Evaluation of the Efficacy of Commercial Disinfectants Against Fusarium oxysporum f. sp. cubense Race 1 and Tropical Race 4 Propagules.

Panama disease caused by Fusarium oxysporum f. sp. cubense has devastated banana production worldwide. This work aimed to determine effective disinfectants against two races of F. oxysporum f. sp. cubense, race 1 and tropical race 4 (TR4), for implementation with on-farm biosecurity procedures against this disease following the outbreak of TR4 in North Queensland in 2015. A total of 32 commercial disinfectants were screened and their activity was assessed after ≤30 s, 5 min, 30 min, and 24 h of contact with an F. oxysporum f. sp. cubense suspension containing 105 chlamydospores/ml without and with soil added (0.05 g/ml). Of the disinfectants tested, the quaternary ammonium compounds containing ≥10% active ingredient were found to be the most effective against both F. oxysporum f. sp. cubense races. These products, when used at a 1:100 dilution, completely inhibited the survival of all F. oxysporum f. sp. cubense propagules across all the contact times regardless of the absence or presence of soil. The bioflavonoid product EvoTech 213 and bleach (10% sodium hypochlorite) used at a 1:10 dilution also eliminated all F. oxysporum f. sp. cubense propagules across all the contact times. None of the detergent-based or miscellaneous products tested were completely effective against both F. oxysporum f. sp. cubense races even used at a 1:10 dilution. Soil decreases the efficacy of disinfectants and therefore must be removed from contaminated items before treatments are applied.

Short communication: Lysine retained among 2 lipid-coated lysine products after exposure to alfalfa or corn silage with different amounts of acidity.

We conducted 2 experiments to determine lysine loss from 2 lipid-coated lysine products after mixing with silage. In our first experiment, we mixed 2 lipid-coated lysine products, crystalline lysine or crystalline lysine and amounts of lipid identical to amounts included in lipid-coated lysine products, with alfalfa or corn silage that had 2 different amounts of acidity. Lysine appeared to disassociate from lipid-coated lysine products in a nonlinear manner after mixing with either alfalfa or corn silage at different amounts of acidity. Additionally, silage source and acidity affected amounts of lysine released from lipid-coated lysine products after mixing. In a corresponding experiment, in vitro estimates of lysine available to ruminal microbiota after mixing with alfalfa or corn silage at different amounts of acidity were measured by ammonia release. In vitro measures were conducted with or without monensin to allow estimates of effects of monensin on amounts of lysine released from the 2 lipid-coated lysine products. It is unclear whether in vitro estimates of lysine fermentation from lipid-coated lysine are truly reflective of ruminal degradation of lysine from lipid-coated lysine because amounts of time needed to measure differences between different lysine sources were greater than typical estimates of mean ruminal particulate retention time. Nonetheless, monensin apparently reduced ammonia release from lysine, but ammonia release from lipid-coated lysine did not differ from crystalline lysine. Clearly, methods of manufacture together with physical and chemical characteristics of diet can affect amounts of lysine provided from lipid-coated lysine products to ruminants.

Study protocol for an evaluation of the effectiveness of 'care bundles' as a means of improving hospital care and reducing hospital readmission for patients with chronic obstructive pulmonary disease (COPD).

