Genomic Consequences of Ecological Speciation in Astyanax Cavefish

The cave environment is consistently radically different than the surface environment because it lacks light, and animals adapting to cave life are subject to strong selective forces much different than those experienced by their ancestors who evolved in the presence of light. As such, their divergence from surface ancestors and eventual speciation is likely to be driven by the shift in ecology. We report here that hybrids between cave and surface Astyanax mexicanus fishes produce offspring with allelic frequencies that differ significantly from Mendelian expectations both for transmission ratios and for independent assortment of unlinked markers. Comparison of allelic content of DNA from fin clips and sperm pools show that the transmission ratio distortion likely occurs during spermatogenesis. Departures from expectations of independent assortment are essentially epistatic phenomena generating linkage disequilibrium. A novel analysis of the epistatic interactions reveals an apparent network of interactions among genes known or suspected to be involved in cave adaptation, implying that the epistasis arose as a “by product” of the divergence due to cave adaptation.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

<Superscript>15</Superscript>N natural abundance of fossil peat reflects the influence of animal-derived nitrogen on vegetation

'¹⁵N signatures of fossil peat were used to interpret past ecosystem processes on tectonically active subantarctic Macquarie Island. By comparing past vegetation reconstructed from the fossil record with present-day vegetation analogues, our evidence strongly suggests that changes in the '¹⁵N signatures of fossil peat at this location reflect mainly past changes in the proportion of plant nitrogen derived from animal sources. Associated with uplift above sea level over the past 8,500 years, fossil records in two peat deposits on the island chronicle a change from coastal vegetation with fur and elephant seal disturbance to the existing inland herbfield. Coupled with this change are synchronous changes in the '¹⁵N signatures of peat layers. At two sites ¹⁵N-enriched peat '¹⁵N signatures of up to +17‰ were associated with a high abundance of pollen of the nitrophile Callitriche antarctica (Callitrichaceae). At one site fossil seal hair was also associated with enriched peat '¹⁵N. Less ¹⁵N enriched '¹⁵N signatures (e.g. -1.9‰ to +3.9‰) were measured in peat layers which lacked animal associated C. antarctica and Acaena spp. Interpretation of a third peat profile indicates continual occupation of a ridge site by burrowing petrels for most of the Holocene. We suggest that ¹⁵N signatures of fossil peat remained relatively stable with time once deposited, providing a significant new tool for interpreting the palaeoecology.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant

Summary Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n=23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1–3 months, n=5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.     

Reduced proliferation in T lymphocytes in aged humans is predominantly in the CD8+ subset, and is unrelated to defects in transmembrane signaling which are predominantly in the CD4+ subset

Peripheral blood lymphocytes (PBL) from elderly donors have a reduced proliferative response to phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies (mAb) compared to those from young donors. To examine whether this is due to intrinsic deficiencies in proliferative potential of T-cell subsets, we compared the growth of unsorted PBL vs sorted CD4+ or CD8+ CD11- cells after anti-CD3 mAb or PHA stimulation. Unsorted PBL of elderly donors (greater than 65 years) showed a significant decrease in proliferation compared to young donors (20-30 years) when stimulated with anti-CD3 mAb or PHA. Sorted CD4+ and CD8+ cells were grown in culture in the absence of accessory cells under optimized growth conditions (CD28 mAb, interleukin 2 and beta-mercaptoethanol present). CD4+ cells from elderly donors showed no reduced growth after anti-CD3 mAb stimulation and only slightly decreased growth after stimulation with PHA. CD8+ CD11- cells from elderly donors, however, showed a 20-30% reduction in the proportion of cells proliferating in response to the mitogens and up to 40% reduction in the rate of cell-cycle progression of the responding cells. We examined whether this reduced proliferation is related to decreased efficiency of signal transduction by comparing this to the mobilization of intracellular free calcium ([Ca2+]i) and calcium channel activity after stimulation with anti-CD3 mAb or PHA. [Ca2+]i was measured in CD4 and CD8 subsets of young and elderly donors using a flow cytometric assay with the dye indo-1. Compared to cells from young donors, CD4+ cells from elderly donors showed a [Ca2+]i response which was up to 26% lower after stimulation with CD3 and 10% lower after stimulation with PHA. This appeared to be related to decreased calcium channel activity in elderly donors, rather than mobilization of intracellular Ca2+ stores. CD8+ cells from elderly donors, however, had a slightly, but significantly, greater [Ca2+]i response to CD3 mAb and PHA than did cells from young donors. Since the age-dependent defect in proliferation is mainly in CD8+ cells, but the [Ca2+]i decline is predominantly in the CD4+ subset, these results suggest that the reduced proliferation of T cells from older donors is not related to decreased efficiency of transmembrane signal transduction.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.     

Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.