The relationship of conceptus number and fetal sex to maternal serum testosterone concentration in the rat

On Day 8 of pregnancy, the number of implantation sites in pregnant rats was adjusted to 1, 2, 4, 6, or greater than 10. Blood was collected on Days 11, 12, 15, 18, and 20 for the determination of serum testosterone, progesterone, and androstenedione. Serum testosterone levels exhibited a direct linear relationship with implantation number, increasing from 1 through greater than 10 implants. This linear relationship was particularly evident at Days 12, 15, and 18 of pregnancy. Serum progesterone levels increased from one to four conceptuses and plateaued above this number. There was no apparent relationship between the number of conceptuses and serum androstenedione levels, which may reflect the multiple origins of this steroid in the pregnant rat. In a separate group of rats in which the number of conceptuses was adjusted to three on Day 8 of pregnancy, blood was collected on Days 11, 12, 15, 18, and 20. Fetal sex was determined between the last bleeding and the day of parturition. Serum testosterone was determined and results were examined with regard to the number of male/female fetuses in the litter of three. There was no relationship between maternal serum testosterone levels and the number of male fetuses, indicating that the fetal testis does not make a significant contribution to circulating maternal testosterone levels.     

Trans-translation and protein synthesis inhibitors

Trans-translation is a process found in all bacteria, which contributes to the release of ribosomes that are stalled through a variety of causes, for example when the 3' end of a truncated mRNA lacking a stop codon is reached or at internal clusters of rare codons. Trans-translation requires tmRNA. Trans-translation is not essential for cell viability under laboratory conditions, but recently it has been shown that it can contribute to cell viability in the presence of protein synthesis inhibitors. In this minireview, we consider the connection between trans-translation and antibiotics and the potential of using trans-translation as a therapeutic target.     

Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis.To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.     

Optical properties of P3HT and N2200 polymers: a performance study of an optimally tuned DFT functional

The optical properties of systems composed of the polymers PolyeraActivInk? N2200 and P3HT are experimentally and theoretically investigated using UV-Vis spectroscopy and time-dependent density functional theory calculations, respectively. From a theoretical point of view, we carried out an analysis considering several functionals and model oligomers of different sizes to mimic the polymers. As our studies were performed with and without solvents, a first important result regards the fact that, by considering solvent effects, a better agreement between theoretical and experimental results could be achieved. Our findings also show that an optimally tuned functional is better suited to describe the experimental absorption profile than a hybrid one for the flexible polymer (P3HT). For the almost rigid polymer considered here (N2200), on the other hand, hybrid functionals may perform better than tuned functionals.     

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.     

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non‐human primate models

Antibody based manipulation of the CD137 (4‐1BB) co‐signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti‐human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti‐CD137 mAb (4B4‐1), recognized activated PBMCs from both human and non‐human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species (～95% identical), with the extracellular domain differing by only 9–10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co‐stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes. Copyright © 2009 John Wiley & Sons, Ltd.     

Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

Suitability of edaphic arthropods as prey for <Emphasis Type="Italic">Proctolaelaps bickleyi</Emphasis> and <Emphasis Type="Italic">Cosmolaelaps brevistilis</Emphasis> (Acari: Mesostigmata: Melicharidae,Laelapidae) under laboratory conditions

Soils are often complex habitats inhabited by a wide range of organisms, some harmful to plants and others beneficial, for example by attacking harmful organisms. Beneficial organisms include predatory mites, some of which have been commercialized for biological control of pest insects and mites. The objective of this work was to evaluate under laboratory condition the suitability of representative soil insect and mite pests, especially Aceria tulipae (Keifer), as prey to the soil-inhabiting predatory mites Proctolaelaps bickleyi (Bram) and Cosmolaelaps brevistilis (Karg). Predation, oviposition and survivorship of recently molted adult females of the predators were assessed in the dark in rearing chambers at 25 ± 1 °C and 75 ± 3% RH. Predation rate by P. bickleyi on A. tulipae was significantly higher than that by C. brevistilis (196.3 vs. 71.0 specimens/day). About 482 A. tulipae were preyed by each P. bickleyi at each day, when 500 A. tulipae were made available daily to the predator. Oviposition rate on that prey was also higher for P. bickleyi (4.2 eggs/day). For C. brevistilis, the highest level of oviposition was on Caliothrips phaseoli (Hood) (1.2 eggs/day). Survivorship was always higher for C. brevistilis (≥ 70%), given its ability to remain alive relatively long even in the absence of prey. High rates of survivorship of P. bickleyi were observed on A. tulipae, Bradysia matogrossensis (Lane) and Protorhabditis sp. Promising results were obtained for P. bickleyi on A. tulipae and even on other prey, justifying the conduction of complementary studies under field condition.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.