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Tissue-engineered constructs have great potential in many intervention strategies. In order for these constructs to function optimally, they should ideally mimic the cellular alignment and orientation found in the tissues to be treated. Here we present a simple and reproducible method for the production of cell-laden pure fibrin micro-fibers with longitudinal topography. The micro-fibers were produced using a molding technique and longitudinal topography was induced by a single initial stretch. Using this method, fibers up to 1 m in length and with diameters of 0.2-3 mm could be produced. The micro-fibers were generated with embedded endothelial cells, smooth muscle cell/fibroblasts or Schwann cells. Polarized light and scanning electron microscopy imaging showed that the initial stretch was sufficient to induce longitudinal topography in the fibrin gel. Cells in the unstretched control micro-fibers elongated randomly in both the floating and encapsulated environments, whereas the cells in the stretched micro-fibers responded to the introduced topography by adopting a similar orientation. Proof of concept bottom-up tissue engineering (TE) constructs are shown, all displaying various anisotropic organization of cells within. This simple, economical, versatile and scalable approach for the production of highly orientated and cell-laden micro-fibers is easily transferrable to any TE laboratory.

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force‐unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection., Cells are constantly subjected to mechanical stress. This review discusses how chaperone and protein degradation systems handle force‐unfolded proteins to preserve protein homeostasis in mechanically stressed cells and tissues.

Any cell within a tissue is constantly confronted with a variety of mechanical stimuli. Sensing of these diverse stimuli plays an important role in cellular regulation. Besides shear stress, cells of the vascular endothelium are particularly exposed to a permanent cyclic straining originating from the interplay of outwards pushing blood pressure and inwards acting contraction by smooth musculature. Perpendicular alignment of cells as structural adaptation to this condition is a basic prerequisite in order to withstand deformation forces. Here, we combine live cell approaches with immunocytochemical analyses on single cell level to closely elucidate the mechanisms of cytoskeletal realignment to cyclic strain and consolidate orientation analyses of actin fibres, microtubules (MTs) and vimentin. We could show that strain-induced reorientation takes place for all cytoskeletal systems. However, all systems are characterized by their own, specific reorientation time course with actin filaments reorienting first followed by MTs and finally vimentin. Interestingly, in all cases, this reorientation was faster than cell body realignment which argues for an active adaptation mechanism for all cytoskeletal systems. Upon actin destabilization, already smallest alterations in actin kinetics massively hamper cell morphology under strain and therefore overall reorientation. Depolymerization of MTs just slightly influences actin reorientation velocity but strongly affects cell body reorientation.

The Eph-ephrin system plays pivotal roles in cell adhesion and migration. The receptor-like functions of the ephrin ligands allow the regulation of intracellular processes via reverse signaling. γ-Secretase mediated processing of ephrin-B has previously been linked to activation of Src, a kinase crucial for focal adhesion and podosome phosphorylation. Here, we analyzed the role of γ-secretase in the stimulation of reverse ephrin-B2 signaling in the migration of mouse embryonic stem cell derived microglia. The proteolytic generation of the ephrin-B2 intracellular domain (ICD) by γ-secretase stimulates Src and focal adhesion kinase (FAK). Inhibition of γ-secretase decreased the phosphorylation of Src and FAK, and reduced cell motility. These effects were associated with enlargement of the podosomal surface. Interestingly, expression of ephrin-B2 ICD could rescue these effects, indicating that this proteolytic fragment mediates the activation of Src and FAK, and thereby regulates podosomal dynamics in microglial cells. Together, these results identify γ-secretase as well as ephrin-B2 as regulators of microglial migration.

BackgroundA diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood.ObjectivesEfficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis.MethodsNew SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples.ResultsA diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian-derived), “hu” (European human-derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping.ConclusionsThese new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

Contents Slow-release GnRH agonist implants have shown to be an effective and reversible alternative to surgical castration. Testicular function is downregulated with an arrest of spermatogenesis on the level of spermatogonia/primary spermatocytes but is fully restored after abolition of downregulation. Aim of this study was to assess the quality of ejaculates after active abolishment of downregulation by implant removal and to follow recrudescence of spermatogenesis. Five dogs – which served as their own controls – were treated with a slow-release implant containing the GnRH agonist azagly-nafarelin. Implants were removed during full downregulation (testosterone

