A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.     

μ3-S bonding mode in H2M3(CO)9S clusters of Ru and Os. An UVPES and theoretical study

The electronic structure of H₂M₃(CO)₉S clusters (M = Ru, Os) is discussed on the basis of their He I and He II excited gas-phase photoelectron spectra and on the basis of CNDO quantum mechanical calculations. The PE data clearly demonstrate the cleavage of two direct MM interactions by operation of the bridging hydrides, giving rise to three-center two-electron MHM levels. The μ₃-S bonding mode has been described in detail and compared with previous results on related μ₃-CY cluster derivatives. The CNDO results on Ru₃(CO)₉S⁼, HRu₃(CO)₉S^? and H₂Ru₃(CO)₉S indicate that the μ₃-S—cluster interaction is mostly independent of the presence of the bridging hydrides.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

Early steps of clathrin-mediated endocytosis involved in phagosomal escape of Fcgamma receptor-targeted adenovirus

Adenovirus type 2 (Ad2) and Ad5 enter epithelial cells via the coxsackievirus B Ad receptor (CAR) and alpha(v) integrin coreceptors. In the absence of CAR, they can be directed to the Fcgamma receptor 1 of hematopoietic cells by an adaptor comprising the extracellular CAR domain and the Fc portion of a human immunoglobulin G (CARex-Fc). This gives rise to Ad aggregates and single particles which together enhance gene delivery up to 250-fold compared to adaptor-less viruses. A small interfering RNA knockdown of the clathrin heavy chain and quantitative electron microscopy of hematopoietic leukemia cells showed that the majority of Ads were phagocytosed as clusters of 1 to 3 microm in diameter and that about 10% of the particles entered cells by clathrin-mediated endocytosis. The clathrin knockdown did not affect phagocytosis but, surprisingly, inhibited viral escape from phagosomes. Similarly, blocking an early stage of clathrin-coated pit assembly inhibited phagosomal escape and infection but not aggregate uptake, unlike blocking of a late stage of clathrin-coated pit formation. We propose a cooperative interaction of clathrin-mediated endocytosis and phagocytosis triggering phagosomal lysis and infection.     

Toxicogenomics in the pharmaceutical industry: hollow promises or real benefit?

Almost 10 years ago, microarray technology was established as a new powerful tool for large-scale analysis of gene expression. Soon thereafter the new technology was discovered by toxicologists for the purpose of deciphering the molecular events underlying toxicity, and the term "Toxicogenomics" appeared in scientific literature. Ever since, the toxicology community was fascinated by the multiplicity of sophisticated possibilities toxicogenomics seems to offer: genome-wide analysis of toxicant-induced expression profiles may provide a means for prediction of toxicity prior to classical toxicological endpoints such as histopathology or clinical chemistry. Some researchers even speculated of the classical methods being superfluous before long. It was assumed that by using toxicogenomics it would be possible to classify compounds early in drug development and consequently save animals, time, and money in pre-clinical toxicity studies. Moreover, it seemed within reach to unravel the molecular mechanisms underlying toxicity. The feasibility of bridging data derived from in vitro and in vivo systems, identifying new biomarkers, and comparing toxicological responses "across-species" was also excessively praised. After several years of intensive application of microarray technology in the field of toxicology, not only by the pharmaceutical industry, it is now time to survey its achievements and to question how many of these wishes and promises have really come true.     

5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones, a new series of potent, broad-spectrum non-nucleoside reverse transcriptase inhibitors belonging to the DABO family

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F(2)-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C(2)-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C(2)-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C(5)-position and the other at the benzylic carbon), being thymine, alpha-methyluracil or alpha-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-alpha-methylthymine derivatives 4d, 5h'-n' showed the highest potency and selectivity among tested compounds, both a properly sized C(2)-NH side chain and the presence of two methyl groups (at C(5) and benzylic positions) being crucial for high antiviral action.     

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.     

Redox equilibrium in mucosal T cells tunes the intestinal TCR signaling threshold

Mucosal immune tolerance in the healthy intestine is typified by lamina propria T cell (LPT) functional hyporesponsiveness after TCR engagement when compared with peripheral blood T cell (PBT). When LPT from an inflamed intestine are activated through TCR cross-linking, their responsiveness is stronger. LPT are thus capable of switching from a tolerant to a reactive state, toggling between high and low thresholds of activation. We demonstrate that in normal LPT global tyrosine phosphorylation upon TCR cross-linking or an increase in intracellular H2O2, an inhibitor of protein tyrosine phosphatases, is muted. Thus, we propose that LPT have a greater reducing capacity than PBT, shifting the balance between kinases and protein tyrosine phosphatases in favor of the latter. Surface gamma-glutamyl transpeptidase, an indirect indicator of redox potential, and glutathione are significantly elevated in LPT compared with PBT, suggesting that elevated glutathione detoxifies TCR-induced reactive oxygen species. When glutathione is depleted, TCR-induced LPT tyrosine phosphorylation rises to PBT levels. Conversely, increasing glutathione in PBT attenuates tyrosine phosphorylation. In LPT isolated from inflamed mucosa, TCR cross-linking induces greater phosphorylation, and gamma-glutamyl transpeptidase levels are reduced compared with those from autologous noninflamed tissue. We conclude that the high TCR signaling threshold of mucosal T cells is tuned by intracellular redox equilibrium, whose dysregulation may mediate intestinal inflammation.