Biosynthesis and use of coproporphyrins and uroporphyrins and their metal complexes in immune analysis and diagnostic methods]

Methods of synthesis of coproporphyrin and uroporphyrin by using bacteria of the genus Arthrobacter are proposed. Metal complexes of coproporphyrin and uroporphyrin with Pt, Pd, and Zn were synthesized. Their structures were identified by spectrophotometry, IR spectrometry, 1H-NMR, mass spectrometry, and HPLC. Data showing the possibility to use coproporphyrin III-metal complexes as luminophores for fluorescence detection of tumors. The current and prospective uses of metal complexes of water-soluble natural porphyrins in advanced immunofluorescence assays are discussed.     

Changes in Wheat Leaf Extracellular Proteolytic Activity after Infection with Septoria tritici

The proteolytic activity of the leaf extracellular space of wheat cultivars Pigüé and Isla Verde was estimated after inoculation of either detached leaves or plants with the fungus Septoria tritici. Pigüé is resistant, whereas Isla Verde is susceptible to the disease caused by S. tritici. Viable conidiospores of the fungus caused similar increases in both hydrogen peroxide production and chitinase activity of the cultivars studied. In contrast, they caused a decrease in the extracellular serine proteinase activity of Isla Verde and a significant increase in that of Pigüé. Independently of the cultivar from which it was extracted, the extracellular serine proteinase inhibited the germination of Septoria tritici conidiospores. These results suggest that the proteolytic activity of the leaf extracellular space can participate in the defence of wheat plants against Septoria tritici. Its regulation may be controlled by specific defence components of each cultivar.     

A variable minisatellite sequence in the chloroplast genome of Sorbus L. (Rosaceae: Maloideae).

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.     

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Phylogenetic analysis of the Rhabdocoela (Platyhelminthes) with emphasis on the Neodermata and relatives

Phylogenetic systematic analysis of 24 taxa representing the rhabdocoel platyhelminths, based on a suite of 89 morphological characters, produced two equally parsimonious trees, 181 steps long, with a consistency index (CI) of 0.69 and a rescaled consistency index (RCI) of 0.56, differing only with respect to that portion of the tree containing Umagillidae, Acholadidae, Graffillinae, Pseudograffillinae, Pterastericolidae and Hypoblepharinidae. Our results accommodate all previously proposed sister taxa to the Neodermata in a single clade in which ((Dalyelliidae + Temnocephalida) Typhloplanidae) is the sister group of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))). Bootstrap and jackknife analyses indicate that the groupings of ((Dalyelliidae + Temnocephalida) Typhloplanidae) and of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))) are highly robust, with the latter clade having a CI of 90% and RCI of 82%. Disagreements among previous analyses of these taxa have been due to the influence of missing data for critical characters in key taxa and differences in the taxa analysed, rather than any inherent weakness in the morphological data. Non-phylogenetic systematic approaches to homology assessment and misconceptions regarding phylogenetic systematic methodology are discussed. Recent analyses combining sequence data with a subset of approximately 60% of the morphological characters should be re-assessed using the entire morphological database. Even if Udonella is a monogenean, it is most parsimonious to suggest that the common ancestor of the Neodermata had a vertebrate–arthropod two-host life cycle.     

An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes.

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome-derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p-Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N-terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p-independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.     

Body Talk: Rhetoric, Technology, Reproduction

Body Talk: Rhetoric, Technology, Reproduction. Mary M. Lay. Laura J. Gurak. Clare Gravon. and Cynthia Myntii. eds. Madison: University of Wisconsin Press, 2000. xiii. 307 pp.     

DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids.

Helicases not only catalyse the disruption of hydrogen bonding between complementary regions of nucleic acids, but also move along nucleic acid strands in a polar fashion. Here we show that the Rep52 and Rep40 proteins of adeno-associated virus type 2 (AAV-2) are required to translocate capsid-associated, single-stranded DNA genomes into preformed empty AAV-2 capsids, and that the DNA helicase function of Rep52/40 is essential for this process. Furthermore, DNase protection experiments suggest that insertion of AAV-2 genomes proceeds from the 3' end, which correlates with the 3'-->5' processivity demonstrated for the Rep52/40 helicase. A model is proposed in which capsid-immobilized helicase complexes act as molecular motors to 'pump' single-stranded DNA across the capsid boundary.     

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.