Aim of this paper is to review our present understanding on the endocrine control of luteal function in the bitch and to add some new data generated in our laboratories in support of the hypothesis of a paracrine/autocrine role of corpus luteum (CL) derived steroid hormones. Luteal lifespan in non-pregnant dogs often exceeds that of pregnant dogs, where luteal regression terminates in a rapid luteolysis, immediately prior to parturition. In non-pregnant dogs, luteal regression occurs independently of a uterine luteolysin and in spite of increased gonadotropic support during the last third of dioestrus. The CL is the only source of progesterone (P(4)) maintaining pregnancy, and they have the capacity to synthesize oestrogens as substantiated by expression of the CYP19 (aromatase) gene observed in this study. Our data demonstrated that lutein and non-lutein cells of the canine CL express in a rather constant manner the progesterone receptor (PR) and the oestrogen receptor, classifying them as targets for an autocrine/paracrine activity of CL-derived steroids. Therefore, a functional role of P(4) within a positive loop feedback system, including StAR and 3β-hydroxysteroid dehydrogenase, has been postulated.

Bu çalışma, 9-11 Temmuz 2008 tarihlerinde Viyana Üniversitesi[Avusturya]'da düzenlenen 6. International Symposium on Canine and Feline Reproduction'da bildiri olarak sunulmuştru. The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH-agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross-sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig-cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [(g) (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre-treatment values following implant removal.

Oestrogens are essential for male fertility targeting the testicular-epididymal compartment. However, the underlying mechanisms are only vaguely known and species specificities must be considered. The boar has a remarkably high testicular-oestrogen output, with the biologically inactive oestrone-sulphate being the major oestrogen occurring in the testicular vein. In the boar testis and epididymis, activity of steroid sulphatase (StS) and oestrogen sulphotransferase has been demonstrated. Thus apart from their synthesis in Leydig cells, provision of biologically active free oestrone seems also to depend on the activity and localization of these enzymes. Our aim was to establish expression patterns and activity of StS in boar testis. Testes were randomly collected from healthy boars and allotted to five age groups, five animals in each, aged 50, 100, 150, 200 and 250 days. Three extra boars aged over 250 days were castrated to obtain fresh tissue for enzyme activity tests. Immunohistochemistry detected StS only in the cytoplasm of Leydig cells and - except for day-50 group in which 65.1 +/- 4.9% (X +/- SD) of the cells were positive - expression was constant with virtually all the cells staining positive. RT-PCR and in situ hybridization confirmed expression and localization of StS mRNA. The V(max) and K(m) value (X +/- SD) for StS was 24.05 +/- 0.3 fmol/s/microg protein and 2.15 +/- 0.12 microM. These data suggest that StS within the Leydig cells of the boar is involved in modulation of testicular oestrogen bioavailability and that the site of sulpho-conjugation is not the testis but a different compartment of the testes-epididymidis complex.

The aim of the study was to evaluate a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for routine diagnosis of classical swine fever (CSF) in wild boar by using 1000 spleen homogenates that were tested previously negative by virus isolation and 26 homogenates from which CSF virus could be isolated. All 26 positive samples in the virus isolation assay also were found to be positive in real-time RT-PCR. Additionally further 10 samples were detected by real-time RT-PCR out of the 1000 negative samples in the virus isolation. With a commercial CSF antigen-ELISA only 14 out of the 36 real-time RT-PCR positive samples could be detected. The sequence analysis of all ten samples that tested positive by real-time RT-PCR and negative by virus isolation revealed CSF virus-specific sequences. Based on the assumption that all samples with a CSF virus-specific sequence or positive in the virus isolation test originated from truly CSF virus-infected wild boar, the following sensitivity values were calculated as antigen-ELISA, 39%; virus isolation, 72% and real-time RT-PCR, 100%. The use of real-time RT-PCR instead of antigen-ELISA and virus isolation as a routine tool for control and eradication of CSF in wild boar populations is recommended.

Contents Specific immunohistochemical methods were applied to detect the presence of CD4-, CD8- and major histocompatibility complex II (MHC II)-expressing immune cells and of endoglin in the canine corpus luteum between days 15 and 75, after ovulation. Corpora lutea were obtained from groups of three clinically healthy beagle bitches, ovariohysterectomized at the respective days. For all four parameters, the effect of time was highly significant. Quantitative evaluation yielded high values on day 15, followed by a decrease on day 30 (CD4, CD8 and endoglin) and day 45 (MHC II). While there were no further changes for cells staining positive for CD4 and endoglin, CD8-positive immune cells increased from day 45 to day 60 to drop again on day 75; MHC II-positive staining increased from day 45 to days 60–75. These data suggest an involvement of the immune system in control of luteal function also in the dog that may have both stimulatory and inhibiting effects.