BACKGROUND: Chronic Obstructive Pulmonary Disease is one of the commonest respiratory diseases in the United Kingdom, accounting for 10% of unplanned hospital admissions each year. Nearly a third of these admitted patients are re-admitted to hospital within 28 days of discharge. Whilst there is a move within the NHS to ensure that people with long-term conditions receive more co-ordinated care, there is little research evidence to support an optimum approach to this in COPD. This study aims to evaluate the effectiveness of introducing standardised packages of care i.e. care bundles, for patients with acute exacerbations of COPD as a means of improving hospital care and reducing re-admissions. METHODS / DESIGN: This mixed-methods evaluation will use a controlled before-and-after design to examine the effect of, and costs associated with, implementing care bundles for patients admitted to hospital with an acute exacerbation of COPD, compared with usual care. It will quantitatively measure a range of patient and organisational outcomes for two groups of hospitals - those who deliver care using COPD care bundles, and those who deliver care without the use of COPD care bundles. These care bundles may be provided for patients with COPD following admission, prior to discharge or at both points in the care pathway. The primary outcome will be re-admission to hospital within 28 days of discharge, although the study will additionally investigate a number of secondary outcomes including length of stay, total bed days, in-hospital mortality, costs of care and patient / carer experience. A series of nested qualitative case studies will explore in detail the context and process of care as well as the impact of COPD bundles on staff, patients and carers. DISCUSSION: The results of the study will provide information about the effectiveness of care bundles as a way of managing in-hospital care for patients with an acute exacerbation of COPD. Given the number of unplanned hospital admissions for this patient group and their rate of subsequent re-admission, it is hoped that this evaluation will make a timely contribution to the evidence on care provision, to the benefit of patients, clinicians, managers and policy-makers. TRIAL REGISTRATION: International Standard Randomised Controlled Trials - ISRCTN13022442 - 11 February 2015.

Critical role of NKT cells in posttransplant alloantibody production.

We previously reported that posttransplant alloantibody production in CD8-deficient hosts is IL-4+ CD4+ T cell-dependent and IgG1 isotype-dominant. The current studies investigated the hypothesis that IL-4-producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8-deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon-γ-dependent and IL-4-independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL-4+ CD4+ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli.

Alloprimed CD8(+) T cells regulate alloantibody and eliminate alloprimed B cells through perforin- and FasL-dependent mechanisms.

While it is well known that CD4(+) T cells and B cells collaborate for antibody production, our group previously reported that CD8(+) T cells down-regulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8(+) T cell-mediated down-regulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8(+) T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to WT transplant recipients. Furthermore, CD8(+) T cells require FasL, perforin and allospecificity to down-regulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8(+) T cells directly induce apoptosis of alloprimed IgG1(+) B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8(+) T cells down-regulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1(+) B cells.

Effects of high-sulphur water on hepatic gene expression of steers fed fibre-based diets.

Sulphur-induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is frequently associated with the consumption of high-sulphur (S) water and subsequent poor performance. Currently, there is no economical method for S removal from surface water sources, and alternative water sources are typically neither readily available nor cost-effective. Determination of genes differentially expressed in response to high-S water consumption may provide a better understanding of the physiology corresponding to high dietary S and ultimately lead to the development of treatment and prevention strategies. The objective of this study was to determine changes in gene expression in the liver, an organ important for S metabolism, of fibre-fed steers consuming high-S water. For this study, liver tissues were collected on the final day of a trial from yearling steers randomly assigned to low-S water control (566 mg/kg SO4 ; n = 24), high-S water (3651 mg/kg SO4 ; n = 24) or high-S water plus clinoptilolite supplemented at either 2.5% (n = 24) or 5.0% (n = 24) of diet dry matter (DM). Microarray analyses on randomly selected healthy low-S control (n = 4) and high-S (n = 4; no clinoptilolite) steers using the Affymetrix GeneChip Bovine Genome Array revealed 488 genes upregulated (p < 0.05) and 154 genes downregulated (p < 0.05) in response to the high- vs. low-S water consumption. Real-time RT-PCR confirmed the upregulation (p < 0.10) of seven genes involved in inflammatory response and immune functions. Changes in such genes suggest that ruminant animals administered high-S water may be undergoing an inflammation or immune response, even if signs of sPEM or compromised health are not readily observed. Further study of these, and other affected genes, may deliver new insights into the physiology underlying the response to high dietary S, ultimately leading to the development of treatments for high S-affected ruminant livestock.

Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging.

Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research.

Dose comparison of single versus double dose in contrast-enhanced magnetic resonance angiography of the renal arteries: intra-individual cross-over blinded trial using Gd-DTPA.

