Aging in the post-genomic era: simple or complex?

A report on the Third Genetic Effects on Aging Meeting, The Jackson Laboratory, Bar Harbor, Maine, August 4-8, 2000.     

Host-associated differences in fitness within and between populations of a seed beetle (Bruchidae): effects of plant variability

Summary Experiments were conducted with the sexually reproducing seed beetle Stator limbatus and its hosts in north-central Arizona to determine if it was substructured into units, each specialized for higher fitness on a specific host species. Unlike many studies, we incorporated scale, i.e., conducting experiments between and within beetle populations on seeds from within and between plant species. Of particular interest was whether intraspecific plant variability prevented beetle specialization within beetle populations. Results suggest that S. limbatus is specialized to certain hosts. On the palo verde Cercidium floridum, beetles originally reared from this host had significantly higher emergence compared to beetles transferred from other hosts. We did not test directly for a genetic basis for this. Alternative hypotheses of variation in symbiotic microorganisms in larval guts and maternal effects were assessed. Essentially no bacteria, yeast or protozoa were found, and maternal effects as expressed by varying egg weights were not detected; however, other microorganisms might have been present and maternal effects through inducible enzymes was possible. Caution, then, is needed in any genetic interpretations of our results. The differences on C. floridum were detected from tests between and within beetle populations. Evidence for specialization was not detected on the other hosts, Cercidium microphyllum and Acacia greggii. On the other hosts, beetles performed well regardless of their source. Significant differences were detected among individual plants of C. floridum as to the suitability of their seeds for deveoopment of S. limbatus. No such differences were detected among the other host plants. These patterns of conspecific plant variability are opposite of what is expected if plant variability prevents specialization of beetles to particular species of hosts. Thus, the data suggest seed variability among plants does not prevent specialization to host species in this system. We discuss how the patterns of host use in this study relate to the hypothesis of sympatric host race formation.     

Physiology and costs of resistance to herbivory and disease in Brassica

We used artificial selection experiments to study genetic allocation costs and physiological mechanisms of resistance to herbivory and fungal disease. Genetic costs to resistance were present in some instances and absent in others. Genetic resistance to the fungal pathogen, Leptosphaeria maculans was cost-free, while resistance to Peronospora parasitica showed a negative genetic correlation between disease resistance and growth rate. Leptosphaeria resistant genotypes had 13% higher chitinase activity. Genetic increases in myrosinase activity were correlated with increased resistance to flea beetles (Phyllotreta cruciferae), but resulted in lower plant fecundity, presumably due to production costs of myrosinase. Genetic costs of resistance may maintain genetic variation in natural plant populations. These studies demonstrate the predictive and explanatory power of a functional approach to plant-herbivore and plant-pathogen interactions.     

Effect of mitochondrial functions on synthesis of yeast cytochrome c

The effects of the mitochondrial protein synthesis inhibitor chloramphenicol and the mitochondrial F0 adenosine triphosphatase inhibitor oligomycin on the synthesis of nucleus-encoded cytochrome c protein were studied. Both inhibitors stimulated cytochrome c protein synthesis in the derepressed state (growth in media containing 2% raffinose) but had no effect on the synthesis of the cytochrome c protein in the repressed state (growth in media containing 5% glucose). Oligomycin uncoupled the synthesis of the apoprotein from its processing into the hemoprotein. Neither antibiotic had a significant effect on the rate of glucose repression of cytochrome protein synthesis. The kinetics of cytochrome c derepression and the effects of these two antibiotics on these kinetics were also studied. Cells were derepressed by transfer from glucose- to faffinose-containing media, and the rate of cytochrome c synthesis increased from the repressed to the derepressed level during the second hour of derepression. Chloramphenicol delayed this derepression, but after 5 h the rate of cytochrome c protein synthesis increased to twice the rate of synthesis in uninhibited cells. On the other hand, oligomycin inhibited derepression of cytochrome c. These results are discussed with respect to the effects of mitochondrial function in the derepressed and repressed states and during the processes of repression and derepression of cytochrome c.     

