Novel biocatalysts for white biotechnology

White Biotechnology uses microorganisms and enzymes to manufacture a large variety of chemical products. Therefore, the demand for new and useful biocatalysts is steadily and rapidly increasing. We have developed methods for the isolation of new enzyme genes, constructed novel expression systems, and optimized existing enzymes for biotechnological applications by methods of directed evolution. Furthermore, we have isolated and characterized biocatalysts relevant for the preparation of enantiopure compounds.     

Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome

Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.     

The chemical composition of throughfall beneath oak, birch and pine canopies in Northwest Germany

The chemical composition of rainwater is altered upon its passage through tree canopies. In order to investigate how rainwater chemistry is affected by canopy-dependent processes in characteristic forest types of Northwest German sandy lowland regions – oak–birch-forests, Betula pubescens Ehrh. swamp forests, and stands of Pinus sylvestris L. – comparative studies on the chemical composition of throughfall were carried out at seven forest sites, situated in close proximity within a nature reserve. Additionally, rainwater was sampled at three heathland sites for analysis of open-field precipitation and at three sites along an oak–birch-forest edge. Throughfall concentrations of most of the parameters analysed were significantly higher than open-field concentrations, especially with regard to electric conductivity, NH₄-N, K⁺, and KMnO₄-index. Ion concentrations in throughfall were the lowest in a 10-year-old stand of Betula pendula Roth. and Pinus sylvestris and in a Betula pubescens swamp forest and were highest beneath a stand of Pinus sylvestris. Except for Na⁺, Cl⁻, and NO₃⁻, ion concentrations in both throughfall and open-field precipitation increased during the growing season (May–October). In throughfall, Ca²⁺, Mg²⁺, K⁺, and Mn²⁺ were strongly correlated. Enrichment ratios between throughfall and open-field deposition varied among sites and elements and were the highest for K^‰+, Mg^2‰+, and Mn^2‰+. Estimates of canopy leaching indicated high leaching rates of K^‰+ and Mn^2‰+ and moderate leaching of Mg^2‰+. The contribution of foliar leaching to throughfall deposition was higher at the deciduous than at the coniferous stands.     

New group of xylene linker-containing acetylcholinesterase reactivators as antidotes against the nerve agent cyclosarin

Nerve agents such as sarin, cyclosarin and tabun are organophosphorus substances able to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None of the known AChE reactivators is able to reactivate AChE inhibited by all nerve agents used. In this work, reactivation potency of nine newly developed AChE reactivators with an incorporated xylene ring in their structure was measured in vitro. Cyclosarin was chosen as an appropriate member of the nerve agent family. Reactivation potency of the tested AChE reactivators was compared with the gold standard of AChE reactivators--pralidoxime. Two oximes (K107 and K108) surpassed the reactivation potency of pralidoxime. Moreover, from the obtained results it could be deduced that AChE reactivators with a functional oxime group in position-2 are the most potent AChE reactivators in the case of cyclosarin intoxications.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.     

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.     

Sporulation as an effective strategy for the survival of bacteria in a biofilter with discontinuous operation

The microbial population of the biofilter developed for the treatment of air contaminated by solvent vapours was evaluated using plate count techniques. The number of total heterotrophic bacteria and bacteria utilizing contaminant as the only source of carbon and energy was estimated during the exchange of the filter-bed material and start-up of its operation. Spore-forming bacteria (Bacillus spp.) occupied a significant part of the filter-bed niche. It is proposed that sporulation helps bacteria to survive during the breaks in operation of the biofilter.     

One Step Isolation of Bovine Asialoglycoprotein Receptor and Its Characterization by Sequence Analysis and MALDI Mass Spectrometry

The asialoglycoprotein receptor (ASGP-R), which is responsible for the uptake of partially deglycosylated serum glycoproteins was isolated from bovine liver. The receptor was purified in one step from solubilized plasma membranes by affinity chromatography on 6-(-D-lactosyl)-n-hexylamine coupled to N-hydroxysuccinimide activated Sepharose with a coupling degree of 7.6 mol/ml gel. The preparation yielded two distinct polypeptides with apparent molecular weights of 48 and 43 kDa as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. A polyclonal antibody raised against the human ASGP-R recognized the bovine 43 kDa protein in Western blot analysis. The 48 and 43 kDa polypeptides were digested by trypsin and the digests were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Sequence analysis of four tryptic fragments, two each of the 48 kDa and of the 43 kDa polypeptides revealed that these were highly homologous to ASGP-R subunits from man, mouse and rat.