The relationship between the apoplastic pH in young sunflower roots (Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe 2 + into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fern reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe 2 + complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe I I I reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe I I I reduction; ammonium had the reverse effect. Nitrate together with HCO 3 - , a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe I I I reduction in the apoplast, and hence Fe 2 + uptake into the cytosol.

Functional lifespan of the corpus luteum (CL) in non-pregnant dogs exceeds that of pregnant animals and may last for more than 80 days. Prolactin and LH act luteotropic, however, luteolytic mechanisms are poorly understood. Other than in life stock there is no uterine luteolysine and it was postulated that local paracrine/autocrine mechanisms might play a major role. In following this hypothesis the present investigations have clearly demonstrated that up-regulation of prostaglandin synthesis in the CL as indicated by the expression of cyclo-oxygenase II occurs with its formation and not regression, pointing towards a luteotropic rather than luteolytic action. Throughout dioestrus luteal and other cells of the CL express the oestrogen (ERalpha) and progesterone receptor (PR). While ERalpha expression was not cycle-related, PR concentrations were high in the early and late-luteal phase and a regulatory role of both steroids on CL-function is assumed. As in other species also in the dog the immune system seems to participate in the mechanisms regulating CL-function as an increased presence of lymphocytes within the CL could be detected at the beginning [CD4- CD8-, major histocompatibility complex (MHC)II-antigen expressing cells] and during the latter half of dioestrus (CD8- and MHCII-antigen expressing cells). Thus, leucocyte-derived cytokines may be important and the expression of the mRNA for interleukin (IL)8, IL10, IL12, tumour necrosis factor (TNF)alpha and transforming growth factor (TGF) beta1 was observed throughout dioestrus. Electron microscopy confirmed the slow process of luteolysis; first distinct signs of degeneration were seen on day 60, accompanied by some apoptotic events. From these data it is concluded that luteal regression as monitored by the gradual decrease of systemic progesterone concentrations in the dog is not an actively regulated but rather a permissive process. Immune-mediated events may play a key role. Changes in the vascular supply, as indicated by the expression of endoglin, seem to be of lower importance.

Uniformly (13)C,(15)N-labeled MutS, the coenzyme B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of (13)C,(15)N-labeled MutS provided signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with (15)N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B(12)-binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B(12)-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an alpha-helical structure ("nascent helix"). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B(12) and appears to be stabilized as a helix (alpha1) upon trapping of the nucleotide of the B(12) cofactor.

