Nachweis einer Pupillenreaktion im Auge der Schmeißfliege

Zusammenfassung Elektronenoptische Untersuchungen mit der Gefrierätzmethode an Augen der Normalform von Calliphora erythrocephala (Meig.) ergeben, daß im dunkeladaptierten Zustand die Rhabdomere 1–6 in den Retinulazellen dicht von Vesikeln umgeben sind, die wahrscheinlich durch Pinocytose an den Mikrovilli der Rhabdomere entstehen. Diese Bläschen fehlen im helladaptierten Zustand nahezu völlig. Die optische Dichte des umgebenden Mediums der lichtleitenden Rhabdomere wird dadurch im Dunkel-Auge herabgesetzt, was mit Hilfe der Becke-Methode im Lichtmikroskop festgestellt werden konnte.Physikalische Berechnungen mit den Gleichungen der geometrischen Optik ergeben, daß durch diese Brechzahländerung in den Sinneszellen im dunkeladaptierten Auge der physiologisch wirksame Lichtfluß in den Rhabdomeren l–6 im grünen Spektralbereich um etwa 30% erhöht werden kann.

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Evidence for a longitudinal pupil in the blowfly eye from studies with light and electron microscopes

Summary Eyes of the wild-type blowfly, Calliphora erythrocephala (Meig.), were investigated by using freeze-etching and the light microscope. In the dark-adapted eye a layer of vesicles 1 m thick borders on the rhabdomeres in sense cells Nos. 1 to 6. These vesicles may arise by pinocytosis at the bases of the rhabdomeric microvilli and decrease the optical density of the sense cell medium adjacent to the rhabdomeres. In the light-adapted sense cells only a few vesicles could be seen. No differences could be observed to exist between the two states of adaptation in the retinular cells Nos. 7 and 8 and in the axial retinular space.Calculations based on geometrical optics show that when rhabdomeres Nos. 1 to 6 are in the dark-adapted state they can transmit about 1.3 times more light energy than when they are in the light-adapted state. The results show the existence of a light-induced pupil reaction correlated to ultrastructural changes in the photoreceptor cells of the blowfly eye.

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Der Deutschen Forschungsgemeinschaft danke ich für die Gewährung eines Ausbildungsstipendiums.     

A new giant egg-laying onychophoran (Peripatopsidae) reveals evolutionary and biogeographical aspects of Australian velvet worms

Representatives of Ooperipatellus (Peripatopsidae) are among the smallest onychophorans known, commonly varying between 10 and 20 mm in length. Herein, we present a peculiar new species of Ooperipatellus from Tasmania that can exceed twice the length of other representatives of this taxon. Ooperipatellus nickmayeri sp. nov. is comprehensively described based on morphological, molecular, karyological and slime protein profile data. Morphological analyses expose a set of novel features in this species, including a swollen area covered with a modified integument on the posterior border of the male genital pad, modified papillae on the female ovipositor and the presence of pseudoplicae in the dorsal integument. The evolutionary significance of pseudoplicae remains unclear, but similarities between O. nickmayeri sp. nov. and Plicatoperipatus jamaicensis, the only species from which these structures were previously known, suggest they evolved due to functional constraints of the onychophoran integument. Our karyological investigation further revealed that the new species has the largest karyotype known within Peripatopsidae (2n = 50, XY). Finally, the results of our molecular phylogenetic analyses support the recognition of O. nickmayeri sp. nov. and shed light on previously unclear aspects of the biogeographical history of Ooperipatellus in Southern Australia, Tasmania and New Zealand.     

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA‐regulated genes are over‐represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA‐related gene expression could be an important component of the Arabidopsis–aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild‐type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1‐1 mutants, which cannot synthesize ABA, and showed a significant preference for wild‐type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1‐1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild‐type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4‐methoxyindol‐3‐ylmethylglucosinolate was more abundant in the aba1‐1 mutant than in wild‐type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids.     

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.     

Maternally and Naturally Acquired Antibodies to Shiga Toxins in a Cohort of Calves Shedding Shiga-Toxigenic Escherichia coli

Calves become infected with Shiga toxin-producing Escherichia coli (STEC) early in life, which frequently results in long-term shedding of the zoonotic pathogen. Little is known about the animals'' immunological status at the time of infection. We assessed the quantity and dynamics of maternal and acquired antibodies to Shiga toxins (Stx1 and Stx2), the principal STEC virulence factors, in a cohort of 27 calves. Fecal and serum samples were taken repeatedly from birth until the 24th week of age. Sera, milk, and colostrums of dams were also assessed. STEC shedding was confirmed by detection of stx in fecal cultures. Stx1- and Stx2-specific antibodies were quantified by Vero cell neutralization assay and further analyzed by immunoblotting. By the eighth week of age, 13 and 15 calves had at least one stx₁-type and at least one stx₂-type positive culture, respectively. Eleven calves had first positive cultures only past that age. Sera and colostrums of all dams and postcolostral sera of all newborn calves contained Stx1-specific antibodies. Calf serum titers decreased rapidly within the first 6 weeks of age. Only five calves showed Stx1-specific seroconversion. Maternal and acquired Stx1-specific antibodies were mainly directed against the StxA1 subunit. Sparse Stx2-specific titers were detectable in sera and colostrums of three dams and in postcolostral sera of their calves. None of the calves developed Stx2-specific seroconversion. The results indicate that under natural conditions of exposure, first STEC infections frequently coincide with an absence of maternal and acquired Stx-specific antibodies in the animals'' sera.Shiga toxin-producing Escherichia coli (STEC), also known as enterohemorrhagic E. coli (EHEC), is a food-borne pathogen which can evoke life-threatening diseases, such as hemorrhagic colitis and hemolytic-uremic syndrome, in humans (26). Cattle and other ruminants are primary reservoirs for STEC serotypes that are typically associated with human disease, e.g., O157:H7. Calves become infected with STEC early in life via horizontal or vertical transmission (55) and do not develop clinical signs of infection but may shed the bacteria for several months and in great quantities (15, 64). Reduction of persistent STEC shedding in cattle would contribute greatly to preventing human STEC infections.Evidence that vaccination may be a sensible control option has come from studies in which cattle shed E. coli O157 less frequently following immunization with STEC O157:H7 antigens (48). However, several other studies deploying various STEC antigens produced conflicting data regarding the efficacy of vaccines to reduce or prevent STEC shedding by cattle (16, 61). Identification of candidate antigens is hampered by the limited knowledge of the immune responses occurring after bovine STEC infections, their kinetics, and their meaning for the control of STEC shedding. Serological responses against a variety of antigens following E. coli O157 colonization have repeatedly been reported. Infected animals frequently develop antibodies against STEC lipopolysaccharides (LPS), e.g., O157 LPS (25). Such antibodies inhibit STEC O157 adhesion to cells in vitro (45), but shedding is not affected by serum and mucosal O157 titers in vivo (25). Mucosal immune responses are directed mainly against membrane-associated and type III secreted STEC proteins (40). Type III secreted antigens are relatively conserved among non-O157 STEC serotypes and were assumed to be broadly cross-protective (48). Antibodies against Tir (translocated intimin receptor), intimin, and Esps (E. coli secreted proteins) A and B are detectable in calves and adult cattle after natural and experimental STEC infections or after vaccination based on these antigens (9, 16, 48, 60). Nevertheless, they do not limit the magnitude or duration of STEC shedding under field conditions (61), where cattle are confronted with a variety of different STEC strains (19, 55).Shiga toxins (Stx) are potent protein cytotoxins and represent the principal STEC virulence factors in the pathogenesis of human infections (49). Cumulating evidence shows that Stx act as immunomodulating agents during bovine STEC infections. Stx1 alters the cytokine expression pattern in mucosal macrophages (56) and intraepithelial lymphocytes (38) and suppresses the activation and proliferation of mucosal and peripheral lymphocytes in vitro (36, 37). The development of an adaptive cellular immune response is significantly delayed following experimental infection of calves with Stx2-producing STEC O157:H7 compared to that in animals inoculated with Stx-negative E. coli O157:H7 (22). In vitro and in vivo studies showed that Stx act during the early phases of immune activation rather than downregulating an established immunity (22, 57). Consequently, Stx may principally exhibit their immunomodulating activity upon first STEC infection of hitherto immunologically naïve animals.Antibodies against Stx may be essential to protect cattle from Stx-mediated immunosuppression, but only when they are present in sufficient amounts at the time of initial STEC infection. Stx-specific antibodies are detectable in sera and colostrums of naturally infected cows (6, 47). In contrast, naturally exposed calves mostly lack Stx-specific antibodies, and antibodies are barely inducible by repeated experimental STEC infections (22, 25). Maternal antibodies were considered to interfere with the development of an acquired anti-Stx immune response in calves (25), but mother-to-offspring transfer of such antibodies has not been confirmed to date. The objectives of this study were to investigate the dynamics of maternal Stx1- and Stx2-specific antibodies in calves held under conditions of natural exposure and to determine the age at the onset of acquired Stx immunity relative to the time of initial STEC infection.     

Two-faced Fcab prevents polymerization with VEGF and reveals thermodynamics and the 2.15 Å crystal structure of the complex

Fcabs (Fc domain with antigen-binding sites) are promising novel therapeutics. By engineering of the C-terminal loops of the CH3 domains, 2 antigen binding sites can be inserted in close proximity. To elucidate the binding mode(s) between homodimeric Fcabs and small homodimeric antigens, the interaction between the Fcabs 448 and CT6 (having the AB, CD and EF loops and the C-termini engineered) with homodimeric VEGF was investigated. The crystal structures of these Fcabs, which form polymers with the antigen VEGF in solution, were determined. However, construction of heterodimeric Fcabs (JanusFcabs: one chain Fc-wt, one chain VEGF-binding) results in formation of distinct JanusFcab–VEGF complexes (2:1), which allowed elucidation of the crystal structure of the JanusCT6–VEGF complex at 2.15 Å resolution. VEGF binding to Janus448 and JanusCT6 is shown to be entropically unfavorable, but enthalpically favorable. Structure-function relationships are discussed with respect to Fcab design and engineering strategies.     

Enantiopurity Determination of the Enantiomers of the Triple Reuptake Inhibitor Indatraline

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in ¹H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)‐indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high‐performance liquid chromatography (HPLC) method based on a modified β‐cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25–1000 μM for the (1R,3S)‐enantiomer and 1.25‐750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)‐enantiomer and up to 99.67% ee for the (1S,3R)‐enantiomer. Chirality 25:923–933, 2013. © 2013 Wiley Periodicals, Inc.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.