Analyzing paired diagnostic studies by estimating the expected benefit

When the efficacy of a new medical drug is compared against that of an established competitor in a randomized controlled trial, the difference in patient‐relevant outcomes, such as mortality, is usually measured directly. In diagnostic research, however, the impact of diagnostic procedures is of an indirect nature as test results do influence downstream clinical decisions, but test performance (as characterized by sensitivity, specificity, and the predictive values of a procedure) is, at best, only a surrogate endpoint for patient outcome and does not necessarily translate into it. Not many randomized controlled trials have been conducted so far in diagnostic research, and, hence, we need alternative approaches to close the gap between test characteristics and patient outcomes. Several informal approaches have been suggested in order to close this gap, and decision modeling has been advocated as a means of obtaining formal approaches. Recently, the expected benefit has been proposed as a quantity that allows a simple formal approach, and we take up this suggestion in this paper. We regard the expected benefit as an estimation problem and consider two approaches to statistical inference. Moreover, using data from a previously published study, we illustrate the possible insights to be gained from the application of formal inference techniques to determine the expected benefit.     

Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin.

Membrane vesicles derived from the apical side of procine intestinal epithelial cells retain, after demembranation in the presence of calcium, two major proteins (I, II) which are released by the addition of calcium chelators. We have purified and characterized these two calcium-binding proteins. Protein I has a mol. wt. of 85 000 and contains two copies of a 36-K subunit and an additional 10-K subunit. It binds in a calcium-dependent manner to F-actin as well as to non-erythroid spectrin. Immunofluorescence microscopy reveals protein I-related antigens in the terminal web of the intestinal cell and in a submembraneous cortical layer in various tissue culture cells. Biochemical and immunological results document that the 36-K subunit of protein I is identical with the cellular p36K recognized as a major substrate for tyrosine phosphorylation by the sarc gene kinase in Rous sarcoma virus-transformed cells. The biochemical properties of protein I agree with its location seen in immunofluorescence microscopy and cell fractionation and suggest that the actin-spectrin network in the cortical layer may be affected by virus transformation.     

Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on p11, a member of the S100 protein family.

p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II. The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7, 2435-2442). We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography. To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.     

The phosphoenolpyruvate-dependent glucose-phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell

The phosphoenolpyruvate-(PEP)-dependent-carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex transport and sensory system. Such a PTS usually consists of two cytoplasmic energy-coupling proteins, Enzyme I (EI) and HPr, and one of more than 20 different carbohydrate-specific membrane proteins named Enzyme II (EII), which catalyze the uptake and concomitant phosphorylation of numerous carbohydrates. The most prominent representative is the glucose-PTS, which uses a PTS-typical phosphorylation cascade to transport and phosphorylate glucose. All components of the glucose-PTS interact with a large number of non-PTS proteins to regulate the carbohydrate flux in the bacterial cell. Several aspects of the glucose-PTS have been intensively investigated in various research projects of many groups. In this article we will review our recent findings on a Glc-PTS-dependent metalloprotease, on the interaction of EIICB(Glc) with the regulatory peptide SgrT, on the structure of the membrane spanning C-domain of the glucose transporter and on the modeling approaches of ptsG regulation, respectively, and discuss them in context of general PTS research.     

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.     

Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.     

Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency

Sporulation is a well-studied process executed with varying efficiency by diverse yeast strains. We developed a high-throughput method to quantify yeast sporulation efficiency and used this technique to analyze a line cross between a high-efficiency oak tree isolate and a low-efficiency wine strain. We find that natural variation in sporulation efficiency mirrors natural variation in higher eukaryotes: it shows divergence between isolated populations, arises from loci of major effect, and exhibits epistasis. We show that the lower sporulation efficiency of the wine strain results from a failure to initiate sporulation, rather than from slower kinetics of meiosis and spore formation. The two strains differentially regulate many genes involved in aerobic respiration, an essential pathway for sporulation, such that the oak tree strain appears better poised to generate energy from this pathway. We also report that a polymorphism in RME1 that affects sporulation efficiency in laboratory strains also cosegregates with significant phenotypic differences in our cross of natural isolates. These results lay the groundwork for the study of variation in sporulation efficiency among natural isolates of yeast.     

An annexin 1 N-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family

The human N-formyl peptide receptor (FPR) is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. FPR is the founding member of a subfamily of G protein-coupled receptors thought to function in inflammatory processes. The other two members, FPR-like (FPRL)1 and FPRL2, have a greatly reduced affinity for bacterial peptides or do not bind them at all, with FPRL2 being considered an orphan receptor so far. In this study we show that a peptide derived from the N-terminal domain of the anti-inflammatory protein annexin 1 (lipocortin 1) can activate all three FPR family members at similar concentrations. The annexin 1 peptide initiates chemotactic responses in human monocytes that express all three FPR family members and also desensitizes the cells toward subsequent stimulation with bacterial peptide agonists. Experiments using HEK 293 cells stably expressing a single FPR family member reveal that all three receptors can be activated and desensitized by the N-terminal annexin 1 peptide. These observations identify the annexin 1 peptide as the first endogenous ligand of FPRL2 and indicate that annexin 1 participates in regulating leukocyte emigration into inflamed tissue by activating and desensitizing different receptors of the FPR family.