Comparing Trials with Multiple Outcomes: The Multivariate One-Sided Hypothesis with Unknown Covariances

The paper deals with a problem arising for tests in clinical trials. The outcomes of a standard and a new treatment to be compared are multivariate normally distributed with common but unknown covariance matrix. Under the null hypothesis the means of the outcomes are equal, under the alternative the new treatment is assumed to be superior, i.e. the means are larger without further quantification. For known covariance matrix there is a variety of tests for this problem. Some of these procedures can be extended to the case of unknown covariances if one is willing to accept a bias. There is, however, also an efficient unbiased test. The paper contains some numerical comparisons of these different procedures and takes a look on the minimax properties of the unbiased test.     

Insulin-mimetic signaling by the sulfonylurea glimepiride and phosphoinositolglycans involves distinct mechanisms for redistribution of lipid raft components.

The insulin signal transduction cascade provides a number of sites downstream of the insulin receptor (IR) for cross-talk from other signaling pathways. Tyrosine phosphorylation of the IR substrates IRS-1/2 and metabolic insulin-mimetic activity in insulin-responsive cells can be provoked by soluble phosphoinositolglycans (PIG), which trigger redistribution from detergent-insoluble glycolipid-enriched raft domains (DIGs) to other areas of the plasma membrane and thereby activation of nonreceptor tyrosine kinases (NRTK) [Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol. 21, 4553-4567]. Here we describe that stimulation of glucose transport in isolated rat adipocytes by a different stimulus, the sulfonylurea glimepiride, is also based on IRS-1/2 tyrosine phosphorylation and downstream insulin-mimetic signaling involving activation of the NRTK, pp59(Lyn), and pp125(Fak), as well as tyrosine phosphoryation of the DIGs component caveolin. As is the case for PIG 41, glimepiride causes the concentration-dependent dissociation of pp59(Lyn) from caveolin and release of this NRTK and the glycosyl-phosphatidylinositol-anchored (GPI) proteins, Gce1 and 5'-nucleotidase, from total and anti-caveolin-immunoisolated DIGs. This results in their movement to detergent-insoluble raft domains of higher buoyant density (non-DIGs areas). IRS-1/2 tyrosine phosphorylation and glucose transport activation by both glimepiride and PIG are blocked by introduction into adipocytes of the caveolin scaffolding domain peptide which mimicks the negative effect of caveolin on pp59(Lyn) activity. Tyrosine phosphorylation of the NRTK, IRS-1/2, and caveolin as well as release of the NRTK and GPI proteins from DIGs and their redistribution into non-DIGs areas in response to PIG is also inhibited by treatment of intact adipocytes with either trypsin plus salt or N-ethylmaleimide (NEM). In contrast, the putative trypsin/salt/NEM-sensitive cell surface component (CIR) is not required for glimepiride-induced glucose transport, IRS-1/2 tyrosine phosphorylation, and redistribution of GPI proteins and NRTK. The data suggest that CIR is involved in concentrating signaling molecules at DIGs vs detergent-insoluble non-DIGs areas. These inhibitory interactions are relieved in response to putative physiological (PIG) or pharmacological (sulfonylurea) stimuli via different molecular mechanisms (dependent on or independent of CIR, respectively) thereby inducing IR-independent positive cross-talk to metabolic insulin signaling.     

Determination of cholecystokinin tetrapeptide and cholecystokinin octapeptide sulfate in different rat brain regions by high-pressure liquid chromatography with electrochemical detection

A method for the determination of cholecystokinins in biological material, based on high-pressure liquid chromatography with direct electrochemical detection (HPLC-EC), is described. Using this method, the levels of cholecystokinin tetrapeptide and octapeptide sulfate in rat brain cortex, hippocampus, striatum, and brain stem were measured and found to be comparable to those reported using radioimmunoassay methods. We show that HPLC-EC is sensitive enough to accurately determine neuropeptides in brain tissue without prior derivatization and is therefore, due to its simplicity, an attractive alternative to existing methods.     

SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.The mucosal surfaces and skin of the human body are colonized by a large number of bacterial species constituting the normal microbiota. In contrast to pathogens, these commensals usually do not elicit any inflammatory responses in epithelial tissues of the healthy host (1). The Gram-positive coccus Finegoldia magna is part of the anaerobic normal microbiota associated with the skin (2), but it also inhabits the oro-pharynx, gastrointestinal, and urogenital tracts (3). During disturbed homeostasis, this bacterium becomes an important opportunistic pathogen; associated with several clinical conditions, such as soft tissue infections, wound infections, bone/joint infections, and vaginosis (3–5). Among anaerobic cocci of the normal microbiota, F. magna is the species most commonly isolated from clinical conditions (3).Recognition of bacteria and their products by cells residing in the submucosal tissues, for example dendritic cells, triggers an inflammatory response leading to production of host defense molecules, including chemokines. Chemokines comprise a large family of peptides that are key players in inflammation by regulating leukocyte trafficking and activation. They are divided into four groups, XC, CC, CXC, and CX₃C, depending on the arrangement of conserved cysteine residues in their NH₂ terminus (6). The CXC subfamily can be further divided into ELR-positive and ELR-negative respectively, based on the presence or absence of the sequence motif glutamic acid-leucine-arginine (ELR) NH₂ terminal to the first cysteine. IFN-γ, a key cytokine produced during bacterial infection, induces expression of the ELR-negative CXC-chemokine MIG/CXCL9 (Monokine Induced by Gamma-interferon)³ (7). MIG/CXCL9 binds and activates a G-protein-coupled seven transmembrane receptor, CXCR3, which is present on eosinophils, activated T cells (CD8+), and NK cells (8). In addition to its ability to recruit and activate leukocytes, MIG/CXCL9 possesses angiostatic properties through activation of CXCR3 expressed on endothelial cells, and it also exerts potent antibacterial properties (9–11). Upon IFN-dependent inflammation, for example during bacterial infection, this chemokine is produced by epithelial cells and participates in activities of both innate and adaptive immunity (10, 12–14).The finding that epithelial cells recognize important human pathogens, such as Streptococcus pyogenes, leading to an increased MIG/CXCL9 production (10, 12) raised the question whether an opportunistic pathogen like F. magna could be recognized in a similar fashion. In skin F. magna is localized to the epidermis where they adhere to basement membranes through an interaction with the basement membrane protein BM-40 (15). Binding to BM-40 is mediated by the surface protein FAF (F. magna adhesion factor) that is expressed by more than 90% of F. magna isolates (15). Bacteria, both commensals and pathogens, express proteases that are important both during colonization and invasion (16). In the case of F. magna, most strains express a subtilisin-like enzyme, SufA (Subtilase of Finegoldia magna), which is associated with the bacterial surface, but also secreted in substantial amounts during bacterial growth (17). Studies on the proteolytic activity of SufA demonstrated that the enzyme cleaves and inactivates antibacterial molecules like LL-37 and MIG/CXCL9 (17). Here, we show that MIG/CXCL9, produced by human keratinocytes in response to inflammatory stimuli, is degraded by SufA-expressing F. magna. The generated MIG/CXCL9 fragments are still able to activate the MIG/CXCL9 receptor, CXCR3 and kill S. pyogenes, while F. magna is left unaffected. This modulation of the MIG/CXCL9 activities promotes the survival of F. magna during inflammation.     

The effects of exposure and microbes on hatchability of eggs in open-cup and cavity nests

Many avian species initiate incubation before clutch completion, resulting in an asynchronous hatch of their eggs. Several studies suggest that early laid eggs in birds that exhibit synchronous hatching may be more vulnerable to the negative impacts of ambient temperatures and/or trans-shell infection by microbes. However, nearly all of these studies have exposed fertile eggs to environmental conditions in artificial cavity nests, and thus, the effects of exposure of eggs to environmental conditions in open-cup nests remains largely unknown. Therefore, we directly compared hatchability and rates of trans-shell infection in fertile domestic chicken eggs that were exposed for 1–5 days in either open-cup or cavity nests. Eggs in open-cup nests had significantly higher rates of trans-shell infection and lower hatchability than those in cavity nests. These differences may result from different environmental conditions in open-cup nests, as well as in rates of microbial colonization of eggs. Cavity nests maintained slightly higher temperatures than did open-cup nests in the same location; thus, eggs in cavity nests were exposed for a longer period of time to temperatures ≥27°C, the temperature at which antibacterial enzymatic activity is initiated in the albumen. Moreover, microbial growth on eggs was much higher in open-cup nests even when eggs in these nests were cleaned daily with alcohol. It may be that the increased exposure to rain events in open-cup nests may facilitate microbial growth and egg infection. Thus, our data suggest that open-cup nesters may face constraints on reproduction different from those that cavity nesters face, and therefore may make choices regarding incubation that reflect these challenges.     

Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist

The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by gamma-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for 3H-dopamine and 3H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs.     

Interaction of bacteriophage T7 gene 4 primase with its template recognition site

The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.