Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

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.     

In vitro culture of tissue from the tunicateStyela clava

Summary Pharyngeal explants and circulatory hemocytes from the tunicateStyela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharnngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions. This study was supported by the National Science Foundation, Washington, DC (grant DCB 85 19848) and by BRSG funds from UCLA Schools of Medicine and Dentistry. Flow cytometric facilities were sponsored in part by a Johnson Cancer Center Core Grant (CA 16042). David A. Raftos is a Fulbright Postdoctoral Fellow and recipient of a Frederik B. Bang Scholarship in Marine Invertebrate Immunology administered by the American Association of Immunologist. Dan L. Stillman was supported by an REU supplement from the National Science Foundation.     

Thermal stratification, nutrient dynamics, and phytoplankton productivity during the onset of spring phytoplankton growth in Lake Baikal, Russia

Lake Baikal, Russian Siberia, was sampled in July 1990 during the period of spring mixing and initiation of thermal stratification. Vertical profiles of temperature, dissolved nutrients (nitrate and soluble reactive phosphorus), phytoplankton biomass, and primary productivity were determined in an eleven-station transect encompassing the entire 636 km length of the lake. Pronounced horizontal variability in hydrodynamic conditions was observed, with the southern region of the lake being strongly thermally stratified while the middle and north basins were largely isothermal through July. The extent of depletion of surface water nutrients, and the magnitude of phytoplankton biomass and productivity, were found to be strongly correlated with the degree of thermal stratification. Horizontal differences likely reflected the contribution of two important factors: variation in the timing of ice-out in different parts of the lake (driving large-scale patterns of thermal stratification and other limnological properties) and localized effects of river inflows that may contribute to the preliminary stabilization of the water column in the face of intense turbulent spring mixing (driving meso-scale patterns). Examination of the relationships between surface water inorganic N and P depletion suggested that during the spring and early summer, phytoplankton growth in unstratified portions of the lake was largely unconstrained by nutrient supplies. As summer progressed, the importance of co-limitation by both N and P became more apparent. Uptake and regeneration rates, measured directly using the stable isotope ¹⁵N, revealed that phytoplankton in stratified portions of the lake relied primarily on NH₄ as their N source. Rates of NH₄ regeneration were in approximate equilibrium with uptake; both processes were dominated by organisms <2 µm. This pattern is similar to that observed for oligotrophic marine systems. Our study underscores the importance of hydrodynamic conditions in influencing patterns of biological productivity and nutrient dynamics that occur in Lake Baikal during its brief growing season.     

Degenerative Joint Disease and enthesopathies in a Bronze Age population from Sindou cave (Senaillac-Lauzes,Lot, France)

The anthropological characteristics of the people who lived during the cultural period of the Late Bronze Age in South West France still remain practically unknown because very few sites have provided skeletal remains which permit of an exhaustive study. The cave of Sindou is, in that sense, one of the scarce exceptions. Although the sample of Sindou cannot be considered as representative of the whole regional population (N=50), we studied the presence and severity of DJD and enthesopathies of microtraumatic origin with the aim of finding some data which contribute to the knowledge of several biological aspects of this human group. From the results of the comparisons of the Sindou remains with two different medieval samples, a great similarity is deduced for these skeletal markers, but the higher frequency and severity of Achilles tendon enthesopathy in Sindou is a probable index of a higher level of physical stress at this specific localisation.     

Antiprogestagen RU486 prevents the LH-dependent decrease in the serum concentrations of inhibin in the rat

Summary 1. In the rat, the LH-dependent ovarian progesterone rise mediates several actions of the primary surge of LH on the ovary. This experiment was aimed at elucidating the effects of the antiprogestagen RU486 on the LH-dependent decrease in both the serum concentrations and the ovarian content of inhibin.2. All rats in this experiment were treated with an antagonist of LHRH (1 mg/200 µl saline at 0800 h in proestrus) to supress the endogenous release of LH. One group of rats received 32 µg LH/250 µl saline at 1200 h in proestrus. Other group was given 4 mg RU486/200 µl oil at 0800 h in proestrus. The third group was injected with both RU486 and LH. Rats from the control group were injected with 250 µl saline and 200 µl oil. Animals were decapitated at 1700 h in proestrus and trunk blood and ovaries collected to determine the serum concentrations of LH, FSH, progesterone, 17ß-estradiol and inhibin as well as the ovarian content of inhibin.3. The ovulatory dose of LH in LHRHa-treated rats decreased both the serum concentrations and the ovarian content of inhibin and increased the serum concentrations of FSH. The administration of RU486 blocked the effect of LH on the serum concentrations of inhibin but not that on the ovarian content of inhibin.4. Since the antiprogestagen RU486 blocked the effect of LH on the serum concentrations of inhibin, we conclude that ovarian progesterone, besides mediating the effects of the primary LH surge on the ovulatory process and luteinization, participates in the LH-dependent drop in the serum concentrations of inhibin in proestrous afternoon.     

