Immunocytochemical detection of the growth-associated protein B-50 by newly characterized monoclonal antibodies in human brain and muscle

The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissuespecific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.     

Startling Sweet Temptations: Hedonic Chocolate Deprivation Modulates Experience,Eating Behavior,and Eyeblink Startle

Many individuals restrict their food intake to prevent weight gain. This restriction has both homeostatic and hedonic effects but their relative contribution is currently unclear. To isolate hedonic effects of food restriction, we exposed regular chocolate eaters to one week of chocolate deprivation but otherwise regular eating. Before and after this hedonic deprivation, participants viewed images of chocolate and images of high-calorie but non-chocolate containing foods, while experiential, behavioral and eyeblink startle responses were measured. Compared to satiety, hedonic deprivation triggered increased chocolate wanting, liking, and chocolate consumption but also feelings of frustration and startle potentiation during the intertrial intervals. Deprivation was further characterized by startle inhibition during both chocolate and food images relative to the intertrial intervals. Individuals who responded with frustration to the manipulation and those who scored high on a questionnaire of impulsivity showed more relative startle inhibition. The results reveal the profound effects of hedonic deprivation on experiential, behavioral and attentional/appetitive response systems and underscore the role of individual differences and state variables for startle modulation. Implications for dieting research and practice as well as for eating and weight disorders are discussed.     

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.     

SPATIAL POPULATION STRUCTURE IN THE WHIRLIGIG BEETLE DINEUTUS ASSIMILIS: EVOLUTIONARY INFERENCES BASED ON MITOCHONDRIAL DNA AND FIELD DATA

The spatial population structure of the pond-living water beetle Dineutus assimilis (Coleoptera: Gyrinidae) was investigated through a field study of population dynamics and dispersal, with a concurrent assessment of the spatial distribution of mitochondrial DNA (mtDNA) restriction-fragment-length polymorphism (RFLP). A comprehensive 2-yr survey within a 60-km² study area revealed pronounced fluctuations in local abundances, including extinctions and colonizations. The recapture of marked individuals showed that dispersal among ponds is frequent in both males and females and connects populations on a large geographic scale (maximum observed flight distance: 20 km). The population structure of D. assimilis is thus characterized by both pronounced genetic drift and frequent gene flow. Together, these two forces generate a pattern of very local and transient genetic differentiation. Mitochondrial DNA samples collected within a few kilometers indicate highly significant spatial structure, if newly founded demes or those that experienced recent bottlenecks are included. These results based on four demes within the study area were placed into a regional context by further samples collected at distances of 100 km and 200 km. F_st estimates computed on increasing spatial scales were variable but showed no increasing trend. Thus, gene flow exerts a strong homogenizing force over a wide geographic range but is counteracted locally by genetic drift. These findings highlight the need to supplement estimates of F_st with additional data to arrive at valid interpretations of the genetic information. More generally, this study raises questions about how to capture the relevant features of dynamic, subdivided populations to understand their evolutionary dynamics.     

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling

A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, ¹H-, ¹³C-nuclear magnetic resonance and mass spectrometry as -linolenic acid. When applied to detached tendrils, exogenous -linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of -linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.Abbreviations EI-MS electron impact-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - NMR nuclear magnetic resonance - TFA trifluoroacetic acid We are indebted to the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt (literature provision) for their support and to Dr. C. Brückner, Halle, for jasmonic-acid determinations.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.     

Über das Axialorgan („mysterious gland“) vonAsterias rubens L.

