Morphology and function of the proboscis in Bombyliidae (Diptera, Brachycera) and implications for proboscis evolution in Brachycera

Based on serial semithin sections and SEM photographs of representatives of European Bombyliinae and Anthracinae, the mouthparts of Bombyliidae are studied and compared with the relevant data from literature on other families of Diptera Brachycera. The three moving units of the proboscis (clypeo-cibarial region, haustellum-maxillary base region, and labella) and their structures and muscles are described. Functions and possible movements are inferred from the structures observed. Articulations both between the parts of the organ and to the head capsule enable the fly to retract its proboscis into a resting position. Proboscis movement from a resting to a feeding position encompasses the following submovements: rotating of the basal clypeo-cibarial region (= fulcrum) against the head capsule, folding of the haustellum-maxillary base region against the fulcrum, evagination and invagination of the labial base, and the labella movements. This is a novelty as compared to the rigid proboscis of Tabanidae and agrees largely with the conditions in the Cyclorrhapha. The evolution of these novelties and their functional significance are discussed. The fulcrum, as well as the haustellum-maxillary base, as the new moving units are deduced from the plesiomorphic state as present in Tabanidae by fusions of sclerites, shifts of musculature and formation of new articulations. Accepted: 5 April 2000     

Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B.     

Production of zearalenone-4-glucoside,a-zearalenol-4-glucoside and ß-zearalenol-4-glucoside

The work at hand describes the production of the zearalenone (ZON) metabolites zearalenone-4-glucoside (ZON-4G), a-zearalenol-4-glucoside (oc-ZOL-4G) and ß-zearalenol-4-glucoside (ß-ZOL-4G). In a first step a genetically modified yeast strain, expressing theArabidopsis thaliana UDP-glu-cosyltransferase UGT73C6, was treated with ZON to produce ZON-4G. The substance was purified by solid phase extraction and subsequent reversed phase preparative HPLC prior to the reduction with sodium borohydride to yield 0C-ZOL-4G and ß-ZOL-4G. The identity and purity of the substances were confirmed by¹³C-and¹H-NMR as well as by HPLC-UV. In total, 50 mg of ZON were used to produce 5 mg of a-ZOL-4G with a purity of 98%, 6 mg of ß-ZOL-4G with a purity of 99% and 5 mg of ZON-4G with a purity of 99%.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal F?rster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.     

Quantification of isoflavones in red clover by high-performance liquid chromatography

An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

When Mad met Bub

The faithful segregation of chromosomes into daughter cells is essential for cellular and organismal viability. Errors in this process cause aneuploidy, a hallmark of cancer and several congenital diseases. For proper separation, chromosomes attach to microtubules of the mitotic spindle via their kinetochores, large protein structures assembled on centromeric chromatin. Kinetochores are also crucial for a cell cycle feedback mechanism known as the spindle assembly checkpoint (SAC) 1 . The SAC forces cells to remain in mitosis until all chromosomes are properly attached to microtubules. At the beginning of mitosis, the SAC proteins—Mad1, Mad2, Bub1, Bub3, BubR1, Mps1, and Cdc20—are recruited to kinetochores in a hierarchical and interdependent fashion (Fig  1 A). There they monitor, in ways that are not fully clarified, the formation of kinetochore–microtubule attachments 1 . Two studies recently published in EMBO reports by the groups of Silke Hauf 2 and Jakob Nilsson 3 , and a recent study by London and Biggins in Genes & Development 4 , shed new light on the conserved SAC protein Mad1.