Effects of different levels of ergot in concentrates on the growing and slaughtering performance of bulls and on carry-over into edible tissue

The aim of the present study was to examine long-term effects of low levels of ergot alkaloids on growing bulls. Natural grown ergot with a mean total alkaloid concentrations of 633 mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent alkaloids, was used. In a dose-response study 38 Holstein Friesian bulls were fed with three different doses of this ergot (0, 0.45 and 2.25 g/kg concentrate corresponding to an average total alkaloid concentration of the daily ration of 0, 69 and 421 microg/kg DM) over a period of approximately 230 days. Live weight, feed intake and health condition were monitored over the entire test period. The bulls were slaughtered at a live weight of approximately 550 kg. Carcass composition and quality were recorded and samples of liver, muscle, kidneys, fat, bile, urine and blood were analysed for ergot alkaloids. Liver enzyme activities and total bilirubin were measured in the blood. Statistically, no significant differences were detectable between the three feeding groups. Mean live weight gain over all groups was 1.41 kg/d with a mean dry matter intake of 7.35 kg/d. No carry over into tissues could be proved out of the experiment. To derive a no-effect level for beef cattle further research including higher ergot doses will be necessary.     

High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli

The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 A resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.     

Carbon dioxide signalling in plant leaves

The role of carbon dioxide (CO(2)) as a signal in biochemical regulation networks of plants is fathomed. Transport mechanisms of CO(2) and HCO3- are surveyed, which are the prerequisite for signalling. A CO(2) sensor is not known to date, but any reaction where CO(2)/HCO3- is a substrate can be a candidate. Carbon concentrating mechanisms, e.g., in higher plants C(4)-photosynthesis and crassulacean acid metabolism (CAM), generate high internal CO(2) concentrations, important for photosynthesis, but also as a basis for signalling via diffusion of CO(2). Spatiotemporal dynamics of desynchronization/synchronization of photosynthetic activity over leaves can be followed by chlorophyll fluorescence imaging. One example of desynchronization is based on patchiness of stomatal opening/closing in heterobaric leaves due to anatomic constraints of lateral CO(2) diffusion. During CAM, largely different internal CO(2) concentrations prevail in the leaves, offering opportunities to study the effect of lateral diffusion of CO(2) in synchronizing photosynthetic activity over the entire leaves.     

Plant-Associated Methylobacteria as Co-Evolved Phytosymbionts: A Hypothesis

Due to their wall-associated pectin metabolism, growing plant cells emit significant amounts of the one-carbon alcohol methanol. Pink-pigmented microbes of the genus Methylobacterium that colonize the surfaces of leaves (epiphytes) are capable of growth on this volatile C1-compound as sole source of carbon and energy. In this article the results of experiments with germ-free (gnotobiotic) sporophytes of angiosperms (sunflower, maize) and gametophytes of bryophytes (a moss and two liverwort species) are summarized. The data show that methylobacteria do not stimulate the growth of these angiosperms, but organ development in moss protonemata and in thalli of liverworts is considerably enhanced. Since methylobacteria produce and secrete cytokinins and auxin, a model of plant-microbe-interaction (symbiosis) is proposed in which the methanol-consuming bacteria are viewed as coevolved partners of the gametophyte that determine its growth, survival and reproduction (fitness). This symbiosis is restricted to the haploid cells of moisture-dependent “living fossil” plants; it does not apply to the diploid sporophytes of higher embryophytes, which are fully adapted to life on land and apparently produce sufficient amounts of endogenous phytohormones.Key Words: epiphytes, coevolution, symbiosis, methylobacteria, phytohormones, phyllosphere, plant-microbe interaction     

The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.     

Identification of three phases in Onconase refolding

Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJ x mol(-1) in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase, refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases (fast, medium, and slow), with rate constants differing by a factor of about 10 each. None of the phases could be accelerated by peptidyl-prolyl cis-trans isomerases, pointing to the absence of kinetic phases that are limited by Pro isomerization. The detailed analysis by various probes indicates that the burial of the N-terminal Trp3, which is associated with the restoration of the active site, occurs in the slow phase, i.e. in the last step of refolding. Evidently, in contrast to the folding of RNase A, there is no catalytically active native-like intermediate in the folding of Onconase.     

Rapidly reversible chlorophyll fluorescence quenching induced by pulses of supersaturating light in vivo

The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100–400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm′, induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.