Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.     

Effect of vitamin on 2-deoxy-d-glucose uptake in human fibroblast cultures

Summary 2-Deoxy-d-glucose (2-DOG) uptake was tested in human fibroblast cultures in the presence and absence of vitamin E. Addition of 10 μg/ml vitamin E to the culture medium significantly reduced this uptake for 2-DOG concentrations of 0.005, to 10 mmol/liter (P≤0.01). The decrease of 2-DOG uptake was inversely proportional to the rise in 2-DOG concentration (P≤0.01). The presence of vitamin E reduced by 71% the average cellular level of lipid peroxides (expressed as thiobarbituric acid reactive substances) and caused a small but significant decrease in the cholesterol concentration (P≤0.01). These last results might explain the decrease in 2-DOG uptake observed in the presence of vitamin E.     

Comparative character of the deep-water and inshore cottoid fishes endemic to Lake Baikal

Lake Baikal's 29 endemic species of cottoid fishes form three groups: shallow-water species in depths to 350 m; eurybathic species from 50 to 1300 m; and abyssal species from 400 to 1600 m. These groups differ in their abilities to withstand high hydrostatic pressure. As in marine deep-water fishes, abyssal cottoids in Baikal have few or no cones in the retina, and some have tubular eyes. Their seismosensory systems predominate, based chiefly on free neuromasts. The proportion of species with canal systems decreases with depth. Diversity of the predominantly gammarid foods also decreases from 45 species in shallow water to five species in deep water, and the lateral line system plays the dominant role in food detection at all depths. Two abyssal cottoids have become secondary pelagic, achieving close to neutral buoyancy through high lipid levels and reduced skeletal mineralization. These forms take advantage of the abundant pelagic planktonic amphipod populations. The adaptations of abyssal forms parallel those seen in deep-water marine fishes.     

ALLOZYME DIVERSITY AND GENETIC IDENTITY IN SCHIEDEA AND ALSINIDENDRON (CARYOPHYLLACEAE: ALSINOIDEAE) IN THE HAWAIIAN ISLANDS

Genetic diversity of allozymes, genetic identity based on allozyme variability, and phylogenetic relationships were studied with respect to breeding system diversity, population size, and island age in 20 of the 29 species of Schiedea and Alsinidendron (Caryophyllaceae: Alsinoideae), a monophyletic lineage endemic to the Hawaiian Islands. Average levels of genetic variability in Schiedea and Alsinidendron were comparable to or higher than those found in other Hawaiian lineages for which equivalent data are available [Bidens, Tetramolopium, and the silversword alliance (Asteraceae: Madiinae)] and similar to average values for species of dicots. Allozyme variability was strongly dependent on breeding system, which varies widely in the Hawaiian Alsinoideae. Species with autogamous breeding systems showed very low variability, measured as the number of alleles per locus, percent polymorphic loci, and mean heterozygosity per locus. Outcrossing hermaphroditic and dimorphic species (those with gynodioecious, subdioecious, and dioecious breeding systems) showed significantly higher genetic variability. Small population size was associated with lower values for all measures of genetic variability. Nearly half of the species occurring in small populations are also autogamous; thus, both factors may have influenced levels of genetic variability in these species. Founder effect was apparent in one species (Schiedea adamantis), which occurs in a single large population, has a gynodioecious breeding system but a very low genetic variability. Island age appeared to have little effect on genetic variability. Slightly lower values of genetic variability for species occurring on Kaua'i and O'ahu result primarily from the occurrence of autogamous Alsinidendron species on those islands. Values for Nei's genetic identity for different species pairs were 0.201–0.942, a far greater range than in Bidens, the silversword alliance, and Tetramolopium. Using UPGMA clustering, there was only moderate support for relationships detected through cladistic analysis. Nei's unbiased genetic identity (I) was greatest among species with outcrossing breeding systems, which for the most part clustered together. Nei's genetic identities for self-fertilizing species were low, indicating that these species are less similar to one another and to outcrossing species, regardless of their affinities based on cladistic analysis. Parsimony analysis of allele frequency data supported two clades also found in phylogenetic analyses using morphological and molecular data. Clades recognized in parsimony analysis of allele frequencies were those lineages containing selfing species, indicating that conditions favoring fixation of alleles occurred in ancestral species. In contrast, maintenance of high genetic diversity in outcrossing species interferes with recognition of phylogenetic relationships using allozyme variability.     

PERSPECTIVE: INDIRECT MATE CHOICE,COMPETITION FOR MATES,AND COEVOLUTION OF THE SEXES

When Darwin first proposed the possibility of sexual selection, he identified two mechanisms, male competition for mates and female choice of mates. Extending this classification, we distinguish two forms of mate choice, direct and indirect. This distinction clarifies the relationship between Darwin's two mechanisms and, furthermore, indicates that the potential scope for sexual selection is much wider than thus far realized. Direct mate choice, the focus of most research on sexual selection in recent decades, requires discrimination between attributes of individuals of the opposite sex. Indirect mate choice includes all other behavior or morphology that restricts an individual's set of potential mates. Possibilities for indirect mate choice include advertisement of fertility or copulation, evasive behavior, aggregation or synchronization with other individuals of the same sex, and preferences for mating in particular locations. In each of these cases, indirect mate choice sets the conditions for competition among individuals of the opposite sex and increases the chances of mating with a successful competitor. Like direct mate choice, indirect mate choice produces assortative mating. As a consequence, the genetic correlation between alleles affecting indirect choice and those affecting success in competition for mates can produce self-accelerating evolution of these complementary features of the sexes. The broad possibilities for indirect mate choice indicate that sexual selection has more pervasive influences on the coevolution of male and female characteristics than previously realized.     

A unique genomic sequence in the Wolf-Hirschhorn syndrome [WHS] region of humans is conserved in the great apes

The Wolf-Hirschhorn syndrome (WHS) is caused by a partial deletion in the short arm of chromosome 4 band 16.3 (4p16.3). A unique-sequence human DNA probe (39 kb) localized within this region has been used to search for sequence homology in the apes' equivalent chromosome 3 by FISH-technique. The WHS loci are conserved in higher primates at the expected position. Nevertheless, a control probe, which detects alphoid sequences of the pericentromeric region of humans, is diverged in chimpanzee, gorilla, and orangutan. The conservation of WHS loci and divergence of DNA alphoid sequences have further added to the controversy concerning human descent.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Constitutively hyposialylated human T-lymphocyte clones in the Tn-syndrome: binding characteristics of plant and animal lectins

Previously, 1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol 22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAc1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Ac2,6GalNAc1-O-R) or TF (Gal1-3GalNAc1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.     

Sex steroid effects on the development and functioning of the growth hormone axis

Summary 1. The secretory pattern of growth hormone (GH) is sexually dimorphic in the adult rat. However, this difference between the sexes does not become apparent until after the onset of puberty, suggesting that pubertal sex steroids play an important role in the manifestation of this phenomenon.2. We have addressed the question as to whether there exists a sexual dimorphism in the hypothalamic neuropeptides that regulate GH release from the anterior pituitary,i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). In addition, we have investigated whether the developmental changes in the GH secretory pattern are correlated with changes in these neuropeptides. The effect of testosterone treatment on SS and GHRH neurons during both the neonatal period and adulthood have also been studied.3. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are sexually dimorphic at certain times during maturation and can be modulated by changes in testosterone levels, suggesting that sex steroid modulation of these two neuropeptide systems could at least partially account for the sexual dimorphism seen in the adult GH secretory pattern.4. The neonatal steroid environment has also been suggested to be involved in the generation of the final adult GH secretory pattern, although the mechanisms underlying this effect are even less well understood. In support of the hypothesis that the neonatal steroid environment plays an important role in organizing the GH axis, we have found that the number of GHRH neurons in the adult brain, as well as their sensitivity to adult steroids, is modulated by neotatal testosterone treatment. The number of SS neurons in the periventricular and paraventricular nuclei were not modulated by neonatal steroids; however, the synthetic capacity of these neurons does appear to be influenced by the neonatal steroid environment.5. These studies suggest that both the neonatal and adult sex steroid environments influence the adult GH secretory pattern by modulating GHRH and SS neurons.