Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C-lobe of ovotransferrin

The interdomain disulfide bond present in the C-lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C-lobe of ovotransferrin in which the interdomain disulfide bond, Cys478-Cys671 was selectively reduced and alkylated with iodoacetamide were compared with the disulfide intact form at the endosomal pH of 5.6. Pyrophosphate and chloride mediated iron release kinetics showed no difference between the disulfide-intact and disulfide-reduced/alkylated forms; the two protein forms yielded similar observed rate constants showing an apparent hyperbolic dependency for anion concentrations. The conformational stability evaluated by unfolding and refolding experiments was greater for the disulfide-intact form than for the disulfide-reduced/alkylated form: the deltaG(D)H2O values at 30 degrees C obtained by using urea were 9.0+/-0.8 and 6.0+/-0.4 kJ/mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2+/-0.9 and 4.3+/-0.5 kJ/mol. The dissociation constant of iron (kd) was almost the same for the two protein forms, and it varied only subtly with urea concentrations but increased markedly with GdnHCl concentrations. The nonidentical values of deltaG(D)H2O and kd for urea and GdnHCl can be attributed to the ionic nature of the later denaturant, in which chloride anion may influence the structure and iron uptake-release properties of the ovotransferrin C-lobe. Taken together, we conclude that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly decreases the conformational stability in the C-lobe.     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): anatomical and cell differentiation

The ontogeny of the antennal glands was studied during the embryonic and post-embryonic development of Astacus leptodactylus. The future glands arising from undifferentiated columnar cells were detectable at the metanauplius stage EI 150 m (EI: eye index; approximately 440 m at hatching). The tubule and labyrinth differentiated in embryos at EI 190 m, and the bladder and coelomosac at EI 250 m. At EI 350 m, the tubule lengthened and divided into proximal and distal sub-regions. In later stages, the gland retained the same morpho-anatomy but the differentiation and size of each part increased. The cells of the coelomosac displayed the cytological features of podocytes in late embryonic development at EI 440 m. Only small apical microvilli and a few mitochondria were observable in the labyrinth cells at EI 250 m; by EI 440 m, these cells presented well-shaped apical microvilli, formed bodies, basal infoldings and mitochondria. In the cells of the tubules and bladder, mitochondria and basal infoldings occurred at EI 440 m and EI 250 m, respectively. The differentiation of the tubules and bladder cells suggested that they were involved in active transport at EI 440 m. Following hatching, the differentiation of the cells and the size of the glands increased. The ontogeny of the antennal glands thus starts in early embryos, the specific cellular functional features being differentiated in the various parts of the glands by EI 440 m. The antennal glands are probably functional just before hatching, i.e., before the juveniles are confronted with the low osmolality of freshwater.Thanks are due to the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran, for financial aid and support. Special thanks are also extended to the Société Française dExportation des Ressources Educatives (SFERE) for a scholarship to S.K.     

Elemental composition of anatomically distinct regions of rat liver

Experiments were conducted to test the commonly held assumption that analysis of a portion of rat liver is representative of the elemental concentration of the whole organ. Male Sprague-Dawley rats (initial body weight approximately 250 g) fed a chow diet or weanling male Long-Evans rats (initial body weight approximately 50 g) fed a semipurified diet with or without copper in the mineral premix were sacrificed after 4 wk on their respective diets and livers were dissected into seven portions representing major anatomical divisions of this organ. Elemental analyses by atomic absorption spectroscopy (calcium, magnesium, iron, zinc, copper, manganese), atomic emission spectroscopy (sodium, potassium), or colorimetric assay (phosphorus) demonstrated no statistically significant differences in composition of these nine elements among anatomical regions of liver. Dietary copper deficiency led to equivalently reduced copper concentration in all portions of rat liver and did not cause any other significant alterations in liver composition of these nine elements within the 4 wk of these studies. These results confirm the validity of the common assumption that analysis of a portion of rat liver can be representative of the elemental composition of the whole organ. This conclusion will allow more analyses to be performed on fewer animals, thereby reducing animal use and reagent costs without sacrificing analytical accuracy. Publication No. 517 of the Bureau of Nutritional Sciences.     

Ultrastructure and central projections of extraocular photoreceptors in caddiesflies (Insecta: Trichoptera)

Summary Retained larval eyes (stemmata) were studied in the imagines of three species of Trichoptera: Phrygania grandis, Agrypnia varia, and Trichostegia minor. At the light-microscopic level the stemmata of all three species appeared to represent different stages of reduction with respect to size, shape and number of lenses. However, in all three species electron-microscopic studies showed units with monolayered rhabdoms, each formed by four retinula cells. By use of immunocytochemistry the presence of S-antigen was demonstrated in the retinula cells and their axons. This method also revealed the central projections of the axons of the retinula cells, which were found (i) to terminate either in the lamina accessoria or (ii) to penetrate this area to join the fibers of the outer chiasma of the optic lobes and then terminate in the medulla accessoria. The lamina accessoria and the medulla accessoria are the assumed remnants of the larval optic lobes. It is suggested that the imaginal stemmata might still be functioning photoreceptors.     