This study was planned as an open-label, single-centre trial with blinded evaluations by two independent radiologists, aimed at the intra-individual comparison of single-dose and double-dose Gd-DTPA-enhanced MRA in the renal arterial territory. Ten healthy volunteers were included in the study. Renal MRAs were carried out on a clinical 1.5-T MR system using a body phased-array coil. Images were acquired with three-dimensional fast spoiled gradient echo sequence. Contrast agent was injected with a power injector keeping the injection time constant for single dose and double dose. Both readers found at least 96% of vascular segments evaluable. Median overall image quality was rated excellent, and diagnostic confidence was rated confident. No statistically significant difference between the dosage groups could be demonstrated. Signal intensity, SNR and CNR were significantly higher for the double-dose group. Our results demonstrate that while a double dose of contrast agent increases SNR, it does not lead to further improvement in visual and perceptual image quality. A single dosage of approximately 0.1 mmol/kg bw Gd-DTPA may be the preferable dosage to demonstrate the renal arteries.

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor.

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (alpha v beta6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an alpha v beta6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-zeta. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin alpha v beta6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.

Exploration of prostanoid receptor subtype regulating estradiol and prostaglandin E2 induction of spinophilin in developing preoptic area neurons.

The prostaglandin E2 (PGE2) mediates estradiol-induced masculinization of sexual behavior in the rat during a perinatal sensitive period. PGE2 induces formation of dendritic spines on preoptic area (POA) neurons and this synaptic pattern change is associated with the ability to express male sexual behavior as an adult. Whether PGE2 is released from astrocytes or neurons in the developing POA is unknown. To further understanding of how PGE2 induces dendritic spine formation at the cellular level, we have explored the PGE2 receptor subtype mediating this response. There are four receptors for PGE2, EP1, EP2, EP3 and EP4, each having unique but interacting signal transduction profiles. Treatment of newborn female rats with the EP receptor agonists iloprost, butaprost and sulprostone indicated that stimulation of both the EP2 and EP3 receptors significantly increased spinophilin, a protein whose levels positively correlate to the presence of dendritic spines and masculinization of the POA. Use of antisense oligonucleotides against the mRNA for each receptor reveals that either EP2 or EP3 receptor knockdown reduces spinophilin in PGE2- or estradiol-treated females, whereas reducing EP1 or EP4 receptor levels by the same means has a smaller but also significant effect. A developmental profile of EP receptor expression indicates EP1 in particular is elevated for the first few days of life, corresponding to the critical period for masculinization, whereas mRNA levels for the other three receptors remain relatively constant.

Effect of stocking density on performance, diet selection, total-tract digestion, and nitrogen balance among heifers grazing cool-season annual forages.

Grazing annual cool-season forages after oat grain harvest in South Dakota may allow an opportunity to increase efficient use of tillable land. However, data are limited regarding effects of stocking density on diet selection, nutrient digestion, performance, and N retention by cattle grazing annual cool-season forage. Heifers were blocked by initial BW (261 ± 11.7 kg) and randomly assigned to 1 of 12 paddocks (1.1 ha) to graze a mixture of grass and brassica for 48 d. Each paddock contained 3, 4, or 5 heifers to achieve 4 replicates of each stocking density treatment. Ruminally cannulated heifers were used to measure diet and nutrient intake. Effects of stocking density on diet and nutrient selection were measured after 2, 24, and 46 d of grazing, and BW was measured at the beginning, middle, and end of the experiment as the average of d 1 and 2, d 22 and 23, and d 47 and 48 BW, respectively. Measures of DMI and DM, OM, NDF, and ADF digestion were collected from d 18 to 23. Increased stocking density increased intake of brassica relative to grass on d 24 (quadratic, = 0.02), but increased stocking density decreased (linear, ≤ 0.01) intake of brassica compared with grass on d 48 (stocking density × time, < 0.01). Increased stocking density increased DM (quadratic, < 0.01), OM (quadratic, = 0.01), and NDF (quadratic, = 0.05) digestion, and stocking density tended to increase DMI (quadratic, = 0.07). Additionally, increased stocking density quadratically increased ( = 0.05) N retention but did not affect overall BW gains. Increased stocking density did, however, contribute to linearly decreased ( = 0.05) BW gains from d 1 to 22 of grazing, but BW gains during the latter half of the experiment were greater than BW gains from d 1 to 22. Ruminal concentration of acetate:propionate was least on d 24 of grazing, and ruminal nitrate concentration tended to linearly decrease ( = 0.06) with greater amounts of time on pasture. Ruminal liquid and particulate fill and amounts of VFA were less (quadratic, ≤ 0.01) with greater amounts of time on pasture. Apparently, binary mixtures of brassica and grass planted after oat grain harvest can provide an opportunity to increase efficient use of land by providing forage resources. Increased stocking density may facilitate a more rapid adaptation to and intake of brassica among cattle grazing brassica-grass-based pastures.

