Immunocytochemical detection of prolactin or prolactin-like immunoreactivity in epididymis of mature male mouse

Summary Prolactin (PRL) binds to the testis of mice and rats where it increases the number of luteinizing hormone receptors, increases the binding of human chorionic gonadotropin (hCG) to LH receptors, and enhances testosterone synthesis and secretion. PRL also binds to the prostate and seminal vesicles of rats and humans where it increases organ weight and stimulates growth and uptake of testosterone. PRL binds to the epididymis of rats but the effect of PRL on this organ is unknown. In the present study, a standard immunoperoxidase (PAP) technique was used to detect the binding of endogenous and exogenous PRL or PRL-like peptides to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, suggesting PRL or PRL-like binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous PRL were slightly stronger than those testing for binding of endogenous peptides. The significance of such binding to the epididymis is uncertain but PRL may perform the same functions in epididymal principal cells as it does in the testis, prostate, and seminal vesicles.     

Persistent sympathetic nervous system arousal associated with tethering in cynomolgus macaques

The swivel-tether system has been used extensively in biomedical research involving nonhuman primates, yet there has been little or no investigation into potential adverse influences of this form of restraint on research results. In the study described here, a portable electrocardiographic telemetry system was used for continuous monitoring of the heart rate of 26 cynomolgus monkeys while: (a) pair-caged, 8 weeks prior to tethering; (b) singly-caged, tethered; (c) singly-caged, tethered, administered propranolol (30 mg/kg/day) in the diet; (d) group-housed (five monkeys per group), 1 week after group formation; and (e) group-housed (five monkeys per group), 4 weeks after group formation. Tethering resulted in persistent elevations in heart rate relative to the other conditions. Administration of propranolol, a beta-adrenergic antagonist, resulted in an abrupt, sustained decrease in heart rate indicating that the increase in heart rate associated with tethering was due to persistent stimulation of the sympathetic nervous system. Since multiple aspects of cardiovascular function are influenced by the sympathetic nervous system, and other organs and systems (e.g., pituitary-gonadal) also may be affected, investigators using the swivel-tether system should be cognizant of these potential effects when designing experiments and interpreting the results.     

Formation and turnover of long-chain fatty acid esters of 5-androstene-3 beta, 17 beta -diol in estrogen receptor positive and negative human mammary cancer cell lines in culture

Microsomal preparations derived from bovine placenta cotyledons, previously investigated as a convenient source of fatty acyl coenzyme A: estradiol-17 beta-acyl transferase, have been shown to acylate other steroids bearing 3 beta- or 17 beta-hydroxyl groups. In the presence of 0.1 mM oleoyl CoA, the apparent Km values for dehydroepiandrosterone, testosterone, and 5-androstene-3 beta,17 beta-diol (delta 5-DIOL) were 45, 67, and 20 microM, respectively. Acylation of delta 5-DIOL occurred at either the 3 beta- or 17 beta-positions to give monoesters. Testosterone, estradiol-17 beta, and delta 5-DIOL acted as competitive inhibitors for the acylation of the 3 beta-hydroxyl group of dehydroepiandrosterone (Ki values 71, 75, and 41 microM, respectively). Such data indicate that a single enzyme of wide substrate specificity may be involved in these acylation reactions. When estrogen receptor (ER) positive and negative human mammary cancer cell lines were incubated with 10 nM [3H]delta 5-DIOL, intracellular accumulation of delta 5-DIOL long-chain fatty acid esters occurred; rates being higher (p less than 0.001) in ER negative cells (MDA-MB-231 and MDA-MB-330) compared to MCF-7 cells (ER positive), and higher (P less than 0.005) in MDA-MB-231 cells compared to ZR-75-1 cells (ER positive). After exposure to 10 nM [3H]delta 5-DIOL for 16 h, the total labeled steroid fatty acid fraction was composed predominantly of delta 5-DIOL-3 beta- and 17 beta-monoesters (approximately 85%), the remainder containing approximately equal amounts of delta 5-DIOL-diesters and dehydroepiandrosterone-3 beta-esters. Subsequent transfer to medium lacking delta 5-DIOL was accompanied by a breakdown of the labeled esters, which was more rapid in the ER positive cell lines. During this period, intracellular free delta 5-DIOL levels rapidly declined in MDA-MB-330 cells but were maintained in MCF-7 cells, presumably by binding to ER. This behavior parallels that of estradiol-17 beta previously observed in these cell lines and further emphasizes the potential importance of the adrenal-derived estrogen delta 5-DIOL in consideration of a hormone-based etiology of human breast cancer.     

