Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH ATLANTIC1

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5–13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte. The second group of samples, from Nova Scotia and Northern Ireland, provisionally referred to as Gymnogongrus sp., showed little morphological variation. The life history in both areas consists of apomictically reproducing diploid female gametophytes and diploid crustose bisporophytes and tetrasporophytes. Rubisco spacer sequences of the samples were identical, and the plasmid previously described in the Nova Scotian samples was also present in the Northern Ireland population. This species is widely distributed in the western Atlantic, from Newfoundland to Massachusetts. In Europe, gametophytes are known only at one site, but crusts are distributed from Denmark, Scotland (and probably Norway) to France. It is very likely that this species was introduced from one side of the North Atlantic to the other by shipping during the early nineteenth century. Several morphological features are unusual within the genus but are shared with G. leptophyllus J. Agardh from the eastern Pacific Ocean, and further work is necessary to determine whether Gymnogongrus sp. and G. leptophyllus are conspecific.     

Is the Tonal Quality of Birdsong Learned? Evidence from Song Sparrows

Song sparrow (Melospiza melodia) songs are composed largely of pure-tonal sounds. This paper investigates the role that learning plays in the development of the tonal structure of song sparrow songs, as well as the role that tonal quality plays in determining the suitability of songs as models for learning. 20 birds were trained with both normal pure-tonal songs and modified songs that included harmonic overtones. The harmonic-modified songs were obtained from birds singing in a helium atmosphere, the result of which is to perturb vocal tract resonances and thus alter a song's tonal quality. Subjects learned equally well from normal and harmonic models. Birds that learned material from harmonic models reproduced some of this material with harmonic overtones, but the majority of notes learned from harmonic models were subsequently reproduced as pure-tonal copies. Thus, the tonal structure of songs does not influence young song sparrows in their selection of song models, but there is a strong tendency to reproduce songs in a pure-tonal fashion, even if learned from harmonic models.     

Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.     

Ultrastructure of Adult Gonads and Development and Structure of the Larva of Rhabdopleura normani (Hemichordata: Pterobranchia)

Sexually mature adults, embryos and larvae of the pterobranch Rhabdopleura normani from Bermuda were studied with light and electron microscopy. The sexes are separate among the zooids of a colony, but a given colony may contain females and males. In zooids of either sex the single gonad is associated with a large haemal sinus in the trunk sac and is displaced laterally (to the right or to the left). The wall of the gonad is composed of three layers: an outer metasomal peritoneum, an internal lining of germinal epithelium and an intervening genital haemal sinus. The mature gametes lie in the lumen within the gonad. The spermatozoon is characterized by an elongate nucleus, no obvious acrosome, a long mitochondrial filament in a midpiece appendix and a single flagellum with a 9+2 axoneme. Females brood 200 μm eggs and embryos in their distinctive, basally coiled tubes. The yolky eggs undergo radial cleavage and develop into ciliated, lecithotrophic, oblong larvae (400 μm in length) that are characterized by: (1) yellow coloration peppered with black pigment spots; (2) a deep ventral depression; (3) a posterior adhesive organ; (4) an anterior apical sensory organ; (5) an evenly ciliated epitdermis. The ventral depression is not invaginating endoderm, but is instead a glandular epithelium that evidently secretes the larval cocoon and the adult tube. Internally, the peritoneum of the coelomic cavities begins to split from the periphery of a large, central mass of yolky mesenchyme cells. The larva swims using cilia, but also undergoes contractions, evidently powered by the peritoneal cells, which constitute a myoepithelium. The discussion considers pterobranch affinities with other deuterostomes and with lophophorates.     