An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths

Summary In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera.     

The macroglomerular complex of the antennal lobe in the tobacco budworm moth Heliothis virescens : specified subdivision in four compartments according to information about biologically significant compounds

The functional organisation of the male specific macroglomerular complex in Heliothis virescens has been studied by tip recordings of sensilla trichodea type 1 combined with cobalt-lysine stainings and by intracellular recordings of antennal lobe projection neurons combined with neurobiotin stainings. The antennal lobe, the macroglomerular complex and the stained axons/dendrites were reconstructed by camera-lucida. Some were further computer reconstructed in three dimensions. The results showed that: 1) The macroglomerular complex consisted of four anatomically separated compartments; 2) A large compartment (the cumulus) at the entrance of the antennal nerve received input from receptor neurons responding to the major pheromone component; 3) Another large compartment, located dorso-medially of the cumulus (the dorso-medial compartment) received input from receptor neurons tuned to the second pheromone component; 4) Two ventrally located compartments received input from two receptor neuron types, co-localized in the same sensillum. Each neuron type responded strongest to one of two interspecifically acting signals, shown to interrupt the pheromone attraction. 5) The function of the dorso-medial compartment was further verified by selective arborizations in this compartment by a projection neuron showing strong response to antennal stimulation with the second pheromone component. At low concentration, the neuron responded synergistically to stimulation with the binary pheromone mixture. Accepted: 29 July 1998     

Field Studies on Growth and Regenerative Capacity in the Foliose Macrolichen Xanthoria parietina (Teloschistales,Ascomycotina)

Growth and development of the foliose macrolichen Xanthoria parietina and adjacent lichen and bryophyte spp. was photographically monitored over a period of five years. This association developed on a plane quadrangle on the top surface of a sandstone block in the botanical garden of the University of Zürich. Line tracings of the thalline outlines were used for a quantitative estimation of laminal growth by means of gravimetric morphometry. Average radial growth of approximately 6 mm yr^?1 was recorded, with maxima around 7 mm yr^?1. The oldest, apothecia-covered central parts of the thalli lost contact with the substratum and broke off along stress cracks but the blank substratum was rapidly re-colonized by new thallus lobes which regenerated along the wound margins, to our present knowledge a peculiarity of X. parietina and few related species among the Teloschistales. Approximately 50% of the surface area of the experimental plot was covered by X. parietina at the beginning and again at the end of this five years period, with significant fluctuations in the time between. New growth in the range of 87% of the total surface area almost compensated the losses of approximately 90% per surface area in five years. New growth and losses within these five years accounted for more than 170% of the initial coverage. X. parietina gains a significant ecological advantage from its very remarkable regenerative capacity.     

Behavioural evidence for detection of the repellent polygodial by aphid antennal tip sensilla

Abstract. Close-up video was used to record responses of the aphid Myzus persicae (Sulzer) (Homoptera: Aphididae) to the plant-derived repellent polygodial. When 0.1% (±)-polygodial was painted onto one half of a Chinese cabbage leaf disc, and the other half treated with the ethanol solvent, apterous adult aphids avoided walking onto the polygodial-treated area during 15 min access periods. Similarly, when the rim of a small plastic Petri dish was treated with polygodial, aphids took significantly longer to walk off the base of the dish and onto the rim, compared with insects in solvent-treated control dishes. Analysis of video-recordings indicated that aphids were repelled following contact of antennal tips with the polygodial-treated areas of leaf or plastic. In contrast, aphids with both antennal tips surgically removed showed no apparent response to polygodial treatment of leaf or plastic surfaces. Aphids were placed between polygodial- and solvent-treated angled glass cover slips to enable unilateral presentation of polygodial on either the left or right side of the insects. Most aphids with antennae intact moved away from the polygodial-treated side, but removal of both antennal tips again negated the response. When the tip was removed from only one antenna, and the other remained intact, unilateral presentation caused a repellent response only if polygodial occurred on the same side as the intact antenna. Presentation of polygodial on both sides of aphids (bilaterally) caused no response if both antennal tips were intact or removed, but aphids with only one antennal tip removed were repelled from the side where the antenna remained intact. The results indicate that aphids detect polygodial upon contact with sensilla located on the antennal tips, and that the repellent response occurs as a negative chemotropotaxis following unilateral stimulation of these sensilla. Contact between the antennal tips and glass substrates was demonstrated using a fluorescent pigment.     

