Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.     

QUANTITATIVE GENETICS OF RESPONSE TO COMPETITORS IN NEMOPHILA MENZIESII: A GREENHOUSE STUDY

To determine the potential for adaptation to a local biotic environment, we examined the magnitude and nature of genetic variation in response to neighboring plants within a natural population of the native California annual, Nemophila menziesii. A total of 22 plants from a natural population were crossed in three reciprocal factorials. The progeny were grown in a greenhouse in nine treatments that varied in conspecific density and in the density of a naturally co-occurring grass species, Bromus diandrus. Increasing the density of each species significantly reduced individual survival, fruit number, and dry weight. Among survivors, we found small to moderate heritability of dry weight within treatments. Additive genetic correlations (r_A) of dry weight between competitive regimes were generally large and positive. In no case were they significantly different from 1, as expected under the null hypothesis that the relative performance of the genotypes under consideration is the same in all environments. On the basis of these results, we cannot conclude that the structure of genetic covariation within this population would promote genetic differentiation in response to locally varying conditions of density of these two species. Aspects of the experiment that may have compromised our ability to detect r_A differing from 1 are discussed.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Mannuronan C-5 epimerase activity in protoplasts of Laminaria digitata

Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.     

Until death do us part? In-depth insights into Dutch consumers’ considerations about product lifetimes and lifetime extension

Long-lasting electronic products contribute to a sustainable society; however, both expected and actual lifetimes are in decline. This research provides in-depth insights into consumers’ considerations about product lifetimes, barriers to extending lifetimes, and responses to a product lifetime label. Results of interviews (n = 22) with Dutch consumers suggest a positive view on long-lasting products. Nevertheless, their products’ value depreciated during their lifetimes. Consumers consider themselves unable to estimate how long products should last, which can be detrimental as low expectations tend to negatively influence actual lifetimes. Also, use intensity and consumers’ care(less) behavior influence the lifetime. To extend product lifetimes, consumers often disregard the option of repairing malfunctioning products. They have limited knowledge and ability, and believe repair provides poor value for money. Lifetime extension can also be hindered by market-related factors, such as convenient replacement services, new technological developments, and (attractive) deals. We suggest a product lifetime label should contain relevant and reliable information; furthermore, we recommend including (extended) warranty information. When information about repairability is included, potential negative responses should be considered. Finally, raising awareness about the environmental impact of short-lived products via a label may have a positive effect but requires more research attention.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Transmission of poly(dT60) single-stranded DNA through polycarbonate track-etched ultrafiltration membranes

The objective of this study was to examine membrane filtration of a single stranded DNA (ssDNA) with 60 thymine nucleotides, and to elucidate the variables controlling its transmission across track-etched porous membranes. Dead end filtration measurements were performed using different pore size membranes (10, 15, and 30 nm) at different transmembrane pressures in solutions with ionic strength ranging from 0 to 1000 mM NaCl. The diffusivity of the ssDNA was determined using fluorescence recovery after photobleaching, yielding hydrodynamic radii ranging from 1.6 to 2.8 nm, with values decreasing with increasing solution ionic strength. Despite the small ssDNA/membrane pore size, nearly 100% rejection was observed for measurements performed with the 10 and 15 nm pore size membranes under low-ionic strength conditions. These high rejections can be attributed to strong repulsive electrostatic ssDNA-membrane interactions. With increasing ionic strength, electrostatic interactions as well as the effective size of the ssDNA decreases and the flexibility of the ssDNA increases, leading to a reduction in ssDNA rejection. A design of experiments approach was used to plan filtration experiments that adequately covered the variable space with a manageable number of experiments. The results yielded an empirical expression relating ssDNA rejection to pore size, solution ionic strength and transmembrane pressure. There was evidence of flow induced elongation at high-transmembrane pressures in the 30 nm pore size membranes, but not in the smaller pore size membranes. These results are consistent with critical flux estimates developed using a free draining model for the ssDNA.     

Beiträge zur Entwicklungsgeschichte vonSphaerularia bombi Dufour 1837

Ohne ZusammenfassungDiese Arbeit wurde durchgeführt mit freundlicher Unterstützung der Gesellschaft von Freunden und Förderern der Universität Bonn (Geffrub)