Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Integrin-like protein at the invaginated plasma membrane of epidermal cells in mature leaves of the marine angiosperm<Emphasis Type="Italic"> Zostera marina</Emphasis> L.

By immunoblotting with anti-human integrin polyclonal antibodies (1, 3 or 5), a single distinct band of about 60 kDa was detected in total protein extracts from mature leaves of the seagrass Zostera marina L., but no band was detected in total protein extracts from immature seagrass leaves, freshwater grass leaves or Arabidopsis thaliana (L.) Heynh. leaves. This integrin-like protein was detected by indirect immunofluorescence microscopy on the surface of non-spherical protoplasts of epidermal cells isolated from mature seagrass leaves using an anti-integrin 3 polyclonal antibody. Electron-microscopic analyses with the same antibody indicated that this integrin-like protein was localized specifically in the invaginated plasma membrane of epidermal cells in mature seagrass leaves. Therefore, this integrin-like protein of about 60 kDa may be involved in the developmentally regulated invagination of the plasma membrane in epidermal cells of the seagrass Z. marina.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

Erörterungen zur definition und anwendbarkeit der begriffe “ultimate factor”, “proximate factor” und “Zeitgeber”

Summary The use of the terms, which have not always been employed uniformly throughout the literature, is discussed and an exact definition is tried. Application of the terms ultimate and proximate factors should not be restricted to annual and circadian periodicities. Instead they should be used for all temporally or spatially restricted processes for which a difference between selecting and regulating mechanisms is to be found. Ultimate factors are environmental factors which in the course of evolution have led, through natural selection, to the relevant restriction. Proximate factors may be defined as those external stimuli which initiate or maintain biological processes under most favourable ecological conditions. The term Zeitgeber finally, should only be used where endogenous circadian or circennial periodicities are synchronized with environmental changes through external factors. In this sense Zeitgeber is merely a special case of proximate factor.

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Ich danke den Herren Prof. Dr. J. Aschoff, Dr. E. Gwinner und Dr. K. Hoffmann, Erling-Andechs, und Prof. Dr. D. S. Farner, Seattle, für die kritische Durchsicht des Manuskriptes und viele wertvolle Hinweise und Anregungen.     

Cholinesterases of the gall bladder

Summary Cholinesterase histochemistry of the human gall bladder was studied using two specific methods.Distribution of acetylcholinesterase: In the mucosa, nerve fascicles consisting of densely packed parallel single nerve fibres, small ganglia and spot-and glomerule-like concentrations of acetylcholinesterase activity were observed. In the muscle layer, a wide-meshed network of delicate nerves, with occasional areas of very dense innervation, and small ganglia were seen. In the serosa, glomerule-like structures surrounded by dense baskets of delicate nerves were observed. — The general scheme of distribution of non-specific cholinesterases was similar to that of acetylcholinesterase.It seems that the cholinergic innervation of the gall bladder is related to both secretion and absorption and contractility. Some cholinergic nerves are probably sensory, especially because acetylcholinesterase-positive structures, possibly pressure of stretch receptors, supplied with nerves were observed in the mucosa and the serosa. The cholinergic innervation of the gall bladder muscle was scarce except occasional areas of very dense innervation. It may thus be concluded that the intermuscle spread of excitation plays an important role, the majority of the smooth muscle cells receiving their nervous influence via electrotonic coupling.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Landscape ecology as a bridge from ecosystems to human ecology

Landscape as a subject of (terrestrial) ecology can be interpreted: first, as a piece of land composed of different ecosystems; and second, as a holistic entity of aesthetic perception derived from landscape paintings and parks of the 18th and 19th century. Such entities display a characteristic arrangement of landscape elements regarded as a whole and taking them apart for specific investigation will break up and virtually destroy it (e.g. a symphony dissociated into single notes). Landscape as a holistic entity satisfies emotional human needs like identification with regions, and explains the attraction of tourists. Entity features are land-use and land cover combined with openness and a certain naturalness. A key question is whether you call a piece of the earths surface just land or landscape– and why. Such questions touch the interface between landscape ecology and human ecology. But human ecology must not dismiss landscape functions. The most beautiful landscape will be reduced to a mere picture if it does not also provide basic life-support. Therefore, energy and matter flows and transformations between the ecosystems of a landscape have to be determined along with its climate, geomorphology (relief), soils, hydrology, species and ecosystem diversity. These different approaches, however, may never be combined into a unified whole. There is no superscience, and incidentally, its complexity would by far exceed human brain capacity. What we can achieve is bridge-building by approximation of selected facts. A conscious spatial arrangement of diversified land-use units (ecotopes) will promote (bio)diversity and may be perceived as an integral landscape pattern. A spatially and temporally differentiated energy input into land-use units will result in a gradient of utilization intensity and allow more species to thrive, again enhancing both diversity and landscape beauty. Modern humans have deliberately chosen artificial surroundings to achieve complete environmental control, even in rural lifestyles. But as far as emotional needs are concerned, this artificiality seems to be neither human nor ecological. Something natural is lacking, and landscape in its holistic sense can provide it – be it a landscaped open space in a city, a rural scene, a seashore or a mountain range. Maintaining and managing such naturalness requires sound ecological knowledge – not as an aim in itself, but to provide a bridge for humans.     

