A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

Direct Synthesis of Poly(dimethylsiloxane) Copolymers with TPE-Properties via CuAAC (Click Chemistry).

Poly(dimethylsiloxane) copolymers were synthesized directly from AA/BB monomers employing a CuAAC reaction (click chemistry) in a polyaddition approach. Using organic dialkynes and oligo(siloxane)s end-functionalized with azide moieties it was possible to obtain siloxane-based copolymers with TPE properties by click chemistry for the first time. As seen from DSC experiments, properties were strongly dependent on the incorporated organic comonomer.

Fluidic Self-Assembly on Electroplated Multilayer Solder Bumps with Tailored Transformation Imprinted Melting Points.

This communication presents fluidic self-assembly of Si-chip on a sequentially electroplated multilayer solder bump with tailored transformation imprinted melting points. The multilayer solder bump is a lead free ternary solder system, which provides a route to transform the melting point of interconnects for applications in solder directed fluidic self-assembly. The outermost metal layers form a low melting point Bi33.7In66.3 solder shell (72 °C). This solder shell enables fluidic self-assembly and self-alignment of freely in water suspended Si-dies at relatively low temperature (75 °C) leading to well-ordered chip arrays. The reduction of the free surface energy of the shell-water interface provides the driving force for the self-assembly. The lowermost metal layer is a high melting point solder and acts as a core. After the self-assembly is complete, a short reflow causes the transformation of the core and the shell yielding a stable high melting point solder with adjustable melting points. The chosen ternary solder system enables the realization of interconnects with melting points in the range of 112 °C to 206 °C.

Core-Shell Transformation-Imprinted Solder Bumps Enabling Low-Temperature Fluidic Self-Assembly and Self-Alignment of Chips and High Melting Point Interconnects.

We demonstrate the realization of core-shell transformation-imprinted solder bumps to enable low-temperature chip assembly, while providing a route to high-temperature interconnects through transformation. The reported core-shell solder bump uses a lower melting point BiIn-based shell and a higher melting point Sn core in the initial stage. The bumps enable fluidic self-assembly and self-alignment at relatively low temperatures (60-80 °C). The bumps use the high surface free energy of the liquid shell during the self-assembly to capture freely suspended Si dies inside a heated (80 °C) water bath, leading to well-ordered defect-free chip arrays; the molten liquid shell wets the metal contact (binding site) on the chips and yields self-aligned and electrically connected devices. The solid core provides the anchor point to the substrate. After the completion of the assembly, a short reflow raises the melting point, yielding a solid electrical connection. The low melting point liquid diffuses into the high melting point core. The tuning of the material ratios leads to tailored transformation-imprinted solders with high melting points (160-206 °C) in the final structure.

Systematic screening of nuclear encoded proteins involved in the splicing metabolism of group II introns in yeast mitochondria.

Studies of yeast, algae and plants have provided genetic and biochemical evidence that the splicing reaction of organellar localized group II introns either depends on proteins encoded by the introns themselves ('maturases') or encoded by other genes of the host organisms. However, only a few of those proteins have been identified to date and characterized in more detail. In order to find new nuclear encoded proteins that assist group II splicing, we screened a complete knockout library of Saccharomyces cerevisiae strain BY4741 consisting of 4878 viable haploid clones. The strain contains a rho+ mitochondrial genome with a set of 13 introns including the three group II introns (aI1, aI2, aI5gamma) in the gene encoding cytochrome-c-oxidase subunit 1 (COX1) and the single group II intron (bI1) in the gene encoding cytochrome b (CYTB). In our screen and initial molecular analysis, we focus on intron aI5gamma, the last intron in the COX1 gene.

Hibernation model of tau phosphorylation in hamsters: selective vulnerability of cholinergic basal forebrain neurons - implications for Alzheimer's disease.

Neurofibrillar tangles made up of 'paired helical filaments' (PHFs) consisting of hyperphosphorylated microtubule-associated protein tau are major hallmarks of Alzheimer's disease (AD). Tangle formation selectively affects certain neuronal types and systematically progresses throughout numerous brain areas, which reflects a hierarchy of neuronal vulnerability and provides the basis for the neuropathological staging of disease severity. Mechanisms underlying this selective neuronal vulnerability are unknown. We showed previously that reversible PHF-like phosphorylation of tau occurs during obligate hibernation. Here we extend these findings to facultative hibernators such as Syrian hamsters (Mesocricetus auratus) forced into hibernation. In this model, we showed in the basal forebrain projection system that cholinergic neurons are selectively affected by PHF-like phosphorylated tau, while gamma-aminobutyric acid (GABA)ergic neurons are largely spared, which shows strong parallels to the situation in AD. Formation of PHF-tau in these neurons apparently does not affect their function as pacemaker for terminating hibernation. We conclude that although formation of PHF-like phosphorylated tau in the mammalian brain follows a certain hierarchy, affecting some neurons more frequently than others, it is not necessarily associated with impaired neuronal function and viability. This indicates a more general link between PHF-like phosphorylation of tau and the adaptation of neurons under conditions of a 'vita minima'.

Group II introns: structure and catalytic versatility of large natural ribozymes.

Group II introns are large, natural catalytic RNAs or ribozymes that were discovered in organelles of certain protists, fungi, algae, and plants and more recently also in prokaryotic organisms. In vitro, some members were found to self-splice from their pre-RNAs by two consecutive transesterification reactions joining the flanking exons and releasing the intron in a typical lariat form. Apart from self-splicing, a variety of other in vitro activities have been detected for group II introns demonstrating their amazing catalytic versatility. Group II introns fold into a conserved secondary structure consisting of six domains radiating from a central wheel that brings the 5' and 3' splice junction into close proximity. Domain 1 is the largest domain that is assumed to deliver the molecular scaffold assembling the intron in its active structure, while domain 5 is the phylogenetically most conserved part that represents the active site of the ribozyme. In vivo, the splicing reaction of many, if not all group II introns is assisted by proteins either encoded by the introns themselves (maturases), or encoded by other genes of the host organisms. The host proteins known to date have additional cellular functions and seem to have been adapted for splicing during evolution. Some of the protein-encoding group II introns were also shown to act as mobile genetic elements. They can integrate efficiently into intronless alleles of the same gene (homing) and at much lower frequencies into ectopic sites (transposition). The mobility process depends on intron encoded protein functions (endonuclease and reverse transcriptase) and on the intron RNA. This review provides a comprehensive survey of the structure/function relationships and the reaction potential of group II introns, the structurally most complicated, but also most fascinating ribozymes when looking at their catalytic repertoire in vitro and in vivo.

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae.

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).