Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

Controlled experiments of habitat fragmentation: a simple computer simulation and a test using small mammals

Habitat fragmentation involves a reduction in the effective area available to a population and the imposition of hard patch edges. Studies seeking to measure effects of habitat fragmentation have compared populations in fragments of different size to estimate and area effect but few have examined the effect of converting open populations to closed ones (an effect of edges). To do so requires a shift in spatial scope-from comparison of individual fragments to that of fragmented versus unfragmented landscapes. Here we note that large-scale, controlled studies of habitat fragmentation have rarely been performed and are needed. In making our case we develop a simple computer simulation model based on how individual animals with home ranges are affected by the imposition of habitat edges, and use it to predict population-level responses to habitat fragmentation. We then compare predictions of the model with results from a field experiment on Peromyscus and Microtus. Our model treats the case where home ranges/territories fall entirely within or partially overlap with that of sample areas in continuous landscapes, but are restricted to areas within habitat fragments in impacted landscapes. Results of the simulations demonstrate that the imposition of hard edges can produce different population abundances for similar-sized areas in continuous and fragmented landscapes. This edge effect is disproportionately greater in small than large fragments and for species with larger than smaller home ranges. These predictions were generally supported by our field experiment. We argue that large-scale studies of habitat fragmentation are sorely needed, and that control-experiment contrasts of fragmented and unfragmented microlandscapes provide a logical starting point.     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm² of growth flask). Results show that the apparent expression of extended Le^a-Le^x, Le^a and Le^x, sialyl Le^a, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.Abbreviations Mab monoclonal antibody - FIT fluorescein isothiocyanate - TACA tumor associated carbohydrate antigen     

Black tea,green tea,and tea polyphenols

Previous studies have shown that tea consumption can impair trace element metabolism, particularly iron status, and increase the risk of anemia in humans and animals. More recently, however, evidence has been accumulating to show that, in animals, consumption of green tea or its polyphenols is associated with a reduction of the incidence and severity of a variety of experimentally induced cancers. In this study we have monitored the growth, trace element status, including hematological parameters of weanling rats given either (1) water, (2) 1% black tea, (3) 1% green, tea, or (4) 0.2% crude green tea extract as their sole drinking fluid while consuming diets containing either adequate or low amounts of iron. With the exception of manaanese, none of the trace elements studied (iron, copper, zinc, and manganese) or the hematological indices measured were affected by the type of beverage supplied, even though the polyphenol extract was shown to chelate metals in vitro and all the animals fed the low iron diet were shown to be anemic. There appeared to be an effect of black and green teas on manganese balance in, both the first and last weeks of the study. A lower level of brain managanese was associated with green tea consumption, and a higher level of this element in the kidneys of animals fed black tea. The results demonstrate that both black and green teas and a green tea polyphenol extract do not represent a risk to animals consuming the beverages as their sole fluid intake with respect to iron availability, although the interactions with manganese deserve further study.     

Physiological aspects of Taxus brevifolia seeds in relation to seed storage characteristics

Water relations, desiccation tolerance and longevity of Taxus brevifolia (Nutt.) seeds were studied to determine the optimal stage of development and storage conditions for seeds of this species. Seeds equilibrated to a range of relative humidities (RHs) had unusually low water contents which can be accounted for by the high lipid content of gametophyte tissues (71% of the dry mass). Water relations of embryonic tissue were more typical of those reported for other seed species. The water content below which freezing transitions were not observable in the embryo was ca 0.24 g H₂O (g dry weight)⁻¹ (g g⁻¹) for all maturity classes studied. Embryos did not achieve significant levels of desiccation tolerance (survival to water contents less than 0.5 g g⁻¹) until the latter stages of development when dry matter was maximal. Mature embryos could be dried to 0.025 g g⁻¹ (seed water content of 0.010 g g⁻¹) with no loss of viability. Thus, at the latter stages of development, embryo water content could be optimized to avoid both desiccation and freezing damage. Survival of mature seeds declined over a 2-year period when seeds were stored at temperatures between 5 and 35°C and RHs between 14 and 75%, corresponding to seed water contents between 0.015 and 0.07 g g⁻¹. The deterioration rate was slowest for seeds stored at the lowest RH and temperature. Our data indicate that seeds of Taxus brevifolia show orthodox rather than recalcitrant storage characteristics, but that the optimum water content for storage was extremely low. The results suggest that even if stored at optimal water contents and low temperatures, T. brevifolia seeds will be relatively short lived. The high quantity of lipids or reducing sugars may be contributing factors in the poor storage characteristics.     

Induction of leaf senescence in Arabidopsis thaliana by long days through a light-dosage effect

Given the influence of photoperiod on reproductive development and whole-plant senescence in monocarpic plants, one would suspect that leaf senescence in these plants might be under photoperiodic control. In Arabidopsis thaliana , which is monocarpic and also a nonobligate long-day (LD) plant, LDs (16 h, 300 μmol m⁻² s⁻¹) caused leaves to die earlier than did short days (SDs, 10 h). Since leaf longevity was not paralleled by the reproductive development in the present study, the reproductive structures did not seem to be the primary controls of leaf senescence. The LD effect appeared to depend on the amount of light rather than on day length, for leaves given LDs at reduced light intensity (180 μmol m⁻² s⁻¹) lived longer than those in LDs with full light. In addition, the higher light intensity promoted chlorophyll loss and anthocyanin accumulation in LDs. Thus, senescence of these leaves seems to be governed by light dosage rather than photoperiod. Light may play a natural role in promoting the senescence of A. thaliana leaves.     

The empirical question of thresholds and mechanisms of mate choice

Summary Theoretical discussions concerning how animals might best sample and select mates have suggested that individuals could base decisions either on a sample of mates (sampled-based decisions) or on a threshold of comparison (threshold-based decisions). Recent theoretical work demonstrates that threshold-based mating decisions generate higher expected fitness than sample-based mating decisions when search costs exist. Empirical results from most unmanipulated systems, however, either conclude that females make sample-based decisions or are inconclusive. A few experimental studies designed to detect mating thresholds purport to demonstrate threshold-based choice but an examination of these studies indicates such conclusions were premature. We believe that few examples of threshold-based choice exist because protocols designed to identify mating thresholds were often inconsistent with models of threshold choice. We suggest that future empirical work strive not to document mating thresholdsper se. Rather, future work might best reveal decision rules by manipulating the distribution of quality among potential mates; such manipulations predict uniquely how females using sample-based and threshold-based decision rules should behave.     

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.