Short-term entrainment of ventilation to the walking cycle in humans

We describe a breath-by-breath method to test for entrainment of breathing and walking cycles. Thirty-eight normal subjects walked comfortably on a treadmill while breathing through a pneumotachograph. We analyzed the time intervals between heel strikes and the onset of inspiration (or expiration) for evidence of phase locking between steps and breaths, using Monte Carlo simulation to model the probability that n consecutive inspirations (or expirations) would begin at a constant time interval +/- 0.10 s from heel strikes by chance. We developed empirical criteria for rhythm synchronization during series of four or more breaths, while maintaining an estimated specificity of 95%. The majority of subjects showed some evidence of entrainment (29 +/- 23% of breaths on average), which occurred intermittently, usually lasting less than 10 breaths at a time. The precision of phase locking during spontaneous entrainment was similar to that in 10 subjects who attempted to maintain deliberate entrainment. The results suggest that the walking cadence provides a persuasive, but not dominant, input to the central breathing pattern generator. The present method can detect entrainment even when it occurs sporadically or with varying coupling pattern.     

DIDS decreases CSF HCO3- and increases breathing in response to CO2 in awake rabbits

An inhibitor of the HCO3-/Cl- exchange carrier protein, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or vehicle was infused in mock cerebrospinal fluid (CSF) via the cisterna magna in conscious rabbits at 10 mumol/l for 40 min at 10 microliter/min. Neither treatment had any effect over 2-5 h on the non-CO2-stimulated CSF ion values or blood gases. With CO2 stimulation such that arterial PCO2 (PaCO2) was increased 25 Torr over 3 h, DIDS treatment significantly decreased the stoichiometrically opposite changes in CSF [HCO3-] and [Cl-] that normally accompany hypercapnia and reflect ionic mechanisms of CSF pH regulation. Expressed as delta CSF [HCO3-]/delta PaCO2, DIDS treatment decreased the CSF ionic response by 35%. In a separate paired study design DIDS administration via the same protocol had no effect on resting ventilation but significantly increased the ventilation and tidal volume responses to a 28-Torr increase in PaCO2. Expressed as change in minute ventilation divided by delta PaCO2, DIDS treatment produced a 39.6% increase. The results support the concept of a DIDS-inhibitable anion exchange carrier being involved in CSF pH regulation in hypercapnia and suggest a DIDS-related effect on the ventilatory response to CO2.     

A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Understorey vegetation change in a Picea mariana chronosequence

Eighteen black spruce (Picea mariana) stands, representing postfire ages of 26 to 120 yr, were surveyed for understorey vegetation and site/microsite characteristics at two spatial scales. This enabled comparison of within- versus among-stand compositional variation.Detrended correspondence analysis (DCA) ordination among the 18 stands revealed a complex age/moisture gradient. DCA ordination among 1 800 quadrats within the stands indicated a similar gradient with much compositional overlap. Quadrats were grouped, using two-way indicator species analysis (TWINSPAN), into 9 classes each representing a phase in understorey vegetation composition. These phases shifted in abundance from young to old stands with a high degree of concordance among replicates in the same age class. Understorey succession is strongly linked to the stages in tree growth, mortality and thinning coupled with the accumulation of site moisture.Abbreviations DCA Detrended Corrospondence Analysis