In vitro antimicrobial activity and MIC quality control guidelines of RPR 106972 (RPR 112808/RPR106950): a novel orally administered streptogramin combination. The Quality Control Study Group

Thymidylate synthase (TS) inhibitor effects on growth of human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (2,4-diamino-10-methylpteroylglutamic acid) (MTX) resistance were studied. During 120-h treatment, HNSCC cell lines A253 and FaDu are equally sensitive to MTX, whereas the polyglutamylatable TS inhibitors ZD1694 and BW1843U89 are 5- to 35-fold more potent than MTX and the lipophilic AG331 is approximately 10(2)-fold less potent than MTX. A253 is intrinsically resistant to intermittent (24 h) MTX and BW1843U89 exposure (higher EC50 values and shallower slopes of concentration-response curves relative to FaDu); AG331 and ZD1694 largely overcome this intrinsic resistance to intermittent exposure. Thymidine (TdR) protects against growth inhibition by these inhibitors, confirming that TS is their target in HNSCC; at high AG331 levels, TdR only partially protects, implying that a second site of action exists. Growth inhibition of HNSCC by ZD1694 and BW1843U89 is protected by leucovorin (LV) at > or = 10(-7) and > 10(-3) M, respectively; 10(-4) M LV cannot protect HNSCC cells against AG331. Results similar to protection studies are obtained if LV addition is delayed < or = 24 h after ZD1694 or BW1843U89 exposure. CCRF-CEM sublines with acquired MTX resistance resulting from dihydrofolate reductase (DHFR) overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG331. Cells with defective MTX transport are highly cross-resistant to ZD1694 and BW1843U89, implicating the reduced folate/MTX carrier in their transport. Minor cross-resistance of the DHFR overexpressing line to ZD1694 and BW1843U89 is observed. A subline with highly defective MTX polyglutamylation is cross-resistant to 120-h exposure to ZD1694, but not to BW1843U89, suggesting a profound contribution of polyglutamylation to the mechanism of action of ZD1694.     

Tyrosine phosphorylation of annexin II tetramer is stimulated by membrane binding

In the present article we have examined if the interaction of the Ca2+-binding protein, annexin II tetramer (AIIt) with the plasma membrane phospholipids or with the submembranous cytoskeleton, effects the accessibility of the tyrosine phosphorylation site of AIIt. In the presence of Ca2+, pp60(c-src) catalyzed the incorporation of 0.22 +/- 0.05 mol of phosphate/mol of AIIt (mean +/- S.D., n = 5). The Ca2+-dependent binding of AIIt to purified adrenal medulla plasma membrane or phosphatidylserine vesicles stimulated the pp60(c-src)-dependent phosphorylation of AIIt to 0.62 +/- 0.04 mol of phosphate/mol of AIIt (mean +/- S.D., n = 5) or 0.93 +/- 0.07 mol of phosphate/mol of AIIt (mean +/- S.D., n = 5), respectively. Phosphatidylserine- or phosphatidylinositol-containing vesicles but not vesicles composed of phosphatidylcholine or phosphatidylethanolamine, stimulated the phosphorylation of AIIt. In contrast, the binding of AIIt to F-actin resulted in the incorporation of only 0.04 +/- 0.04 mol of phosphate/mol of AIIt (mean +/- S.D., n = 5). These results suggest that the interaction of AIIt with plasma membrane and not the submembranous cytoskeleton, activates the tyrosine phosphorylation of AIIt by inducing a conformational change in the protein resulting in the enhanced exposure or accessibility of the tyrosine-phosphorylation site.     

Functional analysis of the C2A domain of synaptotagmin 1: implications for calcium-regulated secretion

Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.     

Image processing and spiral CT of the thorax

The data set of the thorax acquired by spiral CT is volumetric. Such data can be processed so that conventional axial sections are supplemented by reconstructed images, in an attempt to answer specific clinical questions. This review considers three reconstruction techniques: multiplanar reformation, three-dimensional rendering and sliding-thin slab reconstruction. Their relative benefits and limitations are considered, as are the implications of image processing in general.     

Comparing risk-adjustment methods for provider profiling

Risk-adjustment and provider profiling have become common terms as the medical profession attempts to measure quality and assess value in health care. One of the areas of care most thoroughly developed in this regard is quality assessment for coronary artery bypass grafting (CABG). Because in-hospital mortality following CABG has been studied extensively, risk-adjustment mechanisms are already being used in this area for provider profiling. This study compares eight different risk-adjustment methods as applied to a CABG surgery population of 28 providers. Five of the methods use an external risk-adjustment algorithm developed in an independent population, while the other three rely on an internally developed logistic model. The purposes of this study are to: (i) create a common metric by which to display the results of these various risk-adjustment methodologies with regard to dichotomous outcomes such as in-hospital mortality, and (ii) to compare how these risk-adjustment methods quantify the 'outlier' standing of providers. Section 2 describes the data, the external and internal risk-adjustment algorithms, and eight approaches to provider profiling. Section 3 then demonstrates the results of applying these methods on a data set specifically collected for quality improvement.     

The effect of point mutations on the free energy of transmembrane alpha-helix dimerization

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins. We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(+/-50) nM, corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal mol-1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.     

A stereotaxic template atlas of the macaque brain for digital imaging and quantitative neuroanatomy

A stereotaxic brain atlas of the longtailed macaque (Macaca fascicularis) is presented in a format suitable for use as a template atlas of the macaque brain. It includes most of the brain segmented to show the boundaries of landmark structures such that every point in the brain can be represented by a unique set of coordinates in three-dimensional space and ascribed unambiguously to one and only one primary structure. More than 400 structures are represented, including 360 volumetric structures, which constitute the substance of the brain, and 50 superficial features. To facilitate use with ventriculography, magnetic resonance imaging, and other noninvasive imaging techniques, the stereotaxic space is referenced to internal landmarks, viz., the anterior commissure and posterior commissure; the center of the anterior commissure at the midline is the origin of the stereotaxic axes. Reference of stereotaxis to this bicommissural space facilitates structural comparison with human brain atlases, which are commonly referenced to the biocommissural line. It also facilitates comparison of brains of different nonhuman primate species by providing a template brain against which to compare size and internal variability. Thirty-three coronal sections at 1-mm intervals from the spinomedullary junction to the rostral extreme of the caudate nucleus show most structures of the hindbrain, midbrain, and subcortical forebrain. Separately, four side views and 16 coronal sections show cortical structures. Structures are represented by outlines of their boundaries and labeled according to NeuroNames, a systematic English nomenclature of human and nonhuman primate neuroanatomy. Abbreviations are based on a protocol designed to facilitate cross-species comparisons. Instructions are provided for: (1) locating sites from the Template Atlas in the conventional stereotaxic space of an experimental animal, (2) locating sites identified by conventional stereotaxis in the Template Atlas, and (3) using the Template Atlas to collate, compare, and display image information (e.g., labeled cells, recording sites, stimulation sites, lesions) from multiple animals.