Neuronal architecture of the antennal lobe in Drosophila melanogaster

Summary Computer reconstruction of the antennal lobe of Drosophila melanogaster has revealed a total of 35 glomeruli, of which 30 are located in the periphery of the lobe and 5 in its center. Several prominent glomeruli are recognizable by their location, size, and shape; others are identifiable only by their positions relative to prominent glomeruli. No obvious sexual dimorphism of the glomerular architecture was observed. Golgi impregnations revealed: (1) Five of the glomeruli are exclusive targets for ipsilateral antennal input, whereas all others receive afferents from both antennae. Unilateral amputation of the third antennal segment led to a loss of about 1000 fibers in the antennal commissure. Hence, about 5/6 of the approximately 1200 antennal afferents per side have a process that extends into the contralateral lobe. (2) Afferents from maxillary palps (most likely from basiconic sensilla) project into both ipsi-and contralateral antennal lobes, yet their target glomeruli are apparently not the same as those of antennal basiconic sensilla. (3) Afferents in the antennal lobe may also stem from pharyngeal sensilla. (4) The most prominent types of interneurons with arborizations in the antennal lobe are: (i) local interneurons ramifying in the entire lobe, (ii) unilateral relay interneurons that extend from single glomeruli into the calyx and the lateral protocerebrum (LPR), (iii) unilateral interneurons that connect several glomeruli with the LPR only, (iv) bilateral interneurons that link a small number of glomeruli in both antennal lobes with the calyx and LPR, (v) giant bilateral interneurons characterized by extensive ramifications in both antennal lobes and the posterior brain and a cell body situated in the midline of the suboesophageal ganglion, and (vi) a unilateral interneuron with extensive arborization in one antennal lobe and the posterior brain and a process that extends into the thorax. These structural results are discussed in the context of the available functional and behavioral data.Abbreviations AC antennal commissure - AMMC antennal mechanosensory and motor center - iACT, mACT, oACT inner/middle/outer antenno-cerebral tract - bACTI, uACTI bilateral/unilateral ACT relay interneuron - AN antennal nerve - AST antenno-suboesophageal tract - FAI fine arborization relay interneuron - GSI giant symmetric relay interneuron - LI local interneuron - LPR lateral protocerebrum - SOG suboesophageal ganglion - TI thoracic relay interneuron - bVI bilateral V-relay interneuron     

The dynamics of H+ efflux from the trap lobes of Dionaea muscipula Ellis (Venus's flytrap)

Abstract. H⁺ efflux from the trap lobes of Dionaea muscipula Ellis (Venus's flytrap) was measured in vitro. FC, IAA, and 2,4-D markedly increase the rate of H⁺ efflux within minutes of their addition to the incubation medium whereas ABA and DES cause the rate to decrease. Consequently, the H⁺ efflux mechanism of Dionaea is considered to be similar to the H⁺ extrusion pumps of other higher plants in this respect. However, the H⁺ extrusion mechanism of Dionaea may be unusual in that long-term exposure of the trap lobes to known secretion elicitors— bactopeptone, NH₄⁺, Na ⁺, urea, thiourea, glycine or xanthine—also causes a large increase in the steady-state rate of H⁺ efflux from the trap lobes. Since the observed H⁺ effluxes primarily correspond to the adaxial surface of the trap lobes and show similar time- and secretion elicitor-dependencies to the responses seen in situ, it appears that the H⁺ effluxes measured in vitro bear a direct relationship to those observed in the intact, actively secreting plant. Three of the secretion elicitors that were tested— K⁺, NH₄⁺, and urea—have rapid effects on the rate of H⁺ extrusion in addition to their long-term effects. K⁺ and NH₄⁺ cause a rapid acceleration of H⁺ efflux whereas urea causes a rapid deceleration or, at high external concentrations, reversal of the net flux. The effect of K⁺ is inferred to result from K⁺ -H⁺ exchange between the tissue and bathing medium. Studies with structural analogues of NH₄⁺ and urea and inhibitors of the assimilation of reduced nitrogen suggest that the effects of NH₄⁺ and urea result from the pH-perturbing consequences of their metabolism subsequent to their absorption. These effects are considered to be auxiliary to the elicitation of secretion. It is proposed that H⁺ efflux from the trap lobes is mediated by a K⁺-H⁺ exchange mechanism, the activity of which is modified by long-term exposure to secretion elicitors and/or short-term exposure to factors which alter the availability of endogenous H⁺ ions.