Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism

Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY-P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by alpha4, beta5, alpha5, and alpha1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.     

Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production

Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation.     

Maternal Lineage of Warmblood Mares Contributes to Variation of Gestation Length and Bias of Foal Sex Ratio

Maternal lineage influences performance traits in horses. This is probably caused by differences in mitochondrial DNA (mtDNA) transferred to the offspring via the oocyte. In the present study, we investigated if reproductive traits with high variability—gestation length and fetal sex ratio—are influenced by maternal lineage. Data from 142 Warmblood mares from the Brandenburg State Stud at Neustadt (Dosse), Germany, were available for the study. Mares were grouped according to their maternal lineage. Influences on the reproduction parameters gestation length and sex ratio of offspring were analyzed by simple and multiple analyses of variance. A total of 786 cases were included. From the 142 mares, 119 were assigned to six maternal lineages with n≥10 mares per lineage, and 23 mares belonged to smaller maternal lineages. The mean number of live foals produced per mare was 4.6±3.6 (±SD). Live foal rate was 83.5%. Mean gestation length was 338.5±8.9 days (±SD) with a range of 313 to 370 days. Gestation length was affected by maternal lineage (p<0.001). Gestation length was also significantly influenced by the individual mare, age of the mare, year of breeding, month of breeding and sex of the foal (p<0.05). Of the 640 foals born alive at term, 48% were male and 52% female. Mare age group and maternal lineage significantly influenced the sex ratio of the foals (p<0.05). It is concluded that maternal lineage influences reproductive parameters with high variation such as gestation length and foal sex ratio in horses. In young primiparous and aged mares, the percentage of female offspring is higher than the expected 1:1 ratio.     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.     

Birth month associations with height,head circumference,and limb lengths among peruvian children

Associations between season of birth and body size, morbidity, and mortality have been widely documented, but it is unclear whether different parts of the body are differentially sensitive, and if such effects persist through childhood. This may be relevant to understanding the relationship between early life environment and body size and proportions. We investigated associations between birth month and anthropometry among rural highland (n = 162) and urban lowland (n = 184) Peruvian children aged 6 months to 8 years. Stature; head‐trunk height; total limb, ulna, tibia, hand, and foot lengths; head circumference; and limb measurements relative to head‐trunk height were converted to internal age‐sex‐specific z scores. Lowland and highland datasets were then analyzed separately for birth month trends using cosinor analysis, as urban conditions likely provide a more consistent environment compared with anticipated seasonal variation in the rural highlands. Among highland children birth month associations were significant most strongly for tibia length, followed by total lower limb length and stature, with a peak among November births. Results were not significant for other measurements or among lowland children. The results suggest a prenatal or early postnatal environmental effect on growth that is more marked in limb lengths than trunk length or head size, and persists across the age range studied. We suggest that the results may reflect seasonal variation in maternal nutrition in the rural highlands, but other hypotheses such as variation in maternal vitamin D levels cannot be excluded. Am J Phys Anthropol 154:115–124, 2014. © 2014 The Authors. American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.     

Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.