The main objective of the present work was to clarify the causal relationship between leaf apoplastic pH increase and Fe chlorosis under alkaline growth conditions. It has been shown that nitrate supply in contrast to ammonium supply induced a pH increase in the apoplast of young green leaves of Helianthus annuus which was followed within 12 hours by leaf yellowing. Hence nitrate nutrition is the primary cause of a high leaf apoplastic pH which induces Fe deficiency chlorosis and not the impaired provision of ATP for plasmalemma H+ pumps in yellow leaves. Supply of bicarbonate in physiological concentrations had virtually no influence on leaf apoplastic pH. Spraying leaves with diluted acids (citric acid, sulphuric acid) resulted in a decrease of apoplastic pH followed by leaf re-greening. Interestingly, the Fe concentrations remained the same in the yellow control leaves and in the sprayed green leaves. From this it follows that Fe efficiency in leaves is mainly related to the Fe distribution between apoplast and symplast. It was demonstrated that Fe chlorosis induced by nitrate nutrition begins from the base of the youngest leaves, presumably from growing interveinal microsites showing high nitrate uptake rates. Leaf yellowing spread gradually from the leaf base to the tip and after seven days of nitrate supply the leaf was almost completely yellow (98%). Leaf yellowing was measured by means of a video imaging technique. Leaf apoplastic pH recordings were conducted after loading the fluorescent dye FITC-Dextran (4000 D) into the leaf apoplast of intact plants thus simulating in vivo conditions. It was also shown using the new loading technique that the fluorescent dye did not penetrate the leaf symplast. Die herausragende Wirkung von Nitrat auf die Erhohung des Apoplasten-pH in jungen Blattern von Sonnenblumen und damit auf das Auslosen von Eisenmangelchlorose sowie die Wiederergrunung nach Bespruhung mit Sauren Das wesentliche Ziel der vorliegenden Untersuchung war, die kausalen Zusammenhange der pH-Erhohung im Blattapoplasten und das Auftreten von Fe-Chlorose unter alkalischen Bedingungen zu klaren. Es wurde gezeigt, dass Nitraternahrung, im Unterschied zur Ernahrung mit Ammonium, eine pH-Erhohung im Blattapoplasten gruner Blatter von Helianthus annus auslost, gefolgt von einer Gelbfarbung des Blattes innerhalb weniger Stunden (12 h). Deshalb ist Nitraternahrung die primare Ursache der pH-Erhohung im Blattapoplasten, die dann Eisenmangelchlorose auslost; die pH-Erhohung im Apoplasten ist damit nicht die Folge einer unzureichenden Versorgung der H+-Pumpen mit ATP durch das bereits chlorotische Blatt. Die Zugabe von HCO3— in physiologisch vertretbaren Konzentrationen hatte keinen Einfluss auf den pH-Wert im Blattapoplasten. Blattbespruhungen mit verdunnten Sauren (Citronensaure, Schwefelsaure) bewirkten einen pH-Abfall im Apoplasten, gefolgt von einer Wiederergrunung der Blatter. Interessanterweise waren die Fe-Konzentrationen in den wiederergrunten und in den chlorotischen Kontrollblattern identisch. Hieraus wird gefolgert, dass die Fe-Effizienz in den Blattern im wesentlichen von der Fe-Verteilung zwischen Apoplast und Symplast abhangt. Es wurde gezeigt, dass die durch Nitraternahrung ausgeloste Fe-Chlorose an der Basis des jungsten Blattes beginnt, ausgehend vermutlich von „microsites“, die hohe Nitrat-aufnahmereraten zeigen und zum meristematischen Gewebe des wachsenden Blattes gehoren. Die Gelbfarbung breitet sich allmahlich von der Blattbasis zur Blattspitze hin aus, und nach sieben Tagen Nitraternahrung war das Blatt fast vollstandig gelb (98%). Die Gelbfarbung der Blatter wurde mittels Video-Imaging Technik gemessen. Zur pH-Messung im Blattapoplasten unter in vivo Bedingungen wurde der Fluoreszenzfarbstoff FITC-Dextran (4000 D) in den Blattapoplasten intakter Pflanzen geladen. Es konnte gezeigt werden, dass auch bei dieser neuen Beladungstechnik der Fluoreszenzfarbstoff nicht in den Symplasten eindringt.

Amino acid limitation results in impaired sexual fruit body formation in filamentous fungi such as Aspergillus nidulans. The starvation signal is perceived by the cross-pathway regulatory network controlling the biosynthesis of translational precursors and results in increased expression of a transcriptional activator encoded by a c-Jun homologue. In the presence of amino acids, the gene product of the mammalian RACK1 homologue cpcB is required to repress the network. Growth under amino acid starvation conditions permits the initiation of the sexual developmental programme of the fungus, but blocks fruit body formation before completion of meiosis. Accordingly, arrest at this defined control point results in microcleistothecia filled with hyphae. Addition of amino acids results in release of the block and completion of development to mature ascospores. The same developmental block is induced by either overexpression of c-Jun homologues or deletion of the RACK1 homologue cpcB of A. nidulans in the presence of amino acids. Therefore, the amino acid starvation signal regulates sexual development through the network that also controls the amino acid biosynthetic genes. Expression of the RACK1 gene suppresses the block in development caused by a deletion of cpcB. These data illuminate a connection between metabolism and sexual development in filamentous fungi.

Contents For long-term culture of primary bovine luteal cells a continuous bioreactor using hollow-fibre and fluidized-bed modules was developed. Luteal cells were successfully cultured in continuous bioreactors over 21 days and their progesterone production in vitro was traced and quantified under stimulation by hCG and inhibition by PGF2α. Considerable progesterone production rates of about 102–103 molecules progesterone per cell and hour could be estimated.

