.     

Fast phototransformation of the 636 nm-emitting protochlorophyllide form in epicotyls of dark-grown pea (Pisum sativum)

The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m⁻² s⁻¹) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m⁻² s⁻¹) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.     

<Emphasis Type="Italic">Rhodiola rosea</Emphasis> L.: from golden root to green cell factories

Rhodiola rosea L. is a worldwide popular plant with adaptogenic activities that have been and currently are exploited in the traditional medicine of many countries, as well as, examined in a number of clinical trials. More than 140 chemical structures have been identified which belong to several natural product classes, including phenylpropanoid glycosides, phenylethanoids, flavonoids and essential oils, and are mainly stored in the rhizomes and the roots of the plant. A number of mechanisms contribute to the adaptogenic activities of R. rosea preparations and its phytochemical constituents. Among them, the intrinsic inducible mammalian stress responses and their effector proteins, such as heat shock protein 70 (Hsp70), are the most prominent. Due to its popular medicinal use, which has led to depletion of its natural habitats, R. rosea is now considered as endangered in most parts of the world. Conservation, cultivation and micropropagation are all implemented as potential preservation strategies. A number of in vitro systems of R. rosea are being developed as sources of pharmaceutically valuable secondary metabolites. These are greatly facilitated by advances in elucidation of the biosynthetic pathways and the enzymes, which catalyse the production of these secondary metabolites in the plant. In addition, biotechnological approaches show promise towards achieving sustainable production of R. rosea secondary metabolites.     

Evidence for the localization of hydrogen peroxide-stimulated cyclooxygenase activity in rat brain mitochondria: a possible coupling with monoamine oxidase

The distribution of basal and of H2O2-stimulated cyclooxygenase activity in the primary fractions of rat brain homogenates and in the subfractions of crude mitochondrial fraction was studied. For comparison, the localization of H2O2-generating monoamine oxidase (MAO) as well as that of the mitochondrial marker succinate dehydrogenase (SDH) was also examined. H2O2 was generated by MAO using 5 x 10(-4) M noradrenaline (NA) or 2 x 10(-4) M 2-phenylethylamine (PEA) as substrates, or by 25 micrograms glucose oxidase (GOD) per ml in the presence of 1 mM glucose. For nonstimulated (basal) cyclooxygenase, the relative specific activity (RSA) was high in microsomes (1.79) and in the free mitochondria-containing subfraction of the crude mitochondrial fraction (1.94). Parallel distribution of MAO and H2O2-stimulated cyclooxygenase was observed in all fractions studied in the presence of NA. The highest RSA was found in the purified mitochondria for both enzymes (1.85 for MAO and 1.97 for H2O2-stimulated cyclooxygenase). The enrichment of SDH (RSA = 2.21) indicated a high concentration of mitochondria in this fraction. The same distribution of H2O2-stimulated cyclooxygenase was obtained when, instead of the MAO-NA system, hydrogen peroxide was generated by GOD in the presence of glucose. H2O2 generated by deamination of NA or PEA by MAO, or during the enzymatic oxidation of glucose by GOD, caused a threefold increase in mitochondrial endoperoxide formation. Indomethacin (2 x 10(-4) M), catalase (50 micrograms/ml), and pargyline (2 x 10(-4) M) eliminated the MAO-dependent mitochondrial synthesis of PG endoperoxides. The GOD-dependent cyclooxygenase activity in this fraction was abolished by indomethacin or catalase, but not by pargyline. The results show the existence of a mitochondrial cyclooxygenase in brain tissue. The enzyme is sensitive to H2O2 and produces prostaglandin endoperoxides from an endogenous source of arachidonic acid. The identical localization of H2O2-producing MAO and H2O2-sensitive cyclooxygenase suggests a possible coupling between monoamine and arachidonic acid metabolism.     

Synthesis, and structural and biological studies of efrapeptin C analogues

A series of analogues of efrapeptin C (1), with variations in the central tripeptide epitope (positions 6-8), were prepared by a combination of solid- and solution-phase peptide syntheses. The conformations of the modified compounds 2-6 were investigated by circular-dichroism (CD) spectroscopy to differentiate between 3(10)- and alpha-helical secondary structures. The inhibitory activities of the new compounds towards F(1)-ATPase from E. coli were determined. The modified congeners 3-5 were less active by one order of magnitude compared to 1 (K(i) 10 microM), and 6 was completely inactive. Our experiments demonstrate that the flexible, central tripeptide epitope, comprising positions 6-8 in 1, is crucial for molecular recognition, even slight sequence modifications being hardly tolerated.     

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for “antler” genes that encode α-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.     

Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila

The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) proteins in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans‐Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. Here we show that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, we show that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation‐independent mannose 6‐phosphate receptor. Finally, we find that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells.