A Single-Neuron Chemosensory Switch Determines the Valence of a Sexually Dimorphic Sensory Behavior

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Fast and cost‐effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next‐generation Random Amplicon Sequencing (RAMseq)

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost‐effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.     

Breast cancer inhibiting diastereomeric diacetato[1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) derivatives: synthesis and studies on the relationship between reactivity and antitumor activity

Antitumor active [1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) diastereoisomers containing acetic acid derivatives as ‘leaving groups’ (acetate: meso/rac-4F-Pt(Ac)₂; monochloroacetate: meso/rac-4F-Pt(ClAc)₂; dichloroacetate: meso/rac-4F-Pt(Cl₂Ac)₂; trichloroacetate: meso/rac-4F-Pt(Cl₃Ac)₂; glycolate: meso/rac-4F-Pt(OHAc)₂; phenylacetate: meso/rac-4F-Pt(PhAc)₂) were synthesized and characterized by IR and ¹H NMR spectroscopy. In all complexes except meso/rac-4F-Pt(PhAc)₂, which exist as [meso/rac-4F-PtPhAc]⁺PhAc⁻, both carboxylic acid residues are coordinated to platinum. Kinetic studies on the reaction behavior of the title compounds with nucleophiles were performed by using iodide as nucleophile. The studies show that the new complexes react with nucleophiles predominantly via the ‘solvent path’ (i.e. via the reactive intermediates = Pt(X)(OH₂)⁺ and =Pt(OH₂)₂²⁺. Therefore the rates of reactions in which the reactive species are formed affect the antitumor activity of the complexes as well as their inactivation by bionucleophiles during the transport to the tumor. The extent of accumulation in the tumor cell, too, influences the antitumor activity of a complex. The rate constants are discussed in view of the activities of the respective complexes on the human MCF-7 breast cancer cell line. From the title compounds the Cl₂Ac and Cl₃Ac derivatives do not come close to the standard cisplatin, neither in chemical reactivity nor in biological activity. Meso/rac-4F-Pt(Ac)₂ and meso/rac-4F-Pt(ClAc)₂, respectively, show similar hydrolysis rates but lower antitumor activities than cisplatin, presumably due to a reduced drug uptake by the tumor cell. Meso/rac-4F-Pt(PhAc)₂ compare well with their standard carboplatin in respect to both properties. Other than the remaining, poorly water soluble title compounds, meso/rac-4F-Pt(OHAc)₂ equal their standard cisplatin in terms of water solubility and antitumor activity rac-4F-Pt(OHAc)₂ > meso-4F--Pt(OHAc)₂). However, they are markedly faster hydrolyzed than cisplatin. By use of rac-4F-Pt(Ac)₂ as an example it was confirmed that, in contrast to the parent compound rac-4F-PtCl₂, the new complex type is also active under in vivo conditions owing to its markedly lower reactivity (mainly due to the lack of a direct substitution by strong nucleophiles), which entails a reduced inactivation of the drug on its way to the tumor. The in vitro testing on tumor cell lines combined with the evaluation of the water solubility and with kinetic studies on the reaction with nucleophiles is a useful method for the preselection of potent platinum complexes deserving further thorough in vitro and in vivo investigations.     

The problem of optimal clutch size in a tritrophic system: the oviposition strategy of the thistle gallfly Urophora cardui (Diptera,Tephritidae)

The problem of optimal clutch sizes is a central theme in life history theory. Optimal allocation of eggs is especially complicated for insects in tritrophic systems. In this study we analyze some of the processes determining clutch sizes of the thistle gallfly Urophora cardui, a monophagous tephritid fly associated with Cirsium arvense. U. cardui forms multilocular shoot galls, which vary broadly in their size and number of their gall cells. We investigate various fitness consequences of gall size. An analysis of the number of cells per gall (which is correlated with gall diameter and gall weight) showed that in U. cardui there is mutual facilitation rather than larval competition. Increasing numbers of larvae per gall led to a decreasing mortality and increasing larval weight. Larval weight in turn was positively correlated with the probability of survival to adulthood and with adult weight and fecundity. Thus, all fitness parameters measured favoured large galls. Clutch sizes in oviposition experiments were distinctly larger than the number of gall cells of field populations and in cage experiments, suggesting high mortality of eggs and/or early larval instars. There was a significant relationship between the internal structure (i.e., the size of the growing point) of the bud and clutch size, suggesting that U. cardui females are able to measure bud quality and adapt clutch sizes accordingly. Clutch size was positively correlated with the female's age at first oviposition and negatively with the number of previous ovipositions and previously laid eggs. Since the potential egg capacity per female is higher than the average number of larvae it is likely to produce during its short adult lifespan, U. cardui females tend to be time-limited rather than egglimited, which might favour large clutches once an appropriate oviposition site has been located. As the development of the gall and hence the fate of a clutch depends on a number of unpredictable factors, exclusive concentration of eggs in a few large clusters would involve risks which could be avoided by increasing the number of clutches. Therefore we interpret the high variation of clutch sizes in U. cardui as a mixed strategy of bet hedging and gambling.     