Average length of gestation on the dog is 63 ± 2 days but may vary between 57-71 days due to the long period of receptivity at oestrus and the extended period of sperm survival in the female genital tract. In contrast to other domestic animals progesterone- and oestrogen concentrations are almost identical in pregnant and non pregnant bitches, except for their rapid decline immediately prior to parturition. Control of luteolysis still poorly understood. Experiments with indomethacin leading to a blockade of the prepartal PgF2α increase, which commences with the decrease of progesterone, point toward a role of PgF2α at this stage of pregnancy, which was extended for several days. At physiological conditions first visible signs of parturition were observed at peak-PgF2α levels, 33.6 ± 17.6 hours after onset of luteolysis, which lasted over 16.8 ± 3.4 hours. Pulse-type releases of oxytocin were only observed after this point of time. To test for the effect of progesterone-withdrawal, four 51-57 days pregnant bitches were treated with the antiprogestin RU 38486 which inhibits the activity of progesterone at the receptor level. In all dogs first visible signs of parturition were observed 33.5 ± 7.5 hours after onset of treatment. However, the process of parturition came to an end after cervical opening and totally only one puppy was born. Different to a normal parturition no increase of PgF2α was observed. Relaxin levels were not influenced by treatment. These observations suggested that treatment with antiprogestin followed by PgF2α might be an adequate method to induce parturition in the dog; first experiences seem to confirm this conclusion.

The architecture of the fetal villous tree and its vasculature in the bovine placentome were studied in the second half of gestation using both conventional histology and histology of ink-filled blood vessels. These were compared with corrosion casts of plastic fillings of the vasculature, prepared for scanning electron microscopy. This combination of morphological methods allows perception of the villous tree throughout gestation from broad-conical to tall-conical form where branch ramification occurs mainly at right angles to the stem. The stem villus typically contains a single central artery and several peripheral veins arranged in parallel. The proximal branches to the stem, the intermediate villi, contain a central arteriole and accompanying venules. The distal branches, the terminal villi, enclose capillary convolutions which consist of an afferent arterial capillary limb, capillary loops and efferent venous capillary limbs. Vascular interconnections exist within the terminal villi, as capillaries or venules between the capillary convolutions, serially bridging them in up to 5 places, and as capillary anastomoses between the capillary loops. Coiling and sinusoidal dilatations of these loops develop near the end of gestation. The intraplacentomal rearrangement of villous trees with progressive gestation and their morphological vascular adaptations are discussed in relation to placental function, including the ever increasing need for transplacental substance exchange. This adaptation allows the blood to traverse the shortest possible arterioarteriolar route to the periphery of the trees where exchange takes place. The need for an increasing blood flow stimulates capillary growth and at the same time optimises the blood flow reaching the placental barrier represented by the vessel cast surface. The capillaries also carry the blood back into the very voluminous system of venules and veins where back diffusion may occur. The total volume of terminal villi of bovine placentome, the ‘working part’ of villous trees, hence distinctly increases with respect to the stem and intermediate villi, the ‘supplying part’ of the villous tree. In morphological terms the efficiency of the bovine transplacental diffusional exchange is higher than in the closely related ‘co-ruminants’ sheep and goats and distinctly higher when compared with the human placenta.

The liquid in the free space of leaf cell walls, the apoplast, is in direct contact with the plasma membrane and its nutrient uptake systems. Therefore, the pH of the apoplast is of utmost interest. We have elaborated a non-destructive method by which excised sunflower leaves (Helianthus annuus cv. Erika) were perfused with fluorescein isothiocyanate-dextran (FITC-dextran) (4 000 Da) via the transpiration stream. We showed that leaf apoplast pH can be measured by using the fluorescence ratio technique together in conjunction with this dye. Evidence is provided that FITC-dextran does not penetrate the plasma membrane over a period of ca 17 h from the beginning of dye perfusion. Dye enrichment in the leaf apoplast did not cause an ‘inner filter effect’ and thus the fluorescence ratio was only dependent on pH. In vivo calibration yielded a pKa of 5.92, which was virtually identical to the pKa of 5.93 calculated for dye solutions. Hence, FITC-dextran can be detected in complex environments and covers a pH range prevailing in the leaf apoplast. Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells.

Structurally Modified Isokalafungins and Isonanaomycins The total synthesis of structural analogues of the unnatural, C-6-hydroxylated isokalafungins and isonanaomycins is described. The new compounds show improved antibiotic activity and support theories for a mechanism of action which base on the formation of reactive quinone methides. Calculations of the molecular structures show that the chromophore of the most active 9-methyl derivatives 28 and 32 is bended off along its 5-CO-10-CO axis. This corresponds to an unusual shifting of characteristic 13C-NMR signals. Different experiments for the introduction of X-CH2- or sugar residues into 1 (e.g. isolactoquinomycins) demonstrate the limits of the general synthetic pathway. Finally a short overview on significant structure/activity relationships in the (iso)nanaomycin D field is given.

Total Synthesis of Isokalafungins and Isonanaomycins The total synthesis of isokalafungins (2) and isonanaomycins (12), the first members of a nonnatural group of benzoisochromanquinone antibiotics hydroxylated only at C-6, is described. Pathway A (Scheme 1) yields the separated (±)-cis and -trans diastereomers of 2 and 12 and is especially suited for modifications of the dihydropyrane ring. On pathway B the chirality of C-1 is preformed by using 3,4,6-trideoxy-α-L-glycero-hex-3-enopyranosid-2-ulose (22) as a main building block, for the first time synthesized from L-fucose (Scheme 2). According to Scheme 3, condensation of 22 with the intermediate 23 gives the new 6-hydroxylated variants (1S,3S,4S)-isonanaomycin D (30) and (1R,3R,4R)-isokalafungin (2). Pathway B favours modifications of the phenolic ring, which can strongly influence the spectrum of activity. Unexpectedly, the shifting of the hydroxy group from C-9 to C-6 changes the antibacterial properties unessentially.

Contents Catheters were surgically placed in the uterine artery and vein of four control and four dexamethasone (DXMS)-treated cows on days 248 or 249 of gestation; at the same time the corpus luteum was removed and all animals were treated with 10 mg chlormadinone acetate i.m. daily to maintain pregnancy. After one day of blood sampling which began on day 250, animals received either 20 mg DXMS crystalline suspension i.m. or only the vehicle solution. Blood sampling was then continued for five additional days. Plasma was assayed for unconjugated estrone (E1), progesterone (P4), and pregnenolone (P5). Concentrations of El were higher in the venous samples (p < 0.02) and increased over time (p < 0.02). There was a site x time interaction (p < 0.05) and a significant effect of DXMS-treatment on the linear component (p < 0.02) of the slope values for the El concentrations for both sampling sites. Concurrently, there was an effect of time on P4 concentrations (p lt; 0.01), the venous P4 concentrations were higher (p < 0.01), and there was also a site x time interaction (p < 0.01) but not treatment effect. There was no significant effect on concentrations of P5 between treatment goups or sampling sites. It is concluded that DXMS activates the synthetic system(s) involved in production of estrone in the cow during late pregnancy. This change in estrone synthesis occurred at an increasing rate during the 5 day sampling period. The data are consistent with the hypothesis that the placenta must be capable of utilizing precursors in the cholesterol or pregnenolone group for estrone synthesis and that the major pathway could be the delta-5 pathway. Inhalt: Veranderungen in der placentaren Hormonsekretion beim Rind nach Behandlung mit Dexamethason. Bei insgesamt 8 Kuhen wurden am 248./249. Tag der Trachtigkeit Katheter in die Vena und Arteria uterina eingelegt, gleichzeitig wurde das Corpus luteum entfernt und zur Aufrechterhaltung der Graviditat wurden 10 mg Chlormadinonacetat taglich wahrend der gesamten Versuchsdauer i.m. verabreicht. Nach einer ersten Blutentnahmeperiode am Tag 250 erhielten jeweils 4 Tiere entweder 20 mg Dexamethason (kristalline Suspension) bzw. das Vehikel (Kontrollguppe) i.m. verabreicht. Die Blutentnahmen erfolgten uber weitere 5 Tage, erfast wurde der Verlauf von freiem Estron, Progesteron und Pregnenolon. Die in der Vena uterina gemessenen Estronkonzentrationen lagen jeweils hoher (p < 0,02) als in der entsprechenden Arterie und stiegen bis zum Ende des Experiments an (p < 0,02). Weiterhin zeigte sich eine Interaktion zwischen dem Ort der Blutentnahme und dem Zeitpunkt (p < 0,05), der Anstieg der Estronkonzen-trationen nach der Behandlung mit Dexamethason war – bezogen auf die lineare Komponente – sigifikant erhoht (p < 0,02). Fur P4 ergab sich ein deutlicher Abfall (p < 0,01), wobei in der Vena uterina stets hohere Werte gemessen wurden als in der Arteria uterina (p < 0,01). Auch hier war eine Interaktion zwischen dem Zeitpunkt und dem Ort der Blutentnahme gegeben (p < 0,01), allerdings konnte ein Behandlungs-effekt nicht dargestellt werden. Fur Pregnenolon ergaben sich keine Unterschiede zwischen Arteria und Vena uterina sowie den Behandlungsgruppen. Die Ergebnisse zeigen, das eine einmalige Behandlung mit Dexamethason zu einer zunehmenden Steigerung der Estronproduktion fuhrt, im Hinblick auf die unveranderte Progesteronsekretion wird geschlossen, das dabei eher der Delta-5-Syntheseweg zugrundeliegen konnte und das die entsprechenden Precursoren auf der Ebene des Cholesterins oder Prepenolons zu suchen sind.

