An ex vivo co-culture model system to evaluate stromal-epithelial interactions in breast cancer.

Breast cancer is the most commonly diagnosed cancer among women worldwide. High breast cancer incidence and mortality rates, especially in obese patients, emphasize the need for a better biological understanding of this disease. Previous studies provide substantial evidence for a vital role of the local extracellular environment in multiple steps of tumor progression, including proliferation and invasion. Current evidence supports the role of adipocytes as an endocrine organ, which produces steroid hormones, pro-inflammatory cytokines and adipokines, such as leptin. To further define the role of the mammary microenvironment on tumorigenesis, we have developed an adipose-tumor epithelial cell co-culture system designed to reproduce the in vivo mammary environment. We validate this model through use of coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. CARS analysis demonstrates the sustained viability of the adipocytes, and that mammary cancer cell morphology parallels that of tumors in vivo. Also, characterized was the influence of mammary adipose tissue on tumor cell growth and migration. Adipose tissue co-cultured with mammary tumor epithelial cells, in the absence of any serum or supplemental growth factors, resulted in substantial increases in growth and migration of tumor cells. In conclusion, this novel co-culture system provides an ideal model to study epithelial-stromal interactions in the mammary gland. Understanding the relationship between adipose tissue, the most abundant and least studied component of the breast stroma and tumor epithelial cells is critical to clarifying the influence of obesity on the development, progression and prognosis of breast cancer.

Antagonism of physostigmine induced hypothermia and neuroendocrine changes following exposure to different environmental temperatures.

1. The magnitude of physostigmine-induced hypothermia increased with decreasing environmental temperature. 2. The hypothermic response was accompanied by significant changes in plasma levels of corticosterone, glucose and fatty acids. 3. Central cholinergic mediation appears to be a significant component of physostigmine-induced hypothermia and neuroendocrine changes at moderate temperature. 4. At lower ambient temperatures cholinergic blockers produced less antagonism of physostigmine-induced effects. 5. The decreased effectiveness of cholinergic blockers at low environmental temperatures and the increased plasma fatty acid levels under almost all conditions studied may be of importance in considering long term therapy with cholinergic agonists.

Effects of environmental temperature on hypothermia and neuroendocrine changes induced by soman.

The effects of environmental temperature on body temperature and neuroendocrine parameters were evaluated following a single acute dose (60 micrograms/kg) of soman. Plasma levels of corticosterone, glucose, and free fatty acids, as well as acetylcholinesterase activity in plasma, erythrocytes, and brain were determined over a 96-hr time course in rats maintained at 23-25, 14-16, and 3-5 degrees C. Considerable inhibition of plasma and erythrocyte acetylcholine hydrolysis activity was observed after administration of soman at all three environmental temperatures. The degree of hypothermia in all soman-treated rats in each environment tested was associated with the amount of brain acetylcholinesterase inhibition. In animals maintained at 23-25 and 14-16 degrees C, changes in plasma corticosterone levels were influenced by central acetylcholine hydrolysis. Hyperglycemia was found only in rats with greater than 45% brain inhibition regardless of environmental temperature. However, the plasma concentration of glucose over the 96 hr test period varied in relation to the hydrolysis of acetylcholine in soman-treated rats. Recovery of plasma acetylcholinesterase was more rapid at lower environmental temperatures. A greater inhibition of central acetylcholinesterase was found in soman-treated rats exposed to 3-5 degrees C. Soman may be more toxic at low environmental temperatures.

Amphetamine enantiomers and rat consummatory behavior: a new perspective.

The anorectic actions of amphetamine have been known for over forty years, yet the precise relationship(s) between the enantiomeric forms of the drug and anorexia is not clearly understood. Previous studies have utilized primarily racemic amphetamine or its d-isomer in the analysis of feeding behavior. In the present investigation, a detailed examination of the effects of single and repeated equiactive doses of d- and l-amphetamine on food consumption by adult male rats was undertaken with emphasis on aspects of tolerance development. Weight loss and pattern of daily food intake differed depending upon the isomer, dose, and degree of tolerance. Two types of tolerance were seen with both isomers, an initial tolerance with a decrease in efficacy between days 1 and 2, and a later gradual decrease in efficacy over 12 days of repeated dosage. Rats tolerant to the anorectic effects of d-amphetamine were only minimally affected when challenged with an equiactive anorectic dose of l-amphetamine, while rats tolerant to the anorectic effects of l-amphetamine showed a significantly depressed food intake and modified eating pattern when challenged with an equiactive dose of d-amphetamine. Therefore two-way cross tolerance, as previously assumed, does not completely exist between low equiactive doses of d- and l-amphetamine.

Time course of physostigmine effects on neuroendocrine responding at varying environmental temperatures.