The major birch pollen allergen, Bet v 1, shows ribonuclease activity

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units · mg^?1.     

Calculation of IR dichroic values and order parameters from molecular dynamics simulations and their application to structure determination of lipid bilayers

In polarized infrared (IR) absorption experiments, dichroic values are used to study the structure and orientation of lipid molecules. From computer simulations, we obtained angular distributions of IR transition moment (TM) orientations of the stretch vibrations of CH₂ groups of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholin (POPC) lipid bilayers in the gel (L ) and fluid (L ) phases. From these distributions, we calculated dichroic absorption values, as well as order parameters. We established a connection between the dichroic ratio R ^ATR , which is measured in IR-ATR setups, with the dichroic ratio D and the order parameter S _zz . The calculated values compare well with experimental results for the fluid phase. In addition, we computed angular distributions of transition moments with respect to the tail director orientation for the gel and the fluid phases. Only small differences were found between the distributions in the symmetric stretch orientation, the asymmetric stretch orientation, and the C-H bond orientation of CH₂ groups. The distributions of tail directors of POPC showed average tilts of 14.7° in the gel phase and 32.9° in the fluid phase. We developed a theory which makes it possible to calculate average tilt angles of tail directors in the gel phase from dichroic absorption values obtained from IR measurements for a wide range of lipids. Legendre coefficients were calculated from TM distributions. Order parameters, defined as the second Legendre polynomial, were found to closely approximate the TM distribution in lipid bilayers in the fluid phase.Abbreviations MD molecular dynamics - IR infrared - ATR attenuated total reflection - TM transition moment - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-cholin - POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-phatidylglycerol - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholin - NMR nuclear magnetic resonance     

Homodimeric and expanded behaviour of trimethylamine dehydrogenase in solution at different temperatures

Earlier studies using x-ray crystallography have shown that trimethylamine dehydrogenase (TMADH) from methylotropic bacteria exists as homodimers in the crystalline state. In this present hydrodynamic study we show that this is true also in dilute solution conditions and investigate the degree of swelling or relaxation of the protein in solution. Analytical ultracentrifugation was used to determine the molar mass and to investigate whether the homodimeric nature of this molecule in crystal form — as visualized by x-ray crystallography — is reproduced in dilute solution at temperatures between 4 and 40°C. The globular solution structure determined at 4 and 40°C is in good agreement with crystallographic data although trimethylamine dehydrogenase was found to be either more asymmetric in solution — or highly hydrated —, a phenomenon found to increase with temperature. In agreement with the crystallographic structure, the enzyme sediments as a homodimer with a molar mass of (163,000±5,000) g/mol. The concentration dependence of the sedimentation coefficient in the range of 0–1 mg/ml, indicates that no association or dissociation occurs. These findings are additionally supported by sedimentation equilibrium data in the concentration range of 0 to 1.8 mg/ml. Finally, from the sedimentation coefficient distribution at various temperatures, it was concluded that the enzyme is conformationally flexible and assumes an even more expanded structure at higher temperatures which is in good agreement with the hydrodynamic calculations performed. Correspondence to: S. E. Harding     

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.     

Are the most numerous sensilla of terrestrial isopods hygroreceptors? Ultrastructure of the dorsal tricorn sensilla of Porcellio scaber

The ultrastructure of the tricorn sensilla of the woodlouse Porcellio scaber was investigated in cryofixed and freeze-substituted, or chemically fixed specimens. The tricorn sensilla have a foramenized triangular-shaped outer hair and bear a poreless rod-like inner hair. The conical base of the inner hair is connected to the base of the outer hair by a complex cuticular structure. Each sensillum contains three sensory cells. The tip of one of the three dendrites contains a tubular body and is clamped between two bulges of the dendritic sheath. The two other dendrites protrude to the tip of the inner hair, flush against the cuticular wall. The microtubules in the ciliary segments are arranged in nine double tubuli that have neither osmiophilic cores nor arms. The ciliary rootlets are small. The inner segment of the largest dendrite wraps around the two smaller dendrites and one of seven enveloping cells in a mesaxon-like manner. Although this ultrastructure deviates considerably from most crustacean mechanosensitive sensilla, it nevertheless suggests a mechanosensitive function, at least for one of the sensory cells. In many aspects, the tricorn sensilla resemble the thermohygrosensilla of insects. However, our results suggest that the structural criteria for thermo-hygro-sensitivity used in insects cannot simply be applied to crustaceans.     

Local lateral inhibition: a key to spike synchronization?

 Starting from the idea that neural group activity as such is unlikely to be immediately relevant for neural synchronization, we investigate mechanisms that act at the level of individual nerve impulses (spikes). Hence, we consider populations of formal spike-emitting ‘leaky integrate and fire’ neurons instead of networks built from non-spiking oscillators. After outlining the principle of synchronization for basic forms of recurrent impulse coupling by using a pair of simplified formal neurons, we show that local lateral inhibition results in robust impulse synchronization in networks with non-vanishing transmission delays. Received: 12 January 1994/Accepted in revised form: 25 April 1995