Regional ventilation in statically and dynamically hyperinflated dogs

Hubmayr, Rolf D., and Susan S. Margulies. Regionalventilation in statically and dynamically hyperinflated dogs.J. Appl. Physiol. 81(4):1815-1821, 1996.Using the parenchymal marker technique innormal anesthetized dogs, we compared the dynamics of regional lungexpansion between two ventilation strategies designed to increase meanthoracic volume. Dynamic hyperinflation (DH) was produced byventilating the lungs at a rate of 50 breaths/min and with a duty cycleof 0.5. Static hyperinflation (SH) was produced throughthe application of extrinsic positive end-expiratory pressure while thelungs were ventilated at a rate of 15 breaths/min and with a duty cycleof 0.15. Regional tidal volume(VT,r), regional functionalresidual volume, and the time delay between regional expansion and the flow signal at the common airway were computed for upto 100 regions/lobe in 5 animals. Ventilation strategy had no effect onthe overall variance of VT,rwithin lobes. Although the VT,rmeasured during SH correlated withVT,r measured during DH, theaverage correlation coefficient was only 0.69. Ventilation rate-relateddifferences in VT,r and regionalfunctional residual capacity varied with the regional time delay inways qualitatively consistent with parallel inhomogeneity of unit timeconstants. However, a large component of frequency-dependent behaviorremains unexplained by established mechanisms. We conclude that DH and SH should not be considered equivalent lung unit recruitmentstrategies.

     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

High-elevation rock outcrop vegetation of the Southern Appalachian Mountains

Abstract. Species composition patterns and vegetation-environment relationships were quantified for high-elevation rock outcrops of the Southern Appalachian Mountains, an infrequent and insular habitat in a forested landscape. Outcrops occur over a wide geographic range encompassing extensive variation in both geology and climate. Geographic-scale factors interact with site-scale factors to produce variation in vegetation among outcrops. Similarly, site-scale factors interact with micro-scale factors to produce variation in vegetation within outcrops. To provide a quantitatively-based classification of outcrop vegetation we used a TWINSPAN analysis of 154 100-m² plots. We recognized nine communities that primarily correspond to different combinations of elevation, bedrock type, geography, and moisture. Within outcrops of a single bedrock type, vegetation composition of 100-m² plots was consistently correlated with elevation and solar radiation, but relationships to soil nutrients varied with bedrock type. Both site-scale (100 m²) factors (e.g. elevation, slope, aspect, and bedrock type) and plot-scale (1-m²) microsite factors (e.g. soil depth, vegetation height, soil nutrients) were strongly correlated with species composition at the 1-m² level. Environment can be used to predict composition more effectively for 100-m² plots on a single bedrock type than either across bedrock types or at a 1-m² scale. Composition-environment relationships resemble those described for outcrop systems from other regions with pronounced topographic relief more than they do those described for the nearby but flatter and lower-elevation outcrops of the Southeastern Piedmont. There is strong spatial autocorrelation in this community, perhaps owing to dispersal limitation. Consequently, a comprehensive conservation strategy must include reservation of both a range of geologic types and a range of geographic locations.     

Insulin-like growth factor I levels decrease in the development of diabetes inMacaca nigra

Members of the monkey speciesMacaca nigra spontaneously develop impairments in insulin secretion and glucose clearance, and eventually become overtly diabetic. Changes in certain metabolic signals such as clearance of glucose and insulin increment secreted in an intravenous glucose tolerance test have allowed the identification of four stages in the progression from non-diabetes to diabetes in monkeys — non-diabetic, hormonally impaired, borderline diabetic, and diabetic. Recently, another metabolic stage, hyperinsulinemic, was also identified in these animals. In recent years, other factors besides those listed above have been implicated to be correlated with the metabolic progression from a nondiabetic to a diabetic state. One of these factors, is insulin like growth factor I (IGF-I). In diabetic humans who are in poor metabolic control, and in rats with streptozotocin induced ketotic diabetes, serum levels of IGF-I are lowered by as much as 40–50% of control non-diabetics. If indeed decreased IGF-I levels are correlated with the onset of diabetes then changes in IGF-I concentrations prior to the clinically diagnosed disease state would be expected. Using serum samples collected from different animals in a colony ofMacaca nigra in a variety of metabolic states, we have found that IGF-I and insulin levels decrease in each defined metabolic state as the animals progress from nondiabetic to diabetic. Since IGF-I and insulin levels decrease in a similar fashion in the progression of this disease then this maybe indicative of the coordinate expression of these two factors.