Control of calcium accessibility over hydroxyapatite by post-precipitation steps: influence on the catalytic reactivity toward alcohols.

Hydroxyapatites are increasingly used as heterogeneous catalysts since they present atypical behaviours for many acid base reactions. The aim of this study was to discuss the possible involvement of Ca2+ Lewis and/or PO-H Brønsted acid sites belonging to the hydroxyapatite system in the conversion of 2-methylbut-3-yn-1-ol, a model molecule that is known to account for the acid base properties, and of ethanol into n-butanol. A series of hydroxyapatite samples with similar bulk properties was prepared from a lone precipitation batch, but by varying the conditions of the washing and drying steps. Although the surface depth probed by XPS exhibited similar average composition, ISS analysis revealed a gradient of calcium concentration in the first surface layers. In fact, the different conditions of drying and washing resulted in a modulation of the relative amount of Ca2+ and PO-H accessible on the top surface, as revealed by the adsorption of the CO molecule monitored by FTIR. The conversion in the two alcohol molecules is linearly dependent on the nature of the acid base pairs involved: when accessible on the top surfaces, due to their stronger acidity, the Ca2+ Lewis acid sites are preferentially involved, but they are less efficient than PO-H, as illustrated by the linear decrease of the conversion levels with the increasing relative amount of accessible Ca2+ cations. It is thus concluded that PO-H sites enhance the performances of the catalysts for the two reactions, and that washing and drying conditions allowing us to decrease the calcium accessibility at the benefit of PO-H should be favoured.

Molecular structure of isolated MvspI, a variable surface protein of the fish pathogen Mycoplasma mobile.

Mycoplasma mobile is a parasitic bacterium that causes necrosis in the gills of freshwater fishes. This study examines the molecular structure of its variable surface protein, MvspI, whose open reading frame encodes 2,002 amino acids. MvspI was isolated from mycoplasma cells by a biochemical procedure to 92% homogeneity. Gel filtration and analytical ultracentrifugation suggested that this protein is a cylinder-shaped monomer with axes of 66 and 2.7 nm. Rotary shadowing transmission electron microscopy of MvspI showed that the molecule is composed of two rods 30 and 45 nm long; the latter rod occasionally features a bulge. Immuno-electron microscopy and epitope mapping showed that the bulge end of the molecular image corresponds to the C terminus of the amino acid sequence. Partial digestion by various proteases suggested that the N-terminal part, comprised of 697 amino acids, is flexible. Analysis of the predicted amino acid sequence showed that the molecule features a lipoprotein and 16 repeats of about 90 residues; 15 positions exist between residues 88 and 1479, and the other position is between residues 1725 and 1807. The amino acid sequence of MvspI was mapped onto a molecular image obtained by electron microscopy. The present study is the first to elucidate the molecular shape of a variable surface protein of mycoplasma.

Isolation and characterization of P1 adhesin, a leg protein of the gliding bacterium Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a pathogen causing human pneumonia, binds to solid surfaces at its membrane protrusion and glides by a unique mechanism. In this study, P1 adhesin, which functions as a "leg" in gliding, was isolated from mycoplasma culture and characterized. Using gel filtration, blue-native polyacrylamide gel electrophoresis (BN-PAGE), and chemical cross-linking, the isolated P1 adhesin was shown to form a complex with an accessory protein named P90. The complex included two molecules each of P1 adhesin and P90 (protein B), had a molecular mass of about 480 kDa, and was observed by electron microscopy to form 20-nm-diameter spheres. Partial digestion of isolated P1 adhesin by trypsin showed that the P1 adhesin molecule can be divided into three domains, consistent with the results from trypsin treatment of the cell surface. Sequence analysis of P1 adhesin and its orthologs showed that domain I is well conserved and that a transmembrane segment exists near the link between domains II and III.

Triskelion structure of the Gli521 protein, involved in the gliding mechanism of Mycoplasma mobile.

Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a "triskelion" reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule's role in the gliding mechanism is discussed.

Complement-mediated bacteriolysis after binding of specific antibodies to drug-resistant Pseudomonas aeruginosa: morphological changes observed by using a field emission scanning electron microscope.