Lysine bioavailability among 2 lipid-coated lysine products after exposure to silage.

We conducted 2 experiments to determine lysine bioavailability from 2 lipid-coated lysine products. In an in vitro experiment we mixed each lipid-coated lysine product with either alfalfa- or corn-silage at different amounts of acidity. Scanning electron micrographs indicated that surface structure of each lipid-coated lysine particle was eroded after mixing with silage. Additionally, visual evaluation of scanning electron micrographs suggested that peripheral surface abrasion of lipid-coated lysine may be greater when lipid-coated lysine was mixed with alfalfa silage in comparison to corn silage. In a corresponding experiment, in vivo measures of lysine bioavailability to sheep from 2 lipid-coated lysine products and lysine-HCl were determined after mixing in corn silage. Plasma lysine concentrations increased linearly (P < 0.01) in response to abomasal lysine infusion indicating that our model was sensitive to increases in metabolizable lysine flow. Bioavailability of each lipid-coated lysine source and dietary lysine-HCl were calculated to be 23, 15, and 18%, respectively. Even though each dietary source of lysine increased plasma lysine, rates of increases in plasma lysine from one lipid-coated lysine source (linear; P = 0.20) and lysine-HCl (linear; P = 0.11) were not different from plasma lysine levels supported by diet alone. However, the rate of plasma lysine increase in response to lysine from the other lipid-coated lysine source was greater (P = 0.04) than plasma lysine from feed alone. Nonetheless, the rate of plasma lysine increase in response to lipid-coated lysine did not differ (P ≥ 0.70) from the rate of plasma lysine increase from lysine-HCl. Clearly, methods of manufacture, together with physical and chemical characteristics of diet, can impact amounts of metabolizable lysine provided from lipid-coated lysine products. Direct measures of lysine bioavailability from lipid-coated lysine products after mixing with diets should be based on measurements with the products treated similarly to the method of feeding.

Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

Biochemical and endocrine properties of a mechanism-based inhibitor of aromatase.

The conversion of androgens to estrogens by aromatase represents a major alteration in hormonal expression, and its regulation offers a promising method for therapeutic control of disease processes that are hormonally dependent. The design of suicide inhibitors based on enzyme-activated mechanisms provides an attractive approach for regulation of estrogen biosynthesis. MDL 18,962, (10-2(-propynyl)estr-4-ene-3,17-dione), a C-10 substituted analog of the natural substrate androstenedione, was evaluated as a suicide inhibitor of aromatase. Appropriate kinetic evaluations of MLD 18,962 established it to be a highly potent [inhibition constant (Ki) = 4.5 +/- 1.3 nM] irreversible inhibitor of human placental aromatase. The 2-propynyl group was necessary for time-dependent inactivation, as this activity was lost in related compounds. The inactivation process was specific for aromatase, since other P450 enzyme systems are not inactivated by MDL 18,962. This compound exhibited minimal intrinsic hormonal properties, since weak binding affinities were observed for cytosolic androgen, estrogen, or progestin receptors. The stimulation of ovarian aromatase activity by gonadotropins in immature mice was inhibited in animals implanted with 10-mm MDL 18,962 Silastic implants. This inhibition of estrogen biosynthesis suppressed estrogen-dependent uterine growth in these mice.

