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.     

FECAL METHANOGENS AND VERTEBRATE EVOLUTION

It has been assumed that the feeding habits of vertebrates predispose the variety of intestinal differentiations and the composition of the microbial biota living in their intestinal tracts. Consequently, the presence of methanogenic bacteria in the various differentiations of the large intestine and the foregut of herbivorous vertebrates had been attributed primarily to the existence of anaerobic habitats and the availability of carbon dioxide and hydrogen originating from the fermentative microbial digestion of plant-based diets. However, Australian ratites, many murids, and several New World primates lack methanogens, despite their intestinal differentiations and their vegetarian feeding habits. Crocodiles, giant snakes, aardvarks, and ant-eaters on the other hand release significant amounts of methane. A determination of methane emissions by 253 vertebrate species confirmed that competence for intestinal methanogenic bacteria is shared by related species and higher taxa, irrespective of different feeding habits. In “methanogenic” branches of the evolutionary tree, a variety of differentiations of the large intestine evolved and, in some cases, differentiations of the foregut. In contrast, the lack of competence for methanogens in chiropterans/insectivores and carnivores apparently has precluded the evolution of specialized fermenting differentiations of the digestive tract. Our observations reveal that the presence of intestinal methanogenic bacteria is under phylogenetic rather than dietary control: competence for intestinal methanogenic bacteria is a plesiomorphic (primitive-shared) character among reptiles, birds, and mammals. This competence for methanogenic bacteria has been crucial for the evolution of the amniotes.     

Relationships between mesohabitats, ichthyological communities and IBI metrics adapted to a European river basin (The Meuse, Belgium)

We investigated the influence of mesohabitats on fish communities and on attributes of a multimetric index of fish integrity in the River Meuse basin. Three consecutive 150 m sectors in a Meuse tributary (Ourthe, Belgium), each divided in two or three sub-sections presenting various percentages of mesohabitats (riffles, runs and pools), were sampled by electrofishing. In each sub-section, relative and absolute biomasses of each fish species were estimated. The presence of limnophilic cyprinids was inversely correlated (r ² = 0.70 and 0.56 for absolute and relative biomass, respectively) with the percentage of riffles. Salmonids preferred runs and their absolute biomass was highly dependent (r ² = 0.71) on the proportion of this mesohabitat, whereas biomass of limnophilic cyprinids was highly correlated (r ² = 0.75 and r ² = 0.82 for absolute and relative biomass, respectively) with pools. A positive correlation (r ² = 0.58) was established between relative biomass of predators and the percentage of this mesohabitat. An IBI was calculated for the three sectors on the basis of results from the entire Meuse catchment. Scores of most metrics showed low variation among sectors but values of two metrics (% of individuals as tolerant, % of individuals as ubiquitous spawners) were greatest in sector 2, where pools predominated. Further, the lowest IBI score (51/65, integrity class: fair to good) was recorded in sector 2 where pools dominated, while sector 1 (where runs dominated) obtained the highest score (63/65, integrity class: excellent). Sector 3 which has a balanced proportion of riffles and runs obtained an intermediate score (57/65). Considering the response of IBI to the natural variation of mesohabitat proportions, it appears that an accurate sampling requires the prospection of a variety of mesohabitats (with a majority of runs) for the evaluation of river quality.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Isozyme analysis of Galaxias species (Teleostei: Galaxiidae) from the Taieri River, South Island, New Zealand: a species complex revealed

We examined genetic differentiation among 23 samples of non-migratory river galaxias from 17 streams in the Taieri River system, South Island, New Zealand. Four major genetic types were found, two of which occur in narrow sympatry in one location. These were compared with topotypical material representing Galaxias anomalus from the Clutha system (Otago) and G. vulgaris from the Waimakariri system (Canterbury) in order to establish identity. Morphological examination of these four major genetic types revealed consistent concomitant differences. The results suggest that there are at least three species of river galaxias in the Taieri system: G. anomalus, G. vulgaris and at least one previously undescribed species. We propose that the genetic structuring and subsequent speciation of this group has been promoted by the absence of the marine juvenile phase that is found in five other members of the genus native to New Zealand. This structuring may be exacerbated by population fragmentation over the last century owing to the negative influence of introduced trout. The phylogenetic diversity within the river system mirrors the diverse flora and invertebrate fauna of the region, and has conservation implications that parallel those resulting from our improved knowledge of the New Zealand herpetofauna through the application of genetic analysis.     

懒猴属的核糖体DNA变异及其种间分化关系

用１５种限制性内切酶和人２８Ｓ、１８ＳｒＤＮＡ探针构建了懒猴属各物种核糖体ＤＮＡ重复单位的限制性内切酶图谱。在进化速率较高的非转录间隔区,在大、中、小懒猴中分别定位了２３、２４、２４个酶切位点。大懒猴与中懒猴有１２个位点不同,与小懒猴有１４个位点不同,而中、小懒猴间则只有一个位点的差异。经过计算,大懒猴与中懒猴的遗传距离值为１２．６５％,与小懒猴的差异为１４．２４％,中、小懒猴间的差异则仅为０．７     

Fibre length and gibberellins A1 and A20 are decreased in Eucalyptus globules by acylcyclohexanedione injected into the stem

Trinexapacethyl (TriEt), an acylcyclohexanedionetype inhibitor of gibberellin (GA) biosynthesis, was applied to 3-year-old Eucalyptus globules saplings by localised injection near the base of each stem. The objective was to alter cambial region GA levels and to study the effects on secondary xylem fibre development. Seven weeks later wood samples, with bark and cambial region intact, were removed 10 and 30 cm above the point of injection. Fusiform cambial cell dimensions were compared with those of fibre-tracheids in the most recently formed 100 um of secondary xylem. Increasing TriEt applications from 5 to 5 000 mg active ingredient significantly reduced average fibre length, and to a lesser extent average fusiform cambial cell length. Also reduced was the number of cells in the cambial zone and the number of differentiating fibres with primary walls. However, no trends were evident for changes in fibre diameter, the proportion of vessel elements or the ratio of cambial ray cells to fusiform cambial cells. Two gibberellins (GA₁ and GA₂₀), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in cambial region tissues by gas chromatographymass spectrometry using stable isotope labelled internal standards. Increasing TriEt application reduced both GA₁ and GA₂₀ levels. Effects on IAA and ABA were not significant, although their levels tended to be lower at the highest TriEt application rate. The elongation of secondary xylem fibres was positively correlated with higher levels of endogenous GA₁ (r_s= 0.74, P < 0.01) and GA₂₀ (r_s= 0.72, P < 0.01). These results support a causal role for GA₁ in cambial cell division. They are also consistent with the hypothesis that the elongation of differentiating secondary xylem fibres in woody an–giosperms is dependent on GA₁ levels in the cambial region.