Zusammenfassung Das Axialorgan vonAsterias rubens und sein Terminalfortsatz im Dorsalsack wurden licht- und elektronenmikroskopisch untersucht.Die Hämalkanäle des Axialorgans bilden ein labyrinthartiges Konvolut, das in den Axialsinus hineinragt. Die Wandung der Kanäle besteht aus einer Basalmembran, deren Außenfläche von Coelomzellen (Deckzellen, Epizyten) und Muskelzellen überzogen wird. Beide Zellarten enthalten zahlreiche Einschlüsse verschiedener Form und Struktur. Die Deckzellen breiten sich mit vielen zarten Fortsätzen auf der Oberfläche des Organs zu einem zytoplasmatischen Gitterwerk aus, das morphologisch an den Zellüberzug der Glomeruluskapillaren der Vertebratenniere erinnert. Sowohl die Deckzellen als auch die von ihnen teilweise umschlossenen Muskelzellen besitzen Geißeln, die sich in das Lumen des Axialsinus erheben. Auch die Muskelzellen sind mit Ausläufern versehen. Ein Teil dieser Fortsätze enthält Bündel von dicken und dünnen Myofilamenten. Wahrscheinlich handelt es sich um Muskelzellen vom Paramyosintypus.Die Basalmembran wird hie und da durch lockere Bündel von Kollagenfasern mit periodischer Gliederung ihrer Fibrillen bzw. Filamente verstärkt. Diese Bündel sind in die aufgelockerte Innenschicht der Basalmembran eingelagert.Die Hämalkanäle werden nicht von einer endothelartigen Zellschicht ausgekleidet, verhalten sich also ebenso wie die Blutgefäße der Anneliden. Der Innenfläche der Basalmembran schmiegen sich gelegentlich große freie, an Einschlüssen reiche Zellen vom Typ der Coelomozyten an bzw. diese Zellen lösen sich von ihr ab. Das Lumen der Hämalkanäle ist vielfach von freien Zellen ausgefüllt.Die Wandung der Gefäße des Terminalfortsatzes besitzt grundsätzlich den gleichen Aufbau wie die der Hämalkanäle im Axialorgan. Im Gegensatz zu diesen enthält sie jedoch viele nackte Axone, die unter und zwischen den Deckzellen und Muskelzellen verlaufen und mit letzteren engen Kontakt aufnehmen. Regelrechte Synapsen wurden jedoch nicht festgestellt. In den Axonen und ihren Endigungen finden sich Vesikel und massendichte Granula, unter ihnen solche, die von einer Membran umschlossen werden und damit an neurosekretorische Elementargranula erinnern.Die mitgeteilten Befunde stehen mit älteren Lebendbeobachtungen in Einklang, wonach das Axialorgan und sein Terminalfortsatz pulsatorische Bewegungen nach Art eines Herzens ausführten. Die strukturelleÄhnlichkeit des Axialorgans, in demRemane (1959) ein Homologen des Glomerulus von Hemichordaten erblickt, mit den Glomeruli der Vertebratenniere läßt darüber hinaus an eine exkretorische Funktion denken. Auf eine vergleichbare Analogie zwischen der sog. Endblase im Maxillarorgan von Crustaceen und den Nierenglomeruli der Wirbeltiere (Tyson, 1968) wird hingewiesen.

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The axial organ of the starfish, Asterias rubens L.

Summary The axial organ and its terminal process (head piece) in the dorsal sac ofAsterias rubens were investigated with the light-and the electronmicroscope.The hemal channels of the axial organ form a labyrinthine system (spongy body) which is situated within the axial sinus. The walls of the channels consist of a basement membrane, the outer surface of which is covered by coelomic epithelial elements (epicytes) and muscle cells. Either cell type contains numerous inclusions of different shape and structure (phagocytosis, lysosomes). The epicytes spread with many delicate processes on the surface of the organ, thus moulding a cytoplasmic meshwork which is similar to the visceral layer of the glomerular capillaries of the vertebrate kidney. The muscle cells which are also provided with cytoplasmic processes, part of which contains bundles of thick and thin filaments (presumably constituting fibres of the paramyosin-type), are to some extent enclosed by the covering cells. Eithercell type bears cilia projecting into the lumen of the axial sinus.The basement membrane contains occasionally bundles of collagen fibres showing a periodical structure of their individual fibrils. These bundles are embedded in the loose interior layer of the basement membrane.The hemal channels are not lined by an endothelial cellular layer, resembling in this respect the vessels of the annelides, Occasionally, big free coelomocytes rich of inclusion bodies, cling to the inner surface of the basement membrane or become detached of it. The lumen of the hemal channels is frequently crowded with free cells.The structure of the vascular wall in the terminal process is principally the same as in the axial organ proper. In contrast to this it contains, however, many naked axons which run under or between the epicytes and muscle cells and which come into close contact with the latter. Typical synapses, however, have not been detected. Inside the axons and their terminals, vesicles and electron dense granules are to be found. The dense granules are often surrounded by a distinct membrane, thus bearing a close similarity to neurosecretory elementary granules.Our findings are in accordance with older in vivo observations after which the axial organ and its terminal process undergo contractions comparable to the pulsations of a heart. In addition to this, the structural similarity of the axial organ with the glomeruli of the vertebrate kidney suggests an excretory function. As already pointed out byRemane (1959) the axial organ of the Echinoderms might be the homologue to the glomerulus of Hemichordata. A comparable analogy between the terminal sac in the maxillary gland of the crustacea and the glomeruli of the vertebrate kidney (Tyson, 1968) is referred to.

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Die Untersuchungen wurden mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.