Pharmacological induction of 5 alpha-reductase deficiency in the rat: separation of testosterone-mediated and 5 alpha-dihydrotestosterone-mediated effects.

(5 alpha, 20S)-4-Diazo-21-hydroxy-20-methylpregnan-3-one, a mechanism-based inhibitor of testosterone 5 alpha-reductase, produced pronounced and long lasting inhibition of the enzyme in the prostate and preputial glands when administered orally to rats. Administration of the inhibitor to castrate rats bearing testosterone implants produced inhibition of growth of the prostate, seminal vesicles,and preputial glands, but had no effect on growth of the levator ani muscle. (5 alpha-20S)-4-Diazo-21-hydroxy-20-methylpregnan-3-one did not antagonize growth of the accessory sex organs induced by 5 alpha-dihydrotestosterone (5 alpha-DHT). The inhibitor thus produced a pure 5 alpha-reductase deficiency in the rat without detectable receptor-level antagonism, and with consequences similar to those occurring at puberty in the 5 alpha-reductase-deficient human male: reduction of 5 alpha-DHT-mediated processes and maintenance of those mediated by testosterone. The results emphasize the importance of the enzymatic profiles of individual organs in determining selective response to testosterone or 5 alpha-DHT. Evidence is presented which indicates that the testes are the source of circulating 5 alpha-DHT in the immature rat.

Histology of the stomach and duodenum in Crohn's disease.

Crohn's disease (CD) not uncommonly affects the stomach and duodenum, but its histologic appearance is not well described beyond the identification of granulomas. We retrospectively identified 209 upper gastrointestinal biopsy samples from 80 sets of biopsies from 49 patients with CD. Age- and sex-matched control biopsies were selected from recent cases of Helicobacter pylori gastritis (73 biopsy samples from 34 patients), from patients with a known history of nonsteroidal antiinflammatory drug use (18 biopsy samples from 12 patients), and from three patients with ulcerative colitis. Architectural and inflammatory changes were evaluated and compared. Over three fourths of the patients with CD had abnormal biopsy results. Fifty-six percent of patients with CD had acute inflammation, but only 10% of the patients were infected with H pylori. Focal acute inflammation was a characteristic of H pylori-negative CD (stomach, 31%; duodenum, 40%), which was much less common in the non-CD group (stomach, 2%; duodenum, 8%). Surface intraepithelial neutrophils of the duodenum were more common in H pylori-negative patients with CD (25%) than in those who did not have CD (4%), and deep acute inflammation of the duodenum was more likely in H pylori-negative patients with CD (19% vs. 0%). Granulomas were found in only 9% of the CD group. Focal acute inflammation of the gastroduodenum, especially in a background of noninflamed mucosa, is strong evidence for CD in the appropriate clinical context, but the stomach and duodenum must be properly sampled and carefully examined for any evidence of H pylori.

Inhibition of human monocyte antigen presentation, but not HLA-DR expression, by cyclosporine.

The effects of cyclosporine (CsA) on antigen-dependent human T cell proliferation have been studied using tetanus toxoid as the antigen. CsA significantly inhibited antigen-dependent T cell proliferation at concentrations as low as 0.1 microgram/ml. In dissecting this system we found that preexposure of separated monocytes to CsA during the period of antigen processing led to a marked inhibition of proliferation of T cells added subsequently to the monocytes. We investigated whether this suppressive effect on monocyte antigen presentation was related to monocyte HLA-DR expression, interleukin 1 (IL-1) production, or prostaglandin (PG) secretion. None of these functions seemed to be affected by CsA. In particular, human monocyte HLA-DR expression was not inhibited by CsA, even at concentration of 10 micrograms/ml. The addition of exogenous IL-1 or indomethacin did not reverse the inhibitory effects of CsA. These experiments demonstrate that CsA inhibits antigen-dependent human T cell proliferation, at least in part by acting directly on human monocytes to inhibit antigen presentation. The mechanism of action seems to be independent of IL-1 production, PG secretion, and HLA-DR expression.

