Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid.

Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from [2-3H]estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from [2-3H]estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid.     

Effect of the novel ionophore tetronasin (ICI 139603) on ruminal microorganisms.

The antimicrobial activity of the novel ionophore tetronasin (formerly ICI 139603) was compared with that of monensin for the growth of ruminal bacteria, protozoa, and an anaerobic fungus. The potency of tetronasin toward most bacteria and the fungus was an order of magnitude or more greater than that of monensin. Lactobacillus casei was 55 times more sensitive to tetronasin than to monensin, indicating a potential role for tetronasin in reversing lactic acidosis. Bacteria with a gram-positive ultrastructure were generally sensitive to the ionophores and unable to adapt to grow in their presence. The exception was the cellulolytic Ruminococcus flavefaciens, which adapted during successive cultivation on media with increasing ionophore concentrations to grow at 100-fold higher concentrations of tetronasin than were initially lethal to the organism. Gram-negative bacteria were more resistant and generally able to adapt to grow in the presence of both ionophores. An in vivo trial with cattle and in vitro growth experiments indicated that the effect of tetronasin on ciliate protozoa was minor. In vitro experiments measuring hydrogen production by Neocallimastix frontalis suggested that this fungus would be unable to survive in ruminants receiving tetronasin.     

Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.     

The effect of grazing intensity on phosphorus spiralling in autotropic streams

The effect of grazing on primary productivity and phosphorus cycling in autotrophic streams was studied using the snail Goniobasis clavaeformes. Snails were added to each of three replicate laboratory stream channels, receiving once-through flow of groundwater, in densities of 2.1, 3.0, and 4.2 g ash free dry mass (AFDM)/m². A fourth channel received no snails and served as an ungrazed control. Presence of snail grazers resulted in a large reduction in aufwuchs biomass, primary productivity, and biotic phosphorus uptake; a modest reduction in fine particulate organic matter (FPOM); and an increase in the fraction of stream particulate organic matter (POM) exported as seston. Although primary production and aufwuchs biomass continued to decline with increasing snail density, phosphorus uptake increased. This increased phosphorus uptake is attributed to abiotic sorption to inorganic surfaces exposed as a result of efficient removal of aufwuchs at high snail densities. Although snail densities were chosen to bracket the density measured in a natural stream, the experimental densities may result in considerably higher grazing pressure on aufwuchs due to the absence of alternate food sources (e.g., coarse particulate organic matter) usually found in natural streams. Presence of snail grazers increased the spiralling length of phosphorus, primarily by reducing aufwuchs biomass and consequently reducing uptake of phosphorus from the water. Presence of snails also increased downstream transport velocity of phosphorus bound to organic particles. These results follow the patterns predicted in a previous theoretical analysis for mildly phosphorus-limited streams.     

A recessive mutation causing imperforate vagina in mice

A recessive mutation (ipv) causing imperforate vagina was discovered in a line of mice selected for low lean tissue mass as a proportion of body weight. Two full sisters were found to have marked swelling of the perineum and complete closure of the vagina. Crosses of heterozygotes identified by progeny testing produced a female progeny ratio not different from the 3 normal: 1 affected (chi 2 = 0.695; p less than .3) expected on the basis of a recessive allele at a single autosomal locus. As a consequence of the imperforate vagina, the uterus and vagina were greatly distended by fluid. The uterus of affected females displayed a swollen uterine lumen and thin endometrial stroma and muscularis. Ovarian tissue of affected females was similar to that of normal mice, and affected females produced ova that were normal in appearance. The mutation causing an imperforate vagina may present a useful model for studying the basis of abnormal vaginal development in other species and increasing the understanding of normal vaginal development in the mouse.     

Immature mouse uterine tissue in organ culture: Estrogen-induced growth,morphology and biochemical parameters

Summary Although estrogens have been shown to stimulate a variety of morphologic and biochemical changes in the uterus in vivo, no clear consistent demonstration of similar responses in vitro have been made; thus, a defined organ culture system using the immature mouse uterus was established to study the possibility of demonstrating estrogenic responses in vitro. Uterine tissue from immature outbred mice (17 to 24 days of age) were cut crosswise in 1-mm³ coins and cultured in a defined medium in the absence of serum, phenol red, or growth factor supplements. Diethylstilbestrol (DES), a synthetic estrogen, was added to the media at doses ranging from 1 to 100 ng/ml. The effect of DES on uterine cell proliferation was assessed by morphologic changes in uterine epithelial and stromal cells, increase in number of epithelial cells per unit basement membrane, increase in height of luminal epithelial cells, and [³H]thymidine incorporation. Functional changes were determined by measuring the amounts of the estrogen-inducible uterine protein, lactoferrin, that was localized in the epithelial cells and secreted into the media, and the localization of the estrogen receptor in the cultured tissues. Results indicate that under the described conditions of culture, estrogens like DES can induce morphologic and biochemical responses in the uterus that are similar to those seen in vivo. This organ culture system will aid in the investigation of various mechanisms involved in the hormonal regulation of growth and differentiation of estrogen target tissues.     

Properties of ionophore-resistant Bacteroides ruminicola enriched by cultivation in the presence of tetronasin.

Bacteroides ruminicola M384 was grown in the presence of increasing concentrations of tetronasin, an ionophore that has been developed as a feed additive for ruminants. The resulting culture, B. ruminicola M384/TnR, was then maintained in medium containing 0.1 microgram tetronasin/ml. Growth of the parent strain was eliminated by the addition of 0.1 micrograms tetronasin/ml, but the growth rate of B. ruminicola M384/TnR, which grew more slowly than the parent strain, was unaffected by adding tetronasin. Bacteroides ruminicola M384/TnR retained its resistance to tetronasin even after repeated subculture in the absence of the ionophore, suggesting that a mutation had occurred. The absence of plasmids in individual colonies of B. ruminicola M384/TnR implied that the mutation was chromosomal. Bacteroides ruminicola M384/TnR was also more resistant to the ionophores monensin and lasalocid and, to a lesser degree, to the antibiotic avoparcin than B. ruminicola M384. Binding of [14C]tetronasin to B. ruminicola M384/TnR was lower than binding of the ionophore to the parent stain, and this difference was eliminated by washing cells with EDTA. The peptidolytic activity of B. ruminicola M384 towards triphenylalanine (Mr = 460) was unaffected in B. ruminicola M384/TnR, but the rate of breakdown tetraphenylalanine (Mr = 607) was decreased. This difference was also abolished by EDTA. It was concluded that growth of B. ruminicola in the presence of tetronasin resulted in a mutation affecting the permeability of the cell envelope, such that permeation of tetronasin and molecules of a similar size (Mr = 628) was decreased.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

Predictive systems ecology

Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.