Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Contribution to the morphology and biochemistry of mustelide blood. 1. Blood specimen collection in various mustelides with special reference to the use of the tissue adhesive "Fimomed"]

An account is given of various techniques of blood sampling in the context of certain mustelids, including cardiopuncture, amputation of tail tip, talon cutting, incision of the ear vein, puncture of the jugular or femoral vein, and catheterisation of the abdominal aorta. Reference is made to details of use of all techniques, characteristics, advantages and potential setbacks, and preferable use of some of the tested methods to collect blood from mustela and martes species. Blood collection from the abdominal aorta may be helpful in obtaining no-haemolysis and no-additive plasma for biochemical multi-screening. Biochemical, pharmacological, and toxicological follow-up checks may be feasible under certain conditions following surgical exposure of the external jugular vein. The use of "Fimomed" (n-butylcyano-acrylate), a tissue adhesive, may help to reduce effort in terms of time and material and consequently, rationalise veterinary hygiene action as a whole, provided that the conditions for its application are observed. The skin adhesive is properly applicable to skin lesions of mustelids. A combination of suturing with adhesive should be used to close laparotomy wounds for better mechanical strength of the abdominal wall. Possible applications of "Fimomed" should be tested with other species as well.     

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

Ceramide triggers budding of exosome vesicles into multivesicular endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.