A bactericidal mechanism mediated by human serum was investigated by a field emission scanning electron microscope and a strain of drug-resistant Pseudomonas aeruginosa. When the bacteria were treated with meropenem, a carbapenem antibiotic, spheroplasts and bulges (spheroidization) appeared after 1-3 h. When 40% serum was added to the bacteria, the bacteria agglutinated within 2 min and then lysed after 5-30 min. Immunoelectron micrographic analyses showed dispositions of complement component C9 molecules on the cell surface of lysed bacteria by the serum treatment that might suggest formation of a membrane attack complex. Immunoglobulin G (IgG) depletion from the serum diminished the lytic activity and adding human intravenous immunoglobulin (IVIG) restored it, suggesting that lysis was induced by specific IgG binding to the bacteria. IVIG may help patients with less IgG against bacteria to overcome severe infection.

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles.

Virus filtration with nanometer size exclusion membranes ("nanofiltration") is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18-26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.

Sensitivity of common marmosets to detect drug-induced QT interval prolongation: moxifloxacin case study.

INTRODUCTION: Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization studies, but it has not been characterized in common marmosets which are uniquely suited to studies in early-stage development due to their small size and minimal test article requirements. The purpose of this study was to evaluate the sensitivity of the common marmoset to detect moxifloxacin-associated QT interval prolongation. METHODS: Eight telemetered common marmosets were monitored for 24 h following oral administration of moxifloxacin by gavage at 0, 10, 30, and 100 mg/kg using a Latin square design. Concurrently, a pharmacokinetic evaluation in 8 non-telemetered animals was conducted. A rate-corrected QT (QTc) interval was derived using an individual probabilistic QT rate-correction. QTc (placebo-adjusted QTc change from the individual baseline) was calculated and the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) was analyzed. RESULTS: A slight, but not significant, increase in QTc was detected with 10 mg/kg of moxifloxacin. Moxifloxacin at 30 and 100 mg/kg elicited dose-dependent increases in QTc of 14.0+/-3.6 and 35.0+/-6.2 ms, respectively, with associated total moxifloxacin C(max) values of 6.5+/-0.5 and 16.5+/-1.6 microg/mL, respectively. From the PK/PD relationship, the plasma concentration which would attain QTc of 5 to 10 ms was estimated to be 1.67-3.73 microg/mL. The results were consistent with typical clinical trial results (QTc of 6.6-14.8 ms at 2.5-3.5 microg/mL). CONCLUSIONS: The present study demonstrates that the common marmoset is highly sensitive to moxifloxacin-associated changes in cardiac repolarization, assessed as QTc. As such, this species is suitable for precise and reliable detection of small, but significant, drug-associated increases in QTc interval. Thus, the common marmoset should be regarded as a validated animal model for the detection of QT risk in early-stage drug development and represents an important addition to the current in vivo armamentarium.

Morphology of isolated Gli349, a leg protein responsible for Mycoplasma mobile gliding via glass binding, revealed by rotary shadowing electron microscopy.

Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.

Sequence analysis of the gliding protein Gli349 in Mycoplasma mobile.

The motile mechanism of Mycoplasma mobile remains unknown but is believed to differ from any previously identified mechanism in bacteria. Gli349 of M. mobile is known to be responsible for both adhesion to glass surfaces and mobility. We therefore carried out sequence analyses of Gli349 and its homolog MYPU2110 from M. pulmonis to decipher their structures. We found that the motif "YxxxxxGF" appears 11 times in Gli349 and 16 times in MYPU2110. Further analysis of the sequences revealed that Gli349 contains 18 repeats of about 100 amino acid residues each, and MYPU2110 contains 22. No sequence homologous to any of the repeats was found in the NCBI RefSeq non-redundant sequence database, and no compatible fold structure was found among known protein structures, suggesting that the repeat found in Gli349 and MYPU2110 is novel and takes a new fold structure. Proteolysis of Gli349 using chymotrypsin revealed that cleavage positions were often located between the repeats, implying that regions connecting repeats are unstructured, flexible and exposed to the solvent. Assuming that each repeat folds into a structural domain, we constructed a model of Gli349 that fits well the shape and size of images obtained with electron microscopy.

Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos.

The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.