Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Anatomical Studies of Haustorium Ontogeny and the Remarkable Mode of Penetration of the Haustorium in Nuytsiafloribunda (Labill.) R. Br.

The development and structure of secondary haustoria of Nuytsia floribunda are described and compared with other Santalalean haustoria. After establishing contact with the host root, cortical folds of the haustorium grow around the root in separate directions and fuse forming a ring around it. At an early stage of development, meristematic tissue differentiates in the interior proximal part of the haustorium. Zones of collapsed layers are present in the outer cortical region. Subsequently, in the proximal part, two vascular cores, two lysigenous cavities and extensive masses of sclerenchyma develop prior to penetration of the host root. The sclerenchymatous cells form a characteristic structure, described as the sclerenchyma prong. During penetration the intrusive part of the haustorium reaches not only the host xylem but continues growing downwards until it entirely splits the host root. Comparable to a guillotine, the sclerenchyma prong is directly involved in this remarkable process. The sclerenchyma prong finally lies in the distal part of the haustorium. Following this mechanical slicing of the host root, tube-like cells of the intrusive part actively penetrate the host xylem in an axial direction.     

Respiratory neuronal activity during apnea and other breathing patterns induced by laryngeal stimulation

Respiration cycles through three distinct phases (inspiration, postinspiration, and expiration) each having corresponding medullary cells that are excited during one phase and inhibited during the other two. Laryngeal stimulation is known to induce apnea in newborn animals, but the cellular mechanisms underlying this effect are not known. Intracellular recording of ventral respiratory group neurons was accomplished in intact anesthetized, paralyzed, and mechanically ventilated piglets. Apnea was induced by insufflation of the larynx with ammonia-saturated air, smoke, or water. Laryngeal insufflation induced phrenic nerve apnea, stimulation of postinspiratory neurons, and stable membrane potentials in inspiratory and expiratory cells consistent with postinspiratory inhibition. Usually the membrane potential of each neuronal type cycled through an expiratory level before onset of the first recovery breath. Variants of the apnea response, probably reflecting the aspiration reflex or sniffing, sneezing, coughing, and swallowing, were also observed. These latter patterns showed oscillation between inspiration and postinspiration without an apparent intervening stage II expiratory phase. However, stage II expiratory activity always preceded onset of the first ramp inspiration after such a pattern. These findings suggest that activation of postinspiratory mechanisms causes profound alterations in the respiratory pattern and that stage II expiration importantly modulates recovery of ramp inspiratory activity. The mechanism of this latter effect may be inhibition of early inspiratory neurons with consequent postinhibitory rebound.     

Release of iron from ferritin by semiquinone, anthracycline, bipyridyl, and nitroaromatic radicals

The cytotoxicity of many xenobiotics is related to their ability to undergo redox reactions and iron dependent free radical reactions. We have measured the ability of a number of redox active compounds to release iron from the cellular iron storage protein, ferritin. Compounds were reduced to their corresponding radicals with xanthine oxidase/hypoxanthine under N₂ and the release of Fe²⁺ was monitored by complexation with ferrozine. Ferritin iron was released by a number of bipyridyl radicals including those derived from diquat and paraquat, the anthracycline radicals of adriamycin, daunorubicin and epirubicin, the semiquinones of anthraquinone-2-sulphonate, 1,5 and 2,6-dihydroxyanthraquinone, 1-hydroxyanthraquinone, purpurin, and plumbagin, and the nitroaromatic radicals of nitrofurantoin and metronidazole. In each case, iron release was more efficient than with an equivalent flux of superoxide. Introduction of air decreased the rate of iron release, presumably because the organic radicals reacted with O₂ to form superoxide. In air, iron release was inhibited by superoxide dismutase. Semiquinones of menadione, benzoquinone, duroquinone, anthraquinone 1,5 and 2,6-disulphonate, 1,4 naphthoquinone-2-sulphonate and naphthoquinone, when formed under N₂, were unable to release ferrin iron. In air, these systems gave low rates of superoxide dismutase-inhibitible iron release. Of the compounds investigated, those with a single electron reduction potential less than that of ferritin were able to release ferritin iron.     

Two different sets of cis elements regulate scute to establish two different sensory patterns

Summary We have analysed the role of the achaete-scute gene complex in the development of the pattern of campaniform sensilla on the wing blade of Drosophila. We show that the complete pattern results from the superimposition of two independent subpatterns, one of which depends on the achaete gene and the other on scute. The scute subpattern comprises several clusters of sensilla, most of which seem to require the presence of control regions located upstream of the transcribed region. This is in contrast with the pattern of scute-dependent bristles, most of which depends on control elements located downstream of the transcribed region.     

Preventing iron deficiency in preschool children by implementing an educational and screening programme in an inner city practice.

OBJECTIVE--To assess the feasibility and acceptability of screening young children for iron deficiency in a deprived inner city practice and to assess the effects of a programme of dietary education. DESIGN--Prospective study of children in general practice, comparison with historical controls. SETTING--A deprived inner city practice. PATIENTS--127 Children aged 13-24 months. Findings were compared with those in 110 children of the same age studied previously. INTERVENTIONS--All mothers received dietary education antenatally and in the first year after giving birth. Screening for iron deficiency (defined as mean cell volume less than 75 fl and haemoglobin concentration less than 105 g/l) and haemoglobinopathy (when appropriate) was offered for all children attending for immunisation against measles, mumps, and rubella over 12 months; capillary blood samples were taken after immunisation. MAIN OUTCOME MEASURES--Uptake of the screening programme expressed as the percentage of all children eligible for immunisation who were screened, and the effectiveness of the dietary education as shown by the prevalence of iron deficiency in the two groups. RESULTS--Altogether, 122 of the 127 (96%) children who attended for immunisation had their haemoglobin concentration and mean cell volume measured; 90% of all children aged 13-24 months in the practice were screened. Dietary education, clinical procedures, and counselling were incorporated successfully into the clinic''s work. Ten children (8%) were iron deficient, all of whom responded to iron supplements, and eight had a haemoglobinopathy trait. In the previous study 110 children (70%) had been screened and 28 children (25%) had been iron deficient. The two groups were similar in terms of sex, social class, and ethnic group. CONCLUSIONS--Screening young children for iron deficiency, sickle cell disease, and thalassaemia when they attended for immunisation was acceptable and successful in a socially deprived inner city practice. Dietary education may have accounted for some of the reduction in the prevalence of iron deficiency that occurred over the two years.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

Simian virus 40 large T-antigen-dependent DNA replication is activated by protein phosphatase 2A in vitro.

The simian virus 40 large T antigen (T) is a multifunctional phosphoprotein. We found that T-dependent simian virus 40 DNA replication is substantially inhibited by okadaic acid. This result suggests that DNA replication is activated by dephosphorylation in vitro. We show here that the target activated by dephosphorylation, which stimulates DNA replication, is T and that the phosphatase involved is protein phosphatase 2A.     

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.