Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, pregnancy, maturation and after ovariectomy

A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.     

Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey

Summary The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglutinin and concanavalin A suggesting the synthesis of many glycosylated products. Some cells were reactive with antisera to prekeratin, others with antisera to vimentin. The cells also contained actin (showing peculiar intercellular communications), -actinin and tubulin. They were able to metabolize certain steroid precursors, but there was no definitive evidence for the presence of aromatase or ⁵-3-hydroxysteroid dehydrogenase in these cells. This cell line appears to resemble trophectodermal girdle epithelium at a stage of development prior to the onset of eCG production, and may be useful in studies on the control of expression of this substance.Dr. S. Kellie is now at the Imperial Cancer Research Fund Laboratories, Lincoln Inn's Fields, London     

Determination of oestrogen monoglucuronides by the Brown method

In the Brown (Brown, 1955; Brown, Bulbrook & Greenwood, 1957) procedure for the determination of urinary oestrogens, losses may occur during the hydrolysis of the oestrogen conjugates and during the purification of the oestrogens thus formed. Losses during the latter stages were measured previously by adding free oestrogens to the hydrolysed urine. In the present study losses during acid and enzyme hydrolysis were measured by adding synthetic oestrogen monoglucuronides to urine.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.     

Coarse-Scale Distribution Surveys and Occurrence Probability Modeling for Wolverine in Interior Alaska

ABSTRACT We determined wolverine (Gulo gulo) distribution and occurrence probabilities using aerial surveys and hierarchical spatial modeling in a 180,000-km² portion of Interior Alaska, USA. During 8 February-12 March 2006, we surveyed 149 of 180 1,000-km² sample units for wolverine tracks. We observed wolverine tracks in 99 (66.4%) sample units. Wolverine detection probability was ≥69% throughout the survey period. Posterior occurrence probabilities of whether a wolverine track occurred in a sample unit was dependent on survey timing, number of transects flown, number of neighboring sample units with detected tracks, percentage of the sample unit with elevation ≤305 m, and human influences. Our model indicated strong evidence of occurrence (>0.80) in 72% of the 180 survey units, strong evidence of absence (<0.20) in 12%, and weak evidence of occurrence or absence (0.20–0.80) in 16%. Wolverine area of occupancy made up 83% of the study area. Simulations illustrated that 2–4 survey routes were necessary for the survey technique to provide strong evidence of wolverine presence or absence in Interior Alaska if a track was not identified along the first route. The necessary number of survey routes depends on the occurrence probability in a sample unit. We provided managers with a map of wolverine distribution in Interior Alaska and an efficient and lower-cost method to detect coarse-scale changes in wolverine distribution. Our technique was effective in both Interior Alaska and Ontario, Canada, suggesting it would be effective throughout most of the boreal forest range of wolverines where tracks can be readily observed from the air. The technique requires a certain skill level in recognizing tracks; it is essential that tracks are identified correctly and training may be necessary depending on surveyor experience.     

NMR assignments of cd-HO, a 24 kDa heme oxygenase from Corynebacterium diphtheria

We are employing a number of selective in vitro and in vivo methods including NMR to screen compounds that bind to heme oxygenases from pathogenic bacteria. We report the nearly complete HN, N, CO, Cα and Cβ chemical shift assignments of a 215-amino acid HO from Corynebacterium diphtheria in three forms, apo cd-HO-G135A, apo cd-HO and CO-bound ferrous holo cd-HO; these assignments will enable us to identify residues on cd-HO that are perturbed upon binding to selected compounds, and to help with the development of inhibitors specific to the bacterial proteins.     

Does season alter responsiveness of the reproductive neuroendocrine axis to the suppressive actions of cortisol in ovariectomized ewes?

Season can profoundly influence activity of the hypothalamic-pituitary-adrenal axis and alter reproductive neuroendocrine responsiveness to stress and gonadal steroids. Here we tested the hypothesis that the inhibitory effect of a stress-like increment in plasma concentration of the adrenal steroid cortisol on pulsatile LH secretion varies with season. LH pulse patterns were monitored prior to and during the administration of cortisol in the same seven ovariectomized ewes during three stages of the yearly breeding cycle: breeding season, transition to anestrus, and midanestrus. The elevation in cortisol mimicked the rise in plasma level of cortisol in response to an immune/inflammatory stress. During all three seasons, cortisol acutely suppressed the pulsatile release of LH. This inhibition reflected a marked reduction of LH pulse amplitude and a minimal suppression of LH pulse frequency. Of interest, the suppressive effect of this physiologic increment in cortisol did not vary across seasons. This provides initial evidence that, in ovariectomized ewes, cortisol-induced suppression of pulsatile LH secretion differs from that of gonadal steroids in that it is not profoundly influenced by season.     

CD148/DEP-1 association with areas of cytoskeletal organisation in macrophages

In macrophages, tyrosine phosphorylation regulates many signalling pathways leading to growth, differentiation, activation, phagocytosis and adhesion. Protein tyrosine phosphatases (PTPs) represent a biochemical counterbalance to the activity of protein tyrosine kinases, thus regulating the dynamic phosphorylation state of a cell. CD148 is a receptor PTP that is highly expressed in macrophages and is further regulated by pro-inflammatory stimuli. CD148 is normally localised to the plasma membrane of macrophages. Following stimulation with CSF-1 or LPS, there was a re-distribution and concentration of CD148 in areas of membrane ruffling. Treatment of macrophages with anti-CD148 monoclonal antibody inhibited CSF-1-induced macrophage spreading, cytoskeletal re-arrangements and chemotaxis, without affecting cell survival. There were no detectable effects on the CSF-1 receptor-akt signalling pathway. These results are consistent with the hypothesis that CD148 is a regulator of macrophage activity.