Effect of DNA concentration on transgenesis rates in mice and pigs

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10ng/l, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5ng/l and then decreased at 10ng/l for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10ng/l to maximise integration and efficiency rates in pigs.     

The general occurrence of “plastid initials” and their development into plastids in cortical cells of woody plants in winter

Summary Electron microscopic studies revealed that plastid initials, presumed precursors of plastids, occur in cortical cells of the following plants studied in February and March: Betula ermanii Cham.; Prunus sargentii Rhed.; Pyrus communis L.; Ribes sinanense F. Maekawa; Salix matsudana Koidz. forma tortuosa Rhed.; and Sambucus sieboldiana var. miquelii Hara. Since plastid initials were found previously in Malus pumila Mill., Morus bombycis Koidz. and Populus euramericana cv. gelrica (Sagisaka 1991), plastid initials have been found in all woody plants examined to date. In P. euramericana cv. gelrica, at later stages of the development of the initials in March, the conglomerates of plastid initials became heterogeneous in terms of size, extent of thylakoid formation and ability to form starch granules. The formation of prolamellar structures was frequently observed in cells of Magnolia kobus var. borealis Sarg., which was sampled on April 19. These observations suggest the course of events in the development of the plastid initial and the continuity of the life of amyloplasts over a year in the life of woody plants.     

On the meaning and measurement of nestedness of species assemblages

Nestedness of species assemblages occurs when thebiotas of sites with lower numbers of species tend to be subsets of the biotas at richer sites. We develop new quantitative and statistical techniques for measuring, testing, and comparing nestedness, and apply these methods to data from the literature. Significantly nonrandom nestedness was present in all 27 assemblages examined, and tended to be stronger in systems dominated by extinction, such as landbridge islands. Sets of assemblages that were very strongly nested were more likely to have greater species richness on one or a few large sites than on several smaller sites of equivalent total area — that is, to fall toward the single large side of the Single Large Or Several Small (SLOSS) continuum. Our analysis indicates that nestedness, when quantified as a single number for a presence-absence matrix, measures community-wide differences in incidence (the frequency of occurrence or distribution of species). Factors that lead to consistent differences among species in immigration or extinction rates cause strong patterns of nestedness of species assemblages. Nestedness is negatively related to beta diversity: nestedness is low when beta diversity is high, and vice versa. Conservation managers will thus seek to minimize nestedness and the development of nested structure in systems of nature reserves.     

Change of apocytocrhome c translocation across membrane in consequence of hydrophobic segment deletion

Wild-type apocytochrome c and its hydrophobic segment deleted mutants, named 28–39, 72–86 and 28–29/72–86 were constructed, expressed and highly purified respectively. Insertion ability into phospholipid monolayer, inducing leakage of entrapped fluorescent dye fluorescein sulfonate (FS) from liposomes, and translocation across model membrane system showed that the wild-type apoprotein and 28–39 almost exhibited the same characteristics, while mutants with segment 72–86 deletion did not. Furthermore, CD spectra, intrinsic fluorescence emission spectra, and the accessibility of the protein to the fluorescence quenchers: KI, acrylamide and HB demonstrated that the segment 72–86 deletion has a significant effect on the conformational changes of apocytochrome c following its interaction with phospholipid. On the basis of these results it is postulated that the C-terminal hydrophobic segment 72–86 plays an important role in the translocation of apocytochrome c across membrane.     

RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, RS42-6*O, RS137-28*E, and HSY-78*A, derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line RS42-6*O, showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F₂ populations. The recessive gene in line RS42-6*O, was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines RS137-28*E, and HSY-78*A, were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt (RS42-6*O), Mlf (RS137-28*E), and Mlj (HSY-78*A). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.     

