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.