Water-protein NOEs: Optimized scheme for selective water excitation

An alternative scheme for selective water excitation is proposed. The pulse sequence saturates the resonances from the solute, allowing the observation of water-solute NOEs with low artifact levels. The water resonance is subsequently excited by a relatively non-selective 90° pulse. The scheme is compared to other selective water excitation schemes. 2D NOE-NOESY and ROE-NOESY pulse sequences are proposed which afford high sensitivity by efficient water excitation and flip-back by radiation damping, yet allow the use of short mixing times for the buildup of water-solute NOEs.     

Migration studies and histology of injectable microspheres of different sizes in mice

Injectable dermal filler materials consist of either fluids, biological fragments, or suspensions of particles or microspheres. Particles and microspheres are said to "migrate," but migration can occur only when they are injected into blood vessels. To evaluate biocompatibility and transport, five nonresorbable polymethylmethacrylate microspheres of various sizes, suspended in different carriers, as well as resorbable polylactic acid and dextran microspheres were injected subcutaneously into mice. The five implantation sites were the right cheek, right axilla, right groin, urethra, and the right quadriceps muscle of the thigh. These sites were excised along with the local lymph nodes, lungs, liver, and spleen at 1, 3, 6, and 9 months after injection. Polymethylmethacrylate microspheres of 4 microm and 8 microm were phagocytosed but not transported to lymph nodes or distant organs. Larger microspheres of 20, 40, and 100 microm were encapsulated by connective tissue, macrophages, and giant cells. Polylactic acid microspheres caused a mild inflammatory response and had disappeared at 6 months. Dextran microspheres caused a pronounced foreign-body reaction and were phagocytosed at 9 months. The extremely large carbon-coated spheres of 200 to 500 microm in diameter "migrated" up to 1 cm from the implantation site. With the exception of an erroneous intravenous injection, no migration or transportation of any of the injected microspheres to lymph nodes or filter organs was seen. Obviously, the collagen glue released no microspheres. After subdermal injection, the collagen carrier substance kept the microspheres apart as a scaffold for tissue ingrowth, whereas all other carrier substances, such as gelatin, hyaluronic acid, or alginate, separated soon after injection, thereby causing agglomeration of the microspheres.     

<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

Background  

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).     

Use of the mannitol pathway in fructose fermentation of<Emphasis Type="Italic"> Oenococcus oeni</Emphasis> due to limiting redox regeneration capacity of the ethanol pathway

The heterolactic bacterium Oenococcus oeni ferments fructose by a mixed heterolactic/mannitol fermentation. For heterolactic fermentation of fructose, the phosphoketolase pathway is used. The excess NAD(P)H from the phosphoketolase pathway is reoxidized by fructose (yielding mannitol). It is shown here that, under conditions of C-limitation or decreased growth rates, fructose can be fermented by heterolactic fermentation yielding nearly stoichiometric amounts of lactate, ethanol and CO(2). Quantitative evaluation of NAD(P)H-producing (phosphoketolase pathway) and -reoxidizing (ethanol, mannitol and erythritol pathways) reactions demonstrated that at high growth rates or in batch cultures the ethanol pathway does not have sufficient capacity for NAD(P)H reoxidation, requiring additional use of the mannitol pathway to maintain the growth rate. In addition, insufficient capacities to reoxidize NAD(P)H causes inhibition of growth, whereas increased NAD(P)H reoxidation by electron acceptors such as pyruvate increases the growth rate.     

Ketogenic Diet Increases Glutathione Peroxidase Activity in Rat Hippocampus

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost 80 years; however, we know little about the underlying biochemical basis of their action. In this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in antioxidant enzyme activities—glutathione peroxidase, catalase, and superoxide dismutase. In the hippocampus, however, we observed an increase in antioxidant activity accompanied by an increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels. We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocampus might contribute to protect this structure from neurodegenerative sequelae of convulsive disorders.     

NMR structure of Citrobacter freundii AmpD,comparison with bacteriophage T7 lysozyme and homology with PGRP domains

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

Solution structure of the R3H domain from human Smubp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.     

Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.