Interplay between glutathione,Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu₂^I(GS⁻)₂(Atx1)₂, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu₂^I(GS⁻)₂(Atx1)₂ is the major conformation of Atx1 in the cytosol.     

Morphogenetic deficiencies in transplanted gonadal anlagen of the mutant l(3)pl (lethal-polyploid) in Drosophila hydei

Larval gonads of Drosophila hydei, homozygous for the lethal gene l(3)pl (lethal-polyploid), were cultured in normal hosts. Ovaries of the late third larval instar were implanted into metamorphosing larvae. These can attach to the gonoduct system of the host and transform into adult ovarian structures but the spectrum of their capacity to differentiate varies largely. In favourable cases mature oocytes can be formed which are fertile. More frequently mitotic disturbances in the follicle cells and cystocytes lead to the formation of abortive egg chambers and abnormally shaped oocytes. Testes of the middle third larval instar were cultured for 2 weeks in adult females. Primary spermatocytes are able to sustain meiotic divisions and form early spermatids, even though the occurrence of fractionated nuclei in post-meiotic germ cells indicates defective meiotic divisions. Post-meiotic differentiation is blocked in mutant spermatids which fail to elongate. The mutant gene l(3)pl thus, not only affects cell divisions, but also interacts in certain cytodifferentiation processes such as spermatid elongation and egg shaping. All cellular processes found so far to be abnormal in mutant tissues involve microtubular function. This suggests that the gene l(3)pl interacts with the microtubular system and several aspects of this interpretation are discussed.     

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

Hinokinin biosynthesis in Linum corymbulosum Reichenb

Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (−)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (−)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol–lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo , we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase ( PLR-Lc1 ), two phenylcoumaran benzylic ether reductases ( PCBER-Lc1 and PCBER-Lc2 ), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (−)-secoisolariciresinol, which can be further converted to give (−)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (−)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (−)-secoisolariciresinol.     

First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA

Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO(2)(-) to NO(3)(-) in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrA-like sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.     

Microbial activity of suspended biomass from a nitritation–anammox SBR in dependence of operational condition and size fraction

Single-stage nitritation–anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation–anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg?N?m^?3?day^?1). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 μm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH₄ ⁺–N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100–315 μm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.     

Growth and cellular organization of Arabidopsis pollen tubes in vitro

Tricellular pollen tubes of Arabidopsis thaliana were cultured in vitro on solid media and studied with respect to growth, cellular organization and ultrastructure, cytoskeletal organization, organelle movement, deposition and structure of the wall and the occurrence of coated pits, all elements assumed to be relevant for tip growth. For our ultrastructural studies we used freeze fixation and freeze substitution. Although Arabidopsis pollen tubes are broadly similar to those of bicellular species such as Nicotiana tabacum and Lilium spec. and in vivo grown pollen tubes of Arabidopsis, some differences occurred. The density of the equally distributed, relatively small (85 nm) secretory vesicles (SV) in the tip is low (five/µm ²). In between the SV of the tip, membranous material, possibly smooth endoplasmic reticulum, fragments of rough endoplasmic reticulum and loose ribosomes are present. The wall in the tip is not amorphous but layered and a secondary wall is formed already in the flanks of the tip. The general pattern of organelle motion is reverse fountain-like, but individual organelles move in distinct lanes at speeds of up to 2 µm/s, and about half of the organelle population shows a moderate velocity or Brownian movement. These properties are discussed in relation to the low growth rate (10 µm/h) of Arabidopsis pollen grown in vitro. The two similar sperm cells are closely attached and are always found near the vegetative nucleus. No surrounding wall and no cytoskeletal elements were obvious in the sperm cells. The preferential location of the mitochondria at the wall and the large (up to 400 nm) coated pits are unique for angiosperm pollen tubes. The size of the coated pits may allow not only membrane retrieval but also pinocytosis.