Synonymous nucleotide substitution rates of β-tubulin and histone genes conform to high overall genomic rates in rodents but not in sea urchins

Summary Sea urchin and rodent genomes have been posited to evolve rapidly as indicated by divergences in single copy nuclear DNA sequences. We have examined whether the synonymous substitution rates of three highly conserved genes, -tubulin, histone H4, and histone H3, adhere to these high genomic substitution rates by comparing sequences between two sea urchins,Strongylocentrotus purpuratus andLytechinus pictus, and between rodents and humans. Whereas the rate of change between the 3 untranslated regions of the -tubulin cDNA ofS. purpuratus (Sp-1), sequenced in this study, and ofL. pictus (Lp-3) was consistent with the overall rate of change estimated from previous DNA hybridization results between these species, the synonymous substitution rates for the carboxyl domains of these -tubulins, as well as for the late histones H4 and H3, were significantly depressed. In contrast, synonymous nucleotide substitution rates between rodents and between rodent and human for the carboxyl domain proper of identical -tubulin isotypes and for histone H4 and H3.1 did not differ from the overall rate of change for the rodent genomes. Moreover, an analysis of paralogous human and mouse -tubulin sequences supported the conclusion that the synonymous substitution rates in the mouse were higher than those in the human. Differences in constraint on evolutionary change were not evident strictly from the conserved amino acid sequences and base compositions of these genes. Other constraining influences seemed more relevant to the departure of the synonymous substitution rates of the sea urchin -tubulin and histone coding regions from the average genomic rate.     

Genetics of Ia-like alloantigens in chickens and linkage withB major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens (Ia) and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the la antigens are determined by a single codominant locus and that theEa-B and Ia loci are on the same chromosome. Failure to detect a single recombinant between theEa-B and Ia loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The Ia genes are thus included in theB major histocompatibility complex of the chicken.     

Sequence of β<Subscript>2</Subscript>-microglobulin from rhesus macaque (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) includes an allelic variation in the 3′-untranslated region

The rhesus macaque (Macaca mulatta) has become a popular animal model for several human infectious diseases, such as HIV (modeled by SIV infection), hepatitis, and malaria. Investigation of T-cell responses in experimental infectious diseases in rhesus macaques has benefited from an expanding understanding of the diversity of macaque MHC class I heavy chains and the restriction of antigen presentation by macaque class I molecules. Here we add to this understanding with the first nucleotide sequences of M. mulatta ₂-microglobulin (₂m) mRNA, including a portion of the 3-untranslated region (3UTR). In pairwise comparison, the ₂m protein of M. mulatta differs from human and chimpanzee ₂m by nine amino-acid substitutions (92% identity), and from Macaca fascicularis by one amino-acid difference in the signal peptide region (99% identity). Allelic variations were identified at one site in the 3UTR. A structural analysis of human or chimpanzee ₂m and M. mulatta ₂m suggests that the differences cluster in three solvent-exposed clusters and do not involve contacts with the class I heavy chain. We predict that human and macaque ₂m should bind interchangeably with the class I heavy chains of the other species, and show that four M. mulatta class I alleles form cell surface complexes with human ₂m. Further, we predict that W6/32 (a monoclonal antibody that recognizes a combined epitope of some class I heavy chains and ₂m with a subtle species dependence) should bind similarly human or macaque class I molecules that are bound with ₂m of either species, supported by evidence of recognition of both heterologous and homologous complexes of macaque class I heavy chains. Our findings contribute to the growing understanding of rhesus macaque histocompatibility antigens and antigen presentation, and to the phylogeny of ₂m in primates.The nucleotide sequences data reported in this article have been submitted to GenBank under accession numbers AY349163 and AY445843     

Breeding soybeans for the tropics capable of nodulating effectively with indigenousRhizobium spp.

Summary Most soybean varieties fail to nodulate effectively in tropical soils unless inoculated with a competitive strain ofRhizobium japonicum. Developing countries in the tropics, with few exceptions, lack inoculant industries to produce and distribute viable inoculants to small farmers and extension programs to teach them to use inoculant. Several soybean genotypes have been identified that nodulate effectively with many strains of the cowpea inoculation group which is ubiquitous in tropical soils of Africa. Soybean genotypes that nodulate and grow well without inoculant application are called promiscuous. Methodologies for incorporation of the promiscuity character into high-yielding backgrounds are discussed.Supported in part by grant 05-0560 from United Nations Development Program to the International Institute of Tropical Agriculture.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor.