Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

Gliotoxin in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>: an example that mycotoxins are potential virulence factors

Moulds produce several different mycotoxins that may improve their chance of survival in particular environments. For example, Aspergillus fumigatus, an important human pathogen, produces several mycotoxins including gliotoxin. This secondary metabolite, a small lipid soluble dipeptide, exerts toxic effects on phagocytic cells and T-lymphocytes at low concentrations in vitro. A. fumigatus also produces high levels of gliotoxin in vivo, and this suggests that host defense mechanisms might be impaired by this metabolite during host infection. In the past few years, the genes responsible for the production of gliotoxin in A. fumigatus have been identified and more recently gliotoxin-minus mutants have been used in animal experiments to ascertain the biological role of this product. Mycotoxins have also been shown to act as virulence factors in some fungal infections of insects and plants.     

Sperm sex-sorting in the Asian elephant (Elephas maximus)

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.     

Perivascular cells of the supraspinatus tendon express both tendon- and stem cell-related markers

Tendons and ligaments are often affected by mechanical injuries or chronic impairment but other than muscle or bone they possess a low healing capacity. So far, little is known about regeneration of tendons and the role of tendon precursor cells in that process. We hypothesize that perivascular cells of tendon capillaries are progenitors for functional tendon cells and are characterized by expression of marker genes and proteins typical for mesenchymal stem cells and functional tendon cells. Immunohistochemical characterization of biopsies derived from intact human supraspinatus tendons was performed. From these biopsies perivascular cells were isolated, cultured, and characterized using RT-PCR and Western blotting. We have shown for the first time that perivascular cells within tendon tissue express both tendon- and stem/precursor cell-like characteristics. These findings were confirmed by results from in vitro studies focusing on cultured perivascular cells isolated from human supraspinatus tendon biopsies. The results suggest that the perivascular niche may be considered a source for tendon precursor cells. This study provides further information about the molecular nature and localization of tendon precursor cells, which is the basis for developing novel strategies towards tendon healing and facilitated regeneration. H. Tempfer and A. Wagner have contributed equally to this paper.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n = 9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.     

Passerine migration strategies and body mass variation along geographic sectors across East Africa, the Middle East and the Arabian Peninsula

Using the body mass estimates of 12 long-distance migrating Palearctic passerine species monitored at successive sites across the Eastern Africa flyway, we tested whether birds modulate their body mass according to specific seasonal demands across different geographic sectors. We compared body mass estimates across latitudinal distances and geographic sectors in Europe, the desert, Northeast Africa and East Africa. Our results show that, depending on the species and season considered, the average body mass increase or decrease is variable at and among different geographic sectors. By comparing the variation in body mass between different ecological sectors, we were able to show when and where migrants accumulate their migratory fuel reserves during migration.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

γ-Pyrone natural products—A privileged compound class provided by nature

In “Biology Oriented Synthesis” (BIOS), the inherent biological relevance of natural products is employed for the design and synthesis of compound libraries. Towards this end, library generation in BIOS is focused on compound classes from biologically relevant space such as the natural product space or also the drug space and only scaffolds of these areas of proven relevance are employed for synthesis of small focused libraries with limited diversity. We here present a short overview of γ-pyrone natural products, highlighting their biological properties and their potential applicability in a BIOS of a compound library.