Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.     

Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose using MM3

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1 → 2)-, (1 → 3)-- and (1 → 4)--linked -glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20° grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.     

Antiinsectan properties of a novel Fusarium-derived sterol sulfate

The antiinsectan properties of the Fusarium graminearum-derived novel sterol sulfate, 4,4,24-trimethylcholesta-8,14,24(28)-trien-2,3,11,12-tetrol 12 acetate, 3-sulfate were tested on the corn earworm (Heliothis zea (Boddie), the fall armyworm (Spodoptera frugiperda (J. E. Smith), and the driedfruit beetle (Carpophilus hemipterus L.). The sterol sulfate could not be used as a sterol source by any of the insects. The sterol sulfate inhibited growth of S. frugiperda and C. hemipterus larvae at 2500 and 4000 ppm, respectively. Defensive implications of this compound are discussed.     

Phylogeny and taxonomy of the Prenolepis genus‐group of ants (Hymenoptera: Formicidae)

Abstract. We investigated the phylogeny and taxonomy of the Prenolepis genus‐group, a clade of ants we define within the subfamily Formicinae comprising the genera Euprenolepis, Nylanderia, gen. rev. , Paraparatrechina, gen. rev. & stat. nov. , Paratrechina, Prenolepis and Pseudolasius. We inferred a phylogeny of the Prenolepis genus‐group using DNA sequence data from five genes (CAD, EF1αF1, EF1αF2, wingless and COI) sampled from 50 taxa. Based on the results of this phylogeny the taxonomy of the Prenolepis genus‐group was re‐examined. Paratrechina (broad sense) species segregated into three distinct, robust clades. Paratrechina longicornis represents a distinct lineage, a result consistent with morphological evidence; because this is the type species for the genus, Paratrechina is redefined as a monotypic genus. Two formerly synonymized subgenera, Nylanderia and Paraparatrechina, are raised to generic status in order to provide names for the other two clades. The majority of taxa formerly placed in Paratrechina, 133 species and subspecies, are transferred to Nylanderia, and 28 species and subspecies are transferred to Paraparatrechina. In addition, two species are transferred from Pseudolasius to Paraparatrechina and one species of Pseudolasius is transferred to Nylanderia. A morphological diagnosis for the worker caste of all six genera is provided, with a discussion of the morphological characters used to define each genus. Two genera, Prenolepis and Pseudolasius, were not recovered as monophyletic by the molecular data, and the implications of this result are discussed. A worker‐based key to the genera of the Prenolepis genus‐group is provided.     

31P NMR and saturation transfer studies of the effect of Pb2+ on cultured osteoblastic bone cells

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.     

Divergence estimates and early evolutionary history of Figitidae (Hymenoptera: Cynipoidea)

We examine the phylogenetic relationships of Figitidae and discuss host use within this group in light of our own and previously published divergence time data. Our results suggest Figitidae, as currently defined, is not monophyletic. Furthermore, Mikeiinae and Pycnostigminae are sister‐groups, nested adjacent to Thrasorinae, Plectocynipinae and Euceroptrinae. The recovery of Pycnostigminae as sister‐group to Mikeiinae suggests two major patterns of evolution: (i) early Figitidae lineages demonstrate a Gondawanan origin (Plectocynipinae: Neotropical; Mikeiinae and Thrasorinae: Australia; Pycnostigminae: Africa); and (ii) based on host records for Mikeiinae, Thrasorinae and Plectocynipinae, Pycnostigminae are predicted to be parasitic on gall‐inducing Hymenoptera. The phylogenetic position of Parnips (Parnipinae) was unstable, and various analyses were conducted to determine the impact of this uncertainty on both the recovery of other clades and inferred divergence times; when Parnips was excluded from the total evidence analysis, Cynipidae was found to be sister‐group to [Euceroptrinae + (Plectocynipinae (Thrasorinae + (Mikeiinae + Pycnostigminae)))], with low support. Divergence dating analyses using BEAST indicate the stem‐group node of Figitidae to be c. 126 Ma; the dipteran parasitoids (Eucoilinae and Figitinae), were estimated to have a median age of 80 and 88 Ma, respectively; the neuropteran parasitoids (Anacharitinae), were estimated to have a median age of 97 Ma; sternorrhynchan hyperparasitoids (Charipinae), were estimated to have a median age of 110 Ma; the Hymenoptera‐parasitic subfamilies (Euceroptinae, Plectocynipinae, Trasorinae, Mikeiinae, Pycnostigminae, and Parnipinae), ranged in median ages from 48 to 108 Ma. Rapid radiation of Eucoilinae subclades appears chronologically synchronized with the origin of their hosts, Schizophora (Diptera). Overall, the exclusion of Parnips from the BEAST analysis did not result in significant changes to divergence estimates. Finally, though sparsely represented in the analysis, our data suggest Cynipidae have a median age of 54 Ma, which is somewhat older than the age of Quercus spp (30–50 Ma), their most common host.     

Microbiome analysis among bats describes influences of host phylogeny, life history, physiology and geography

Metagenomic methods provide an experimental approach to inform the relationships between hosts and their microbial inhabitants. Previous studies have provided the conceptual realization that microbiomes are dynamic among hosts and the intimacy of relation between micro- and macroorganisms. Here, we present an intestinal microflora community analysis for members of the order Chiroptera and investigate the relative influence of variables in shaping observed microbiome relationships. The variables ranged from those considered to have ancient and long-term influences (host phylogeny and life history) to the relatively transient variable of host reproductive condition. In addition, collection locality data, representing the geographic variable, were included in analyses. Results indicate a complex influence of variables in shaping sample relationships in which signal for host phylogeny is recovered at broad taxonomic levels (family), whereas intrafamilial analyses disclosed various degrees of resolution for the remaining variables. Although cumulative probabilities of assignment indicated both reproductive condition and geography influenced relationships, comparison of ecological measures among groups revealed statistical differences between most variable classifications. For example, ranked ecological diversity was associated with host phylogeny (deeper coalescences among families were associated with more microfloral diversity), dietary strategy (herbivory generally retained higher diversity than carnivory) and reproductive condition (reproductively active females displayed more diverse microflora than nonreproductive conditions). Overall, the results of this study describe a complex process shaping microflora communities of wildlife species as well as provide avenues for future research that will further inform the nature of symbiosis between microflora communities and hosts.