Visualizing biochemical activities in living cells through chemistry

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.     

Correspondence effects with manual gestures and postures: A study of imitation.

In this study, the authors applied methods and theories from research of stimulus–response compatibility (SRC) to action imitation. In 6 experiments, they adopted the logic of the Simon paradigm (B. Hommel & W. Prinz, 1996) to explore interference between task-relevant symbolic stimulus features (color) and task-irrelevant iconic stimulus features (2 hand gestures and 2 postures). The same 2 hand gestures served as responses. Pronounced correspondence effects for both gestures and postures showed up throughout. In line with theories of SRC, the authors account for these correspondence effects in terms of overlap arising between stimulus and response features in a common representational domain. As a specific extension of this approach, they propose 2 functionally independent mechanisms: One operates movement-based when dynamic information is provided, and the other operates state-based with static postures as stimuli. Implications for theories of both SRC and action imitation are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Chemometric Classification of Comb and Cuticular Waxes of the Honeybee Apis Mellifera Carnica

Waxes are important as building material and for the chemical communication of the honeybee Apis mellifera carnica. In this study chemometric tools were established for classifying the different waxes inside the hive. By using gas chromatography in combination with mass spectrometry, components of different types of waxes were analyzed. By considering different substance classes of waxes, discriminant function analyses revealed distinct subtypes of comb waxes and of cuticular waxes. It is shown that the aging of comb wax is in part a spontaneous physicochemical process due to differential volatilities of compound classes with different chain length ranges. On the other hand it is directly influenced by the bees by adding lipolytic enzymes to the comb wax. The data suggest that the varying cuticular wax and comb wax compositions could serve as cues for bees to recognize castes, sexes, or comb age.     

Combination of UDP‐Glc(NAc) 4′‐Epimerase and Galactose Oxidase in a One‐Pot Synthesis of Biotinylated Nucleotide Sugars

The enzymatic epimerization of uridine 5′‐diphospho‐α‐D ‐glucose (UDP‐Glc, 1 ) and uridine 5′‐diphospho‐N‐acetyl‐α‐D ‐glucosamine (UDP‐GlcNAc, 2 ) and the subsequent oxidation of uridine 5′‐diphospho‐α‐D ‐galactose (UDP‐Gal, 3 ) and uridine 5′‐diphospho‐N‐acetyl‐α‐D ‐galactosamine (UDP‐GalNAc, 4 ) were combined with chemical biotinylation with biotin‐ε‐amidocaproylhydrazide in a one‐pot synthesis. Analysis by CE and NMR revealed a mixture (1.0:1.4) of the biotinylated nucleotide sugars uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐α‐D ‐galactose (UDP‐6‐biotinyl‐Gal, 7) and uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐α‐D ‐glucose (UDP‐6‐biotinyl‐Glc, 9 ), respectively, in a reaction started with 1 . One product, uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐N‐acetyl‐α‐D ‐galactosamine (UDP‐6‐biotinyl‐GalNAc, 8) was formed when the reaction was initiated with 2 . It could be demonstrated for the first time that a UDP‐Glc(NAc) 4′‐epimerase (Gne from Campylobacter jejuni) and galactose oxidase from Dactylium dendroides can be used simultaneously in enzymatic catalysis. This is of particular interest since the coaction of an enzyme demanding reductive conditions and an oxygen‐dependent oxidase is unexpected.     

Dioxins,furans, and non-ortho PCBs in Canadian Arctic seabirds

This is the first account of PCDDs, PCDFs, and non-ortho PCBs in Canadian Arctic seabirds. Livers and eggs of thick-billed murres, northern fulmars, and black-legged kittiwakes were collected in 1975 and 1993 from Prince Leopold Island in Lancaster Sound, Canada. Detectable concentrations of PCDDs, PCDFs, and non-ortho PCBs were found in all the Arctic seabird samples analyzed. Of the PCDD congeners assayed, only 2,3,7,8-substituted PCDDs were detected in the samples, whereas non-2,3,7,8-substituted PCDFs were found in addition to 2,3,7,8-substituted PCDFs in some of the samples. The predominant PCDD/F congener found in the livers of all three species was 2,3,4,7,8-PnCDF, both in 1975 and 1993. Concentrations of most dioxins and furans decreased in the fulmars and kittiwakes between 1975 and 1993 but increased in the murres. Of the non-ortho PCBs measured, PCB-126 occurred in the highest concentrations and contributed the majority of the non-ortho PCB-TEQ in all three species in both years. The highest concentrations of dioxins and furans as well as the highest TEQ values were found in the northern fulmar livers in both 1975 and 1993. Concentrations of some of the PCDDs and PCDFs are among the highest reported for Canadian Arctic biota.     

Fabrication of diamond nanowires for quantum information processing applications

We present a design and a top-down fabrication method for realizing diamond nanowires in both bulk single crystal and polycrystalline diamond. Numerical modeling was used to study coupling between a Nitrogen Vacancy (NV) color center and optical modes of a nanowire, and to find an optimal range of nanowire diameters that allows for large collection efficiency of emitted photons. Inductively coupled plasma (ICP) reactive ion etching (RIE) with oxygen is used to fabricate the nanowires. Drop-casted nanoparticles (including Au, SiO₂ and Al₂O₃) as well as electron beam lithography defined spin-on glass and evaporated Au have been used as an etch mask. We found Al₂O₃ nanoparticles to be the most etch resistant. At the same time FOx e-beam resist (spin-on glass) proved to be a suitable etch mask for fabrication of ordered arrays of diamond nanowires. We were able to obtain nanowires with near-vertical sidewalls in both polycrystalline and single crystal diamond. The heights and diameters of the polycrystalline nanowires presented in this paper are ≈ 1 μm and 120–340 nm, respectively, having a 200 nm/min etch rate. In the case of single crystal diamond (types Ib and IIa) nanowires the height and diameter for different diamonds and masks shown in this paper were 1–2.4 μm and 120–490 nm with etch rates between 190 and 240 nm/min.     

Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen^®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ⁰ cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ⁰ cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.