Soil nitrogen dynamics along a gradient of long-term soil development in a Hawaiian wet montane rainforest

I analyzed the rates of net N mineralization and nitrification of soils from seven sites in a Hawaiian wet montane forest. The sites differ in age, ranging from 400 to 4,100,000 yr, but are comparable in other variables (all at 1200 miasl with 4000 mm or more mean annual rainfall), and the chronosequence simulated a development of soils from basaltic lava. Soils were incubated for 20 days at 17.5 °C, which is nearly equivalent to a mean field air temperature of the sites, and at an elevated temperature of 25.5 °C under three treatments: 1) field-wet without amendments, 2) air dried to a permanent wilting point, and 3) fertilized with phosphate (NaH₂PO₄) at the rate of 50 g P per g dry soil. Both mineralization and nitrification rates varied significantly among the sites at the field temperature (p<.00001). Fractions of the mineralized organic matter (indexed by the N produced per g organic C) increased sharply from the youngest to the 5000-yr site before declining abruptly to a near constant value from the 9000 to the 1,400,000-yr sites. Total organic C in the top soils (<15 cm deep) increased almost linearly with age across the sites. Consequently, net NH₄- and NO₃-N produced on an area basis (g m^-2 20 d^-1) increased sharply from 0.2 in the youngest site to 1.2 in the 5000-yr site, then both became depressed once but steadily increased again. The fraction of organic matter mineralized, and the net N turnover rates were outstandingly high in the oldest site where a large amount of organic matter was observed; the topsoil organic matter which was used in this analysis appeared to be highly labile, whereas the subsurface organic matter could be relatively recalcitrant. As suggested by earlier workers, the initial increase in N turnover seemed to correspond to the increasing quantity of N in the soils through atmospheric deposition and biological fixation. The later decline in fraction of organic matter mineralized seemed to relate to increasing soil C/N ratios, increasingly recalcitrant organic matter, and poorer soil drainage with age. The elevated temperature treatment produced significantly higher amounts of N mineralization, except for the youngest site where N was most limiting, and for two sites where soil waterlogging might be severe. P fertilization invariably resulted in slower N turnovers, suggesting that soil microbes responded to added P causing N immobilization. The youngest site did not significantly respond to added P. The magnitude of immobilization was higher in older than in younger soils, suggesting that P more strongly limits microbial populations in the older soils.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Anaerobic acidogenesis of primary sludge: the role of solids retention time

This research investigates the effect of solids retention time (SRT) on the acid-phase anaerobic digestion of primary sludge. A series of experiments were conducted using two continuous-flow 3-L units with the following configuration: a completely mixed reactor (CMR) with clarifier and solids recycle and an upflow anaerobic sludge blanket (UASB) reactor. Results show that C(2) to C(5) volatile fatty acids (VFA) were the predominant compounds formed. At a constant hydraulic retention time (HRT) of 12 h, variation in SRT from 10 to 20 days resulted in a slight increase in VFA production in both systems, but at a shorter SRT (5 days) a drastic drop in acid production was observed. In addition, the percent distribution of VFA was to some extent affected by the change in SRT. On the other hand, organic matter degradation [measured by the chemical oxygen demand (COD) specific solubilization rate or the percent volatile suspended solids (VSS) reduction] appeared to be independent of SRT, at least in the range investigated. The percent soluble COD in the form of VFA, however, increased steadily with increasing SRT, approaching the 90% level at 20 days. The remaining soluble COD in the effluent from these systems may be mainly attributed to metabolic intermediates and unused soluble substrate. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.