Human ovarian surface epithelium in primary culture

The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17 beta-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17 beta-HSD activity, and presence of keratin.     

Human BF*F-subtypes: segregation analysis with inclusion of MHC haplotypes

Summary The segregation of factor B(BF)F subtypes was analyzed in conjunction with other MHC markers in 15 families with 89 offspring. Informative data for BF F subtypes were obtained from 11 families, 6 of them with known recombinant individuals for the HLA-B/DR/GLO region. The subtypes did not contribute further to the localization of the cross-overs, but followed the known segregation of conventional BF allotypes. In 2 families of one kinship, the recognition of heterozygous BF^*FAFB individuals could be established following the inclusion of three generations. The rarer of the two BF F subtype alleles, BF^*FA, is positively associated with the HLA haplotypes BW62, CW3, C4A^*3 and A29, CWX, B44, C4A^*3, B^*1, DR7. BF F subtypes are regarded as a very useful additional tool for studies of MHC organization and disease association.     

Elevated air carbon dioxide concentrations increase dissolved carbon leaching from a cropland soil

Terrestrial sources of nitrogen (N), particularly N-fixing alder, may be important for sustaining production in headwater streams that typically lack substantial subsidies of marine-derived nutrients from spawning salmon yet support upstream-dispersing juvenile salmonids. However, other physiographic characteristics, such as watershed slope and topographic wetness, also control transport of nutrients to streams and may confound apparent linkages between alder and stream N. Seasonal patterns in precipitation and temperature may interact with watershed characteristics to modulate stream N availability. We empirically modeled the effect of alder cover and other watershed physiographic variables on stream N and contrasted these relationships over the growing season among 25 first-order streams from the lower Kenai Peninsula, Alaska. For each date, percent alder cover, mean topographic wetness, and mean slope were used as watershed predictors of NO _x –N concentration (nitrate?+?nitrite) and daily NO _x –N yield using Generalized Additive Models (GAM) and compared using Akaike’s Information Criterion (AIC_c). Alder cover was the only probable model and explained 75–96% of the variation in NO _x –N concentration and 83–89% of the variation in daily NO _x –N yield. The relationship between alder and both NO _x –N concentration and daily NO _x –N yield changed from constant inputs in May across the range of alder cover (linear fit) to increasing inputs in July and September (non-linear fits) implying that high-alder watersheds were N-saturated. The strong linkage between alder and stream N coupled with the concurrent timing of maximum stream N from alder in the spring to salmon fry emergence indicates the potential importance of this subsidy to headwater stream ecosystems.     

Transcriptional response to alcohol exposure in Drosophila melanogaster

Background  

Alcoholism presents widespread social and human health problems. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Genetic factors that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance.     

Affinity Purification of Angiotensin Type 2 Receptors from N1E-115 cells: Evidence for Agonist-Induced Formation of Multimeric Complexes

Abstract: The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (Angll) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT₂-receptor subtype, whereas the density of the AT₁ receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) selectively solubilized AT₂receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (Angll) during affinity purification of AT₂ receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar¹, lle⁸-Angll was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT₂-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar¹, lle⁸-Angll, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT₁-receptor antagonist, was completely ineffective in eluting any Angll receptors from the affinity column, further confirming their AT₂ identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions. The 110-kDa and 66-kDa proteins, but not smaller, affinity-purified proteins, specifically bound ¹²⁵l-Angll as determined by covalent cross-linking of ¹²⁵l-Angll to the receptors with the homobifunctional cross-linker disuccinimidyl suberate, or by size exclusion chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity- purified material with antibodies selective for AT₂ receptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66-kDa protein, as well as a smaller (54-kDa) immunoreactive protein, was detected under reducing conditions. Collectively, these data suggest that CHAPS-solubilized AT₂ receptors from N1E-115 cells may consist of a binding protein of approximately 66 kDa, which in the presence of an agonist readily associates with other smaller proteins to form larger multimeric complexes.