Enzyme-activated inhibitors of steroidal hydroxylases.

Cytochrome P450 monooxygenases (CYP450) of the steroid biosynthetic pathways are highly substrate specific in comparison to the variable specificities of hepatic CYP450 enzymes. Both groups of enzymes catalyze the reductive cleavage of molecular oxygen with transfer of oxygen to the substrate to form hydroxylated derivatives. Those steroids formed in endocrine tissues represent highly specific endocrine/autocrine hormones with enhanced biological potency, while hepatic hydroxylation of steroids reduces their endocrine bioactivities and enhances urinary elimination. Changes of the hormonal milieu of endocrine and peripheral tissues are associated with the development of hyperplastic and/or malignant conditions. Hormone deprivation induces regression of endocrine dependent growth via apoptosis and may also alter growth of hormone insensitive cells by the induction of negative growth factors. Biosynthetic CYP450 enzymes of those steroids that mediate specific disease processes are potential therapeutic targets for selective intervention. This objective can be accomplished by the design of specific pseudo-substrate analogs that will be activated during enzyme-directed catalysis to produce a reactive functional group in the enzyme's active site that will either tightly or irreversibly bind and inactivate the host enzyme. The CYP450 enzymes that hydroxylate the C19 carbon of androgens (aromatase) and the C18 carbon of corticosterone (aldosterone synthase) were selected as target enzymes because they are terminal enzymes of biosynthetic pathways which hydroxylate specific angular methyl groups. Hypersecretion of their respective hormonal products, estrogens and aldosterone, are associated with specific disease conditions. Substrate analogs containing ethynyl, vinyl, or nitrile groups attached to the C19 or C18 methyl groups were enzyme-activated inhibitors. The ethynyl analogs, 19-acetylenic androstenedione (Plomestane) and 18-acetylenic deoxycorticosterone, had nanomolar inhibitory constants (Ki values) and were irreversible inactivators of their target enzymes in animal models.

A-ring bridged steroids as potent inhibitors of aromatase.

The design and syntheses of androstenedione derivatives with bridges spanning the 2,19-, 3,19-, 4,19- and 6, 19-positions are described. 2,19-Bridged compounds bearing hydroxyl groups on the two-carbon bridge (3a and 3b) were designed as stable carbon analogs of potential lactol intermediates in the enzymatic conversion of androgens to estrogens. Compounds 3a and 3b are competitive inhibitors of aromatase. Pyran 25 is a potent, time-dependent inhibitor of aromatase with partial NADPH dependence. These data suggest a mechanism of inhibition for 25 which involves both tight-binding competitive and mechanism-based components, with the former predominating. The sulfur, amino, and all carbon analogs of pyran 25 were prepared. Thiopyran 36, piperidine 42 and the all-carbon analog 47 are also time-dependent inhibitors of aromatase. Compound 47 is the most potent inhibitor and its time-dependent inhibition is not NADPH dependent. The kinetics of piperidine 42 suggest uncompetitive inhibition.

Biological characterization of A-ring steroids.

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar affinities for aromatase. These inhibitory constants (Ki values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (Ki = 35-70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (Ki = 2 nM). Substitution of a methylene group with oxygen and sulfur in this expanded bridge resulted in Ki values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time-dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring residual enzyme activity. Part of this inhibition was NADPH cofactor-dependent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited tau 50 values, the t1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A-ring-bridged androstenedione analogs represent a novel series of potent steroidal aromatase inhibitors. The restrained A-ring bridge containing CH2, O, S, or NH could effectively coordinate with the heme of the P450 aromatase to allow the tight-binding affinities reflected by their nanomolar Ki values.