In search of mammary gland stem cells

Summary The evidence for mammary epithelial stem cells and their phenotypic characteristics in normal and neoplastic development is reviewed. The presence of stem cells in all parts of the mammary parenchyma at all stages of differentiation has been demonstrated by transplantation experiments. The phenotypic characterization of stem cells has been defined by a battery of monospecific antibodies. These studies suggest that a mammary epithelium stem cell compartment exists in the basal layer of the gland as well as in the end bud. Whether these same stem cells are expressed in mammary preneoplasias and neoplasias has not been answered conclusively. Phenotypic markers specific for stem cells and stably expressed in transformed cell populations are needed to follow the fate of stem cells.Dedicated to Professor Stuart Patton on the occassion of his 70th birthday.     

Neuron-specific enolase (NSE)-like and neurofilament protein (NFP)-like immunoreactivities in the rat dorsal root ganglia and sciatic nerve

The presence of neuron-specific enolase (NSF) and neurofilament proteins (NFP) immunoreactivities (IR) was investigated in dorsal root ganglia (DRG) of adult rats at cervical, thoracic, lumbar and sacral levels. All neurons display NSE-like IR with a variable intensity of immunostain which is not related to the neuronal size. Conversely, the antibody against all three proteic subunits of NFP no labelled the primary sensory neurons, whereas the intraganglionic axons and dorsal root of spinal nerves result positives. In the sciatic nerve the immunoreactivity was similar for NSE- and NFP-like IR. No regional differences were found among the different levels of DRG for NSE-like IR. The present results demonstrate heterogeneity in the neurons of the rat. DRG for NSE-like IR, and differences between sensory neurons and fibers in the distribution of NFP-like IR.     

Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ - τ Interaction

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule-associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self-assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin-binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18-residue-long peptide corresponding to the third tubulin-binding motif of τ. This suggests that the ability of τ for self-assembly could be localized in a short sequence of amino acids present in the tubulin-binding repeats of the τ molecule.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.     

Cyanobacteria of Rocks and Soils of the Orinoco Lowlands and the Guayana Uplands,Venezuela

The occurrence of 23 cyanobacterial species, belonging to 9 different genera and 5 cyanobacterial lichen species of 5 different genera on exposed, open rock surfaces of inselbergs and on soil in savannas of the Orinoco lowlands and the Guayana uplands is described. Their distribution patterns and frequency within the different habitats are given. The filamentous procaryotic blue-green algae/cyanobacteria Stigonema ocellatum and Scytonema crassum, together with the unicellular cyanobacterium Gloeocapsa sanguinea were the most frequent species on rocks, whereas the filamentous cyanobacterium, Schizothrix telephoroides, dominated in cyanobacterial mats on the savanna soil. All species showed intensively coloured sheaths, either brown or yellow in the case of Stigonema ocellatum and Scytonema crassum, or red in Gloeocapsa sanguinea and Schizothrix telephoroides. In addition, a number of cyanobacterial lichens occurred.     

The evolution and ecology of multiple antipredator defences

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such “defence portfolios” that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.