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates. Experiments with leaves of sunflower (Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH+4 application resulted in a decrease and NO+3 application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p-trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.

Contents: Placentomes were romoved from 4 to 6 cows before/after treatment with dexamethasone (DXMS) at days 200/205, 220/225, and 250/255 of pregnancy. Preparations of cotyledonary tissue were first perfused with plain HEPES buffered medium 199 (rinsing fraction), followed by the addition of either 3H-androstenedione and 3H-pregnenolone or unlabelled cholesterol and pregnenolone as substrates. As indicated by examining the rinsing fractions from untreted animals estrone concentrations. (output) increased from day 200 to day 250 of pregnancy, treatment with DXMS led to a further significant increase on all days examined. Concomitantly with this increase a decrease in pregnenolone and progesterone was observed. Distinct effects of substrate additon were limited to the transformation of 3H-pregnenolone into 3H-testosterone/epitestosterone (yield ˜ 8%), furtheron the addition of unlabelled pregnenolone (but not of cholesterol), significantly increased progesterone synthesis as determined by radio immunoassay. Neither these nor other results published till now account for the high estrogen production by the bovine placenta on in vivo conditions. It is concluded that the in vitro systems used so far not or only in part reflect the in vivo conditions necessary to allow complete expression of the steroidogenic capacity of the bovine trophoblast. Inhalt: In vitro-Untersuchungen zur Steroidhormonbiosynthese in der Plazenta des Rindes zwischen den Tagen 200 und 255 der Graviditat Von jeweils 46 Kuhen wurden an den Tagen 200, 220 und 2.50 der Graviditat ca. 2–3 Plazentome operativ entfernt. Die Tiere wurden danach mit 10 mg Dexamethason (DXMS) behandelt und weitere Plazentome wurden an den Tagen 205, 225 und 255 gewonnen. Kotyledonengewebe (Zellen und Zellverbande, ca. 50–80 mg) wurden zu-nachst mit reinem HEPES-gepufferten Medium 199 perifundiert (Spulfraktionen). Danach wurden alternierend als Substrate 3H-Androstendion und 3H-Pregnenolon sowie unmarkiertes Cholesterin und Pregnenolon zugesetzt. Die Untersuchung der Spulfraktionen ergab fur Estron hochste Werte an Tag 250, die Behandlung mit DXMS fuhrte zu jedem Zeitpunkt (Tage 205, 225, 255) zu einem weiteren, hoch-signifikanten Anstieg. Zeitgleich mit dem Estronanstieg zeigte sich ein Abfall in den Pregnenolon– und Progesteronwerten. 3H-Pregnenolon wurde zu ca. 8% in 3H–7Testosteron/Epitestosteron umgewandelt, nach Zusatz von nicht markiertem Pregnenolonaber nicht von Cholerterinergab sich eine siynifikante Erhohung der Progerteronproduktion. Weder diese noch andere bisher erhaltene Ergebnisse konnen die hohe, unter in vivo-Bedingungen beobachtete, plazentare Ostrogenproduktion beim Rind erklaren. Es wird gefolgert, daβ die bisherigen in vitro Systeme keine oder nur bedingte Ruckschlusse auf die Gegebenheiten zulassen, die unter in vivo Bedingungen die plazentare Steroidsynthese beim Rind kontrollieren.

