Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zemplén.     

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Haplotype, which is the sequence of SNPs in a specific chromosome, plays an important role in disease association studies. However, current sequencing techniques can detect the presence of SNP sites, but they cannot tell which copy of a pair of chromosomes the alleles belong to. Moreover, sequencing errors that occurred in sequencing SNP fragments make it difficult to determine a pair of haplotypes from SNP fragments. To help overcome this difficulty, the haplotype assembly problem is defined from the viewpoint of computation, and several models are suggested to tackle this problem. However, there are no freely available web-based tools to overcome this problem as far as we are aware. In this paper, we present a web-based application based on the genetic algorithm, named HapAssembler, for assembling a pair of haplotypes from SNP fragments. Numerical results on real biological data show that the correct rate of the proposed application in this paper is greater than 95% in most cases. HapAssembler is freely available at http://alex.chonnam.ac.kr/~drminor/hapHome.htm. Users can choose any model among four models for their purpose and determine haplotypes from their input data.     

Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome‐derived esterases

Enantiomerically pure β-arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate-based screening using a pH-indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.     

Immunocytochemical localisation of the icosapeptide fragment of the PP precursor: a marker for ‘true’ PP cells?

Antisera were raised against the icosapeptide fragment of the pancreatic polypeptide (PP) isolated from the canine pancreas. They were used for the immunocytochemical study of the cellular localisation and distribution of the icosapeptide in the gut and pancreas of various mammals. The results indicate that PP and the icosapeptide coexist in the majority of the PP-immunoreactive cells in the pancreas of cat, dog, pig, monkey and man and in all the PP-immunoreactive cells in the stomach of the cat and dog. The icosapeptide does not seem to occur in cells or nerves containing PP-related peptides, such as peptide YY or neuropeptide Y. PP-immunoreactive cells devoid of the icosapeptide could be demonstrated in the large intestine. These cells are probably distinct from the pancreatic PP cell type, and the PP-immunoreactive material probably represents the homologous peptide YY rather than PP. The present findings support the view that the icosapeptide is part of the PP precursor and hence, only the cells containing immunoreactive icosapeptide in addition to immunoreactive PP are to be considered ‘true’ PP cells. The icosapeptide antisera did not stain PP cells in mouse, rat and guinea-pig, suggesting marked species variation in the amino acid sequence of the icosapeptide portion of the PP precursor.     

Comparing different approaches to calculate the effects of heterogeneous root distribution on nutrient uptake: a case study on subsoil nitrate uptake by a barley root system

Simulation models of nutrient uptake of root systems starting with one-dimensional single root approaches up to complex three-dimensional models are increasingly used for examining the interacting of root distribution and nutrient uptake. However, their accuracy was seldom systematically tested. The objective of the study is to compare one-dimensional and two-dimensional modelling approaches and to test their applicability for simulation of nutrient uptake of heterogeneously distributed root systems giving particular attention to the impact of spatial resolution. Therefore, a field experiment was carried out with spring barley (Hordeum vulgare L. cv. Barke) in order to obtain data of in situ root distribution patterns as model input. Results indicate that a comparable coarse spatial resolution can be used with sufficient modelling results when a steady state approximation is applied to the sink cells of the two-dimensional model. Furthermore, the accuracy of the model was clearly improved compared to a simple zero sink approach assuming both near zero concentrations within the sink cell and a linear gradient between the sink cell and its adjacent neighbours. However, for modelling nitrate uptake of a heterogeneous root system a minimum number of grid cells is still necessary. The tested single root approach provided a computational efficient opportunity to simulate nitrate uptake of an irregular distributed root system. Nevertheless, two-dimensional models are better suited for a number of applications (e.g. surveys made on the impact of soil heterogeneity on plant nutrient uptake). Different settings for the suggested modelling techniques are discussed.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we discovered an unexpected role for yeast dynein light chain (Dyn2) in the NPC. Dyn2 is a previously undescribed nucleoporin that functions as molecular glue to dimerize and stabilize the Nup82-Nsp1-Nup159 complex, a module of the cytoplasmic pore filaments. Biochemical analyses showed that Dyn2 binds to a linear motif (termed DID(Nup159)) inserted between the Phe-Gly repeat and coiled-coil domain of Nup159. Electron microscopy revealed that the reconstituted Dyn2-DID(Nup159) complex forms a rigid rod-like structure, in which five Dyn2 homodimers align like 'pearls on a string' between two extented DID(Nup159) strands. These findings imply that the rigid 20 nm long Dyn2-DID(Nup159) filament projects the Nup159 Phe-Gly repeats from the Nup82 module. Thus, it is possible that dynein light chain plays a role in organizing natively unfolded Phe-Gly repeats within the NPC scaffold to facilitate nucleocytoplasmic transport.