Progesterone production and clearance in cyclic ewes passively immunized against progesterone

The effects of passive immunization of ewes against progesterone on plasma progesterone concentrations and on the metabolic clearance rate (MCR) and production rate (PR) of progesterone were investigated. Three treatment groups were studied: 1) nonimmunized controls, 2) ewes passively immunized with antiprogesterone serum, and 3) immunized progestagen-treated ewes, treated concomitantly with anti-serum and with a synthetic progestagen that is not bound by the antiserum. Progesterone levels in the immunized ewes reached a maximum of 27.7+/-4.8 nmol/l and were significantly higher (P<0.05) than in the nonimmunized controls (9.2+/-1.1 mol/l) or the immunized progestagen-treated ewes (15.6+/-1.6 nmol/l). Mean progesterone MCR in the immunized ewes was 1.6+/-0.5 and 2.1+/-0.3 liter/min on Days 7 and 13 of the estrous cycle, respectively, compared with 0.8+/-0.2 and 1.4+/-0.3 liter/min, respectively, in nonimmunized controls. The progesterone production rate in the immunized ewes was significantly higher than in nonimmunized controls, and reached 12.0+/-2.2 and 19.7+/-1.6 nmol/min on Days 7 and 13 of the estrous cycle, respectively, compared with 4.6+/-0.6 and 10.0+/-2.5 nmol/min in nonimmunized controls (P<0.03 for both comparisons). Treatment with progestagen had no significant effect on progesterone MCR or PR of immunized ewes. The LH pulse frequency on Days 10 to 11 of the cycle was 0.7+/-0.3, 1.8+/-0.3 and 0.0+/-0.0 pulses/6 h in the control, immunized and immunized progestagen-treated groups, respectively (P<0.05). It is concluded that the increased plasma progesterone levels in the immunized ewes are the result of an increased progesterone production rate, which may have been induced by an increase in gonadotrophin secretion or by a direct effect of the anti-progesterone serum on the ovary.     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind⁻) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA⁻ SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.     

In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.     

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-beta-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST-YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6-p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1.     

Patterns and Emerging Trends in Global Ocean Health

International and regional policies aimed at managing ocean ecosystem health need quantitative and comprehensive indices to synthesize information from a variety of sources, consistently measure progress, and communicate with key constituencies and the public. Here we present the second annual global assessment of the Ocean Health Index, reporting current scores and annual changes since 2012, recalculated using updated methods and data based on the best available science, for 221 coastal countries and territories. The Index measures performance of ten societal goals for healthy oceans on a quantitative scale of increasing health from 0 to 100, and combines these scores into a single Index score, for each country and globally. The global Index score improved one point (from 67 to 68), while many country-level Index and goal scores had larger changes. Per-country Index scores ranged from 41–95 and, on average, improved by 0.06 points (range -8 to +12). Globally, average scores increased for individual goals by as much as 6.5 points (coastal economies) and decreased by as much as 1.2 points (natural products). Annual updates of the Index, even when not all input data have been updated, provide valuable information to scientists, policy makers, and resource managers because patterns and trends can emerge from the data that have been updated. Changes of even a few points indicate potential successes (when scores increase) that merit recognition, or concerns (when scores decrease) that may require mitigative action, with changes of more than 10–20 points representing large shifts that deserve greater attention. Goal scores showed remarkably little covariance across regions, indicating low redundancy in the Index, such that each goal delivers information about a different facet of ocean health. Together these scores provide a snapshot of global ocean health and suggest where countries have made progress and where a need for further improvement exists.     

Viscosity and transient solvent accessibility of Trp-63 in the native conformation of lysozyme

We have measured the rates of isotope exchange at the nitrogen of the indole ring of Trp-63 of lysozyme and of L-tryptophan as a function of solution viscosity. We have used two cosolvents, glycerol and ethylene glycol, to modify the relative viscosity. We have derived the appropriate kinetic equations for the alternative possibilities that the exchange takes place either in solution or in the intact protein matrix. Because we chose to study the proton-catalyzed exchange reaction, the rate of it is not expected to be diffusion-limited. We confirmed this by measuring the exchange from tryptophan. These results and the known effects of glycerol and ethylene glycol on the solvation of indole allow us to predict that if the exchange reaction takes place in a protein matrix the effects of the two cosolvents when compared under isoviscous conditions should be identical. This is what we find for Trp-63 in lysozyme at 15, 20 and 26 degrees C. The slope of the linear plot of log k vs. log relative viscosity is 0.6. This strongly supports a model for conformational fluctuations where transient solvation takes place without major changes in protein folding. The most interesting feature of our findings is the fact that a slow reaction admittedly not diffusion-limited shows, when taking place in a protein matrix, a linear dependence on solution viscosity. We suggest that what we observe is the effect of damping of movement of the side chain expressed as a change in the friction along the reaction coordinate in the corresponding phase space. The presence of such effects stresses the validity and usefulness of Kramers model of rate processes for reactions taking place in a protein matrix. Such behavior is predicted by several of the recently proposed general mechanisms of enzyme catalysis.     

Changes in the integrity of large unilamellar vesicles due to their interaction with tobacco cell suspensions

Negatively charged large unilamellar vesicles (LUV) were incubated with tobacco (Nicotiana tabacum var. xanthi) cell suspensions and with the cell-free medium of the cell suspensions. The extent of cell-LUV interaction was determined by the leakage of the LUV contents. Cells enhanced the leakage of LUV contents and this effect increased with cell age. Addition of polylysine to the reaction mixture increased even further the leakage of the LUV contents. The cell-free medium of the cell suspension also affected the integrity of the LUV. Cell-free medium, by itself, promoted leakage of LUV contents and caused a reduction in the leakage exerted by polylysine. Centrifugation (8000g) of the cell-free medium decreased its effect, heat treatment (122°C) did not alter its effect and sonication enhanced it. The effects of the cell-free medium are attributed to the presence of cell wall debris of disintegrated cells.