5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-thiophenecarbonitriles as potent, selective, inhibitors of IKK-epsilon kinase

The identification and hit-to-lead exploration of a novel, potent and selective series of substituted benzimidazole–thiophene carbonitrile inhibitors of IKK-ε kinase is described. Compound 12e was identified with an IKK-ε enzyme potency of pIC₅₀ 7.4, and has a highly encouraging wider selectivity profile, including selectivity within the IKK kinase family.     

The contribution of adhesion signaling to lactogenesis

The mammary gland undergoes hormonally controlled cycles of pubertal maturation, pregnancy, lactation, and involution, and these processes rely on complex signaling mechanisms, many of which are controlled by cell–cell and cell–matrix adhesion. The adhesion of epithelial cells to the extracellular matrix initiates signaling mechanisms that have an impact on cell proliferation, survival, and differentiation throughout lactation. The control of integrin expression on the mammary epithelial cells, the composition of the extracellular matrix and the presence of secreted matricellular proteins all contribute to essential adhesion signaling during lactogenesis. In vitro and in vivo studies, including the results from genetically engineered mice, have shed light on the regulation of these processes at the cell and tissue level and have led to increased understanding of the essential signaling components that are regulated in temporal and cell specific manner during lactogenesis. Recent studies suggest that a secreted matricellular protein, CTGF/CCN2, may play a role in lactogenic differentiation through binding to β1 integrin complexes, enhancing the production of extracellular matrix components and contributions to cell adhesion signaling.     

Empirical Evaluation of Oligonucleotide Probe Selection for DNA Microarrays

DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.     

Molecular analysis reveals lowbush blueberry pest predation rates depend on ground beetle (Coleoptera: Carabidae) species and pest density

Molecular gut-content analysis allows determination of pest predation by field-collected predators. Ground beetles (Coleoptera: Carabidae) common in lowbush blueberries may consume blueberry spanworm, Itame argillacearia (Packard) (Lepidoptera: Geometridae), and blueberry flea beetle, Altica sylvia Malloch (Coleoptera: Chrysomelidae), providing pest suppression. Using newly developed pest specific primers, laboratory feeding trials showed that the median detection time (MDT) for blueberry spanworm in the largest beetle, Carabus nemoralis O.F. Müller, was 3.7 h, whereas Poecilus lucublandus (Say) and Pterostichus mutus (Say) had MDTs between 27.1 and 31.6 h for both pests. At a field-site with high pest abundances, the probability of detecting blueberry spanworm and blueberry flea beetle DNA was greater in P. lucublandus, 26 and 39 % respectively, than in P.mutus, 8 and 20 % respectively. Only 0 and 1 % of P. lucublandus and P. mutus, respectively, tested positive for blueberry spanworm DNA at a second site with low abundance. At the first site, the probability of detecting pest DNA in both ground beetle species was positively related to pest density. Higher pest DNA detection rates and captures of ground beetles corresponded to field areas where significant pest reductions occurred from late May to early June. Conservation of predatory carabid beetles could lead to valuable biological control in lowbush blueberries.     

Human axillary secretions influence women''s menstrual cycles: The role of donor extract of females

Menstrual synchrony in human females has previously been demonstrated among women attending a predominantly female university as well as among women attending coeducational universities. In each of these studies, women who spent the most time together were most likely to show the menstrual synchrony. In this experiment, the possibility that substances in axillary secretions might mediate this effect was tested using a prospective, double-blind research design and a combined axillary extract from a group of female donors. Female subjects who reported themselves to have normal (29.5 +/- 3 day) cycles were exposed to the axillary extracts or blank/ethanol for 10 to 13 weeks. Recipients of the axillary extracts showed a significant reduction in "days' difference in menses onset" relative to the donor cycle, no change was evident for recipients of blank/ethanol. These results demonstrate that constituents from the axillary region of donor females can shift the time of menstrual onset of another group to conform with the donors' cycle and that this effect can occur even in the absence of social contact.     

Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.     

Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b

The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSⁿ (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.     

Peptide ligands that bind IgM antibodies and block interaction with antigen.

We have selected a peptide-display phage library on IgM Abs and identified a panel of phage-expressing peptides that bind to IgM Abs in general, but not to Abs of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM Abs. The peptides bind to both soluble pentameric Abs and to monomeric cell-surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM Abs to Ag. These peptides may create confounding artifacts when IgM Abs are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility.