Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.     

Phenology of Festuca pallescens in relation to topography in north-western Patagonia

Abstract. The phenological changes in populations of Festuca pallescens (St. Yves) Parodi at different topographic positions and exposure along an altitudinal gradient (600 - 1100 m) were investigated during two growing seasons in northwestern Patagonia. Stepwise multiple regression analysis was used to describe the relationship between phenology and environment during the entire growing season. Analysis of variance was also performed at each sample date to detect significant environmental factors influencing phenology at different sites. The sum of maximum air temperatures was identified as the environmental variable best correlated with the seasonal variation of phenological events of Festuca pallescens over the period of two growing seasons, explaining 93.2 % of the total variance. Significant differences between sites were observed at each sample date. Main effects of altitude and topographic position and two-way interactions between altitude and topographic position, and topographic position and exposure were also detected as significant. Phenology was delayed at increased altitude. Differences in phenology between topographic sites at the same altitude were not detected during the entire growing season and were only observed in the reproductive phase. At this time, the phenology was significantly delayed at high topographic positions on the slopes as compared with low and mid positions. At high altitudes in the valley (950 m a. s. 1.), where steep slopes and humid conditions prevail, phenology was delayed on western exposures and low positions. The results adequately summarize and quantify the effect of spatial and temporal environmental variation on the phenological development of Festuca pallescens in northwestern Patagonia.     

Enzyme Variation of Eimeria arizonensis from Peromyscus truei and P. boylii

ABSTRACT. Cricetid rodents, Peromyscus truei and P. boylii , were inoculated with sporulated oocysts of Eimeria arizonensis collected from wild P. truei maintained in the lab. In P. truei the prepatent period was 4–5 days, the patent period was 9–11 days, and sporulated oocysts were 21.5 × 25.0 (20–23 × 24–26) μm with sporocysts 7.7 × 12.0 (6–8 × 10–13) pm. In P. boylii the prepatent period was 6–7 days, the patent period was 8–9 days, and sporulated oocysts were 20.1 × 23.2 (18–22 × 21–24) pm with sporocysts 6.8 × 10.0 (5–8 × 9–12) pm. Sporulated oocysts from both host species were used in direct side-by-side comparison of isozyme banding patterns using protein electrophoresis. The parasite has polytypic loci for leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6-PGD). In oocysts from P. truei , LAP showed one band with fast migration and LDH and 6-PGD each showed two bands, one with fast and one with slow migration. In oocysts from P. boylii , LAP and LDH each had one band with slow migration and 6-PGD had one band with moderate migration. Oocysts of E. arizonensis collected from P. boylii were used to inoculate P. truei. The prepatent and patent periods, structural measurements, and isozyrne banding patterns of the resultant oocysts were the same as those from P. truei when inoculated with oocysts from P. truei.     

Reductions in genetic variation inDrosophila andE. coli caused by selection at linked sites

Selection at linked sites has important consequences for the properties of neutral variation and for tests of the predictions of the neutral theory of molecular evolution. We review the theory of the effect of adaptive gene substitutions on neutral variability at linked sites (hitchhiking or selective sweeps) and discuss theoretical results on the effect of selection against deleterious alleles on variation at linked sites (background selection). InDrosophila melanogaster there is a clear relation between the frequency of recombination in a given region of the chromosome and the amount of natural variability in that region. Attempts to predict this relation have given rise to models of selective sweeps and background selection. We describe possible methods of discriminating between these models, and also discuss the probable strong influence of selective sweeps on variation in largely nonrecombining genomes, with particular reference toEscherichia coll. Finally we present some unresolved questions and possible directions for future research.     

Apolipoprotein E and Low-Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform-Specific Alterations

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ? E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human ¹²⁵I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a K_D of 1.8 nM and a B_max of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade ¹²⁵I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with ¹²⁵I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. ¹²⁵I-ApoE2 binding was significantly lower than that of the other ¹²⁵I-apoE isoforms in both cell types. ¹²⁵I-ApoE4 binding was similar to that of ¹²⁵I-apoE3 in both astrocytes and neurons. On the other hand, ¹²⁵I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.     

Temporal changes in host adaptation in the pea aphid, Acyrthosiphon pisum

Abstract.

• 1 Temporal changes in host adaptation were followed in a local population of the pea aphid, Acyrthosiphon pisum. Aphid clones were collected in one alfalfa and one clover field at three different times. In the spring, first-generation females were collected. Later, in the autumn, females belonging to the last parthenogenetic generation were collected. Lastly, sexual females were collected after mating in autumn and allowed to produce eggs which were hatched. The performance was evaluated on alfalfa and clover. The spring-collected individuals were also assessed on peas.
• 2 On the overwintering hosts clover and alfalfa, the clones performed best on the plant of origin, i.e. negative correlations in performance. Correlations between performance on the temporary summer host, pea, and that on clover/alfalfa were weak or nonsignificant.
• 3 Significant variation in host performance was found within both host fields at spring, which is a prerequisite for changes in clone composition due to selection/migration.
• 4 The clones from alfalfa showed an increase in mean performance on alfalfa between spring and autumn, whereas no changes among the clones from the clover field were observed. This difference in seasonal response between the two fields could have been the result of larger variation in performance among the alfalfa clones and/or a differential tendency to migrate among clones in both fields.
• 5 After sexual recombination in the autumn, mean performance in the alfalfa field returned to the spring level, probably as a result of emergence of new genetic combinations. In the clover field, mean performance did not change significantly over time.

     

Reproductive success and clonal genetic structure of the rareArnica montana (Compositae) in The Netherlands

In a medium-sized population ofArnica montana, a threatened species in The Netherlands, the breeding system, reproductive success and genetic clonal structure were studied. Pollination experiments suggested thatA. montana is largely self-incompatible. Inbreeding depression was observed for seedling weight but not for fruit weight and germination rate. Although genetic variation is rather low in this population, the data suggest an outcrossing mating system. Analysis of the genotype of all mapped rosettes in a plot of 100 m² indicated that dense clusters often consist of identical genotypes, suggesting a clonal structure. Open clusters frequently contained several different genotypes. This may be caused by limited fruit dispersal, since seedlings were found mainly within or in the near surroundings of the clusters.     

Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA -glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wildtype and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.     

In vitro culture selection increases glyphosate tolerance in barley

In vitro culture of barley calluses has been used to produce plants with increased glyphosate tolerance. Calluses from immature embryos of barleyHordeum vulgare L. (Jeff) were cultured on Murashige and Skoog medium with 10^-6, 10^-5, 10^-4, 5×10^-4, 10^-3, or 10^-2M glyphosate for one, four or thirty months. Plants were regenerated from calluses maintained in glyphosate medium at 10^-6, 10^-5 or 10^-4M for four months, at 10^-5 or 5×10^-4M for one month and at 10^-5M for thirty months. The progeny of each regenerated plant was analyzed for response to glyphosate. Some progenies showed increased tolerance to glyphosate.