Contents Cystic ovaries and ovaries with pre-ovulatory follicles (POF) of cows were collected at the slaughter house and progesterone (P4), estradiol-l7β (E2), testosterone (T), IGF-I (selected samples only), glucose and lactate were determined in cystic and follicular fluid. According to the levels of P4 and E2, ovarian cysts were classified as luteinized cysts (LC; P4: 345.14 ng/ml, E2: 3.34 ng/ml), follicular cysts (FC; P4: 25.41 ng/rnl, E2 336.51 ng/ml) and mixed-function cysts (MfC; P4: 59.43ng/ml, E2: 76.62ng/ml). There were no differences in steroid hormone and IGF-I concentrations between FC and POF. In LC, glucose levels were lower and lactate levels were higher than in FC, MfC and POF, indicating a changed metabolic pattern. In polycystic ovaries, five times the combination LC X FC and three times the combination LC x MfC was found. From this, it was concluded that the final events leading to cyst formation are controlled by intrafollicular/intraovarian mechanisms rather than systemic mechanisms.

COMPETITIVE ELISAs for the detection of antibodies to bluetongue virus (BTV) are well established and widely available (World Organisation for Animal Health [OIE] 2009). Recently, sandwich (double antigen) ELISAs have been introduced by two companies. These ELISAs can detect the antibody response to a BTV infection earlier than competitive tests, and are highly sensitive for vaccine-induced antibodies (Eschbaumer and others 2009, Oura and others 2009). Their dependence on IgM (Anon 2010), however, might compromise their sensitivity after its peak early in infection. This short communication describes a study to investigate the ability of sandwich ELISAs to detect antibodies to bluetongue in experimentally infected animals. For this study, 12 cattle, 10 sheep, three alpacas and three llamas were experimentally infected with BTV serotype 8 (BTV-8). All animal experiments were notified to the competent state authority. Whole blood and serum samples were taken at regular intervals after infection. BTV RNA in blood was detected by real-time RT-PCR (Hoffmann and others 2009), and Vero cells were used for virus isolation. Serum samples were analysed with a commercially available competitive ELISA (Bluetongue Virus Antibody Test …

Pseudocoenzyme B12 (=Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamide; 1) and adenosyl-factor A (=Coβ-(5′-deoxy-5′-adenosyl)-(2-methyladenin-7-yl)cobamide; 3) are two natural analogues of coenzyme B12 (=adenosylcobalamin-Coβ-(5′-deoxy-5′-adenosyl)-(5,6-dimethyl-1H-benzimidazolyl)cobamide; 2), where the Co-coordinating 5,6-dimethyl-1H-benzimidazole nucleotide base of 2 is replaced by the purine bases adenine and 2-methyladenine. In contrast to 2, which exists solely in the ‘base-on' form, UV/VIS spectroscopy qualitatively indicates ‘base-off' constitution for 1 and 3 in aqueous solution. (cf. the established ‘base-off' form as unexpected binding mode of B12 cofactors in several B12-dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B12 (1) was synthesized in 85% yield by alkylation with 5′-O-tosyladenosine of (adenin-7-yl)cob(I)amide, which was produced electrochemically from pseudovitamin B12 (Coβ-cyano-(adenin-7-yl)cobamide). Likewise, adenosyl-factor A (3) was prepared in ca. 70% yield from factor A (=Coβ-cyano-(2-methyladenin-7-yl)cobamide; 5). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamides exist predominantly in a ‘base-off' constitution, with minor but significant contributions of the ‘base-on' form. From the UV/VIS spectra, the temperature-dependent equilibrium constants of the ‘base-off'/‘base-on' reconstitution reaction were determined as Kon (1)=0.30 and Kon (3)=0.48 at 25°, corresponding to a contribution of the ‘base-on' forms of 23% for 1 and of 32% for 3.

The kinetics of vacuolar acidification upon addition of ATP and/or pyrophosphate (PPi) has been assayed on single immobilized vacuoles by computer-aided microfluorimetry of 9-aminoacridine, and by acridine orange absorption photometry on vacuole suspensions isolated from green suspension cells of Chenopodium rubrum L. Two proton pumps at the tonoplast, an ATPase and a pyrophosphatase (PPase), operate in parallel to acidify the vacuole with different contributions adding up to a transtonoplast Δ pH of 2.6 pH units at external pH 7.2. The saturable components of proton pumping reach half maximal velocity with 0.32 ± 0.06 mM ATP and 23 ± 2.5 μM PPi, respectively. At saturating substrate concentrations, ATPase and PPase hydrolyse ATP and PPi, respectively, at a ratio of 2.3. The same ratio holds for the corresponding proton fluxes maintaining a given steady-state vacuolar pH. We conclude that both pumps operate at the same stoichiometry.