1. Hypothermia was found to be related to both the dose of physostigmine and the environmental temperature. 2. Plasma corticosterone levels were elevated above controls regardless of dose of physostigmine or environmental temperature. 3. Plasma free fatty acid levels appeared to be inversely related to physostigmine-induced hypothermia. 4. A hyperglycemic response was observed under all experimental conditions at 0.5 hours and 1.0 hour post injection. 5. Significant inhibition of brain acetylcholinesterase was observed, whereas, plasma and erythrocyte acetylcholinesterase activity was inconsistent.

The redox centers of xanthine oxidase are on independent structural domains of the enzyme.

Xanthine oxidase employs four electron transport sites (flavin adenine dinucleotide (FAD), molybdenum, and two FeS centers) in catalyzing a variety of redox reactions. To determine whether the redox sites reside in independent domains of the enzyme, the temperature of heat inactivation of each site's catalytic activity was determined, except that no attempt was made to distinguish between the two FeS sites. In the oxidase form of xanthine oxidase, the order of thermal stabilities was Mo greater than FAD greater than FeS, while after conversion to its dehydrogenase form the above ranking was Mo greater than FeS greater than FAD. The small but reproducible difference in heat inactivation temperatures among the redox sites demonstrated that the sites are located in separate domains of the enzyme. To confirm the above segregation of redox centers, the temperature of heat-induced release of each redox cofactor from its site on the enzyme was examined. These temperatures were found to be different for each redox cofactor and agreed closely with the heat inactivation temperatures measured above. The data thus demonstrate that both heat inactivation and cofactor release derive from thermal unfolding of independent domains. Using a technique termed "thermal digestion analysis," the FAD domain was located in a approximately equal to 42,000-Da tryptic fragment, while the FeS and Mo domains were isolated in a trypsin-resistant 92,000-Da fragment. We conclude that xanthine oxidase is constructed in modular fashion with the redox sites located in independent structural domains.

Brain catecholamine alterations accompanying development of anorexia in rats bearing the Walker 256 carcinoma.

In the present study, the relationship between central catecholamine levels and the anorexia induced by Walker 256 carcinoma was investigated. Results indicate that the anorexia is not due to depletion of central catecholamines. Tumor bearing rats sacrificed at night, when spontaneous food intake is selectively depressed, showed increased norepinephrine levels in the hypothalamus, cortex and hippocampus and increased dopamine levels in the striatum, midbrain, and cortex. Increased nighttime hypothalamic norepinephrine levels were positively correlated with the magnitude of spontaneous food intake in tumor rats.

Effects of certain hallucinogenic amphetamine analogues on the release of [3H]serotonin from rat brain synaptosomes.

The enantiomers of 3,4-(methylenedioxy)amphetamine (MDA), p-methoxyamphetamine (PMA), and N-Me-MDA (MDMA), along with their alpha, alpha-dimethylated derivatives, were evaluated for an effect on the release of [3H]serotonin from rat whole brain synaptosomes. The amphetamine isomers were all potent in inducing the release of [3H]serotonin at bath concentrations of 1 and 10 micrometers but were inactive at 0.1 micrometers. No significant difference in isomer potency was observed at the 10 micrometers concentration. However, at 1 micrometer the (+) isomer of MDMA was more effective in inducing release than was the (-) isomer. Since it is the (+) isomer which is clinically active, this result suggests that transmitter release may play a role in the biological activity of MDMA. By contrast, the alpha, alpha-dimethyl compounds were not effective in releasing serotonin, even at the highest bath concentration.

A new, potent, conformationally restricted analogue of amphetamine: 2-amino-1,2-dihydronaphthalene.

A new stimulant compound, 1,2-dihydro-2-naphthalenamine (2-amino-1,2-dihydronaphthalene, 2-ADN), was prepared as an analogue of amphetamine and of 2-aminotetralin. The optical isomers of 2-ADN were obtained by chemical resolution, and the absolute configuration was determined to be R-(+) and S-(-). Preliminary pharmacological evaluation revealed that racemic 2-ADN is approximately one-fourth as potent as (+)-amphetamine as a stimulant in mice. The S-(-) isomer of 2-ADN was found to be solely responsible for the stimulant effects of the racemate. Both reserpine and alpha-methyl-p-tyrosine antagonized the stimulation produced by 2-ADN.

Synthesis and adrenergic blocking effects of 2-(alkylamino)-3,4-dihydroquinazolines.

Based on the known biological activity of a variety of guanidine-containing agents, several N-substituted 3,4-dihydroquinazolines were synthesized. These compounds can be considered to be rigid analogues of phenylguanidines. In anesthetized rats the compounds decreased blood pressure and were antagonists of the pressor response to norepinephrine.