Characterization of the iron-binding properties of pyoverdine using electron-capture dissociation-tandem mass spectrometry

Pyoverdines (PVD) are a group of siderophores produced by fluorescent Pseudomonads. Identification of PVD variants mostly relies on liquid chromatography-tandem mass spectrometry (LC–MS/MS) using collision-induced dissociation (CID). Here, both CID and the novel dissociation technique electron-capture dissociation (ECD) were applied to characterize PVD succinamide and its Fe(III)-chelated complex. The results clearly showed that ECD produced diagnostic side chain fragmentation of the PVD peptide chain and preserved the labile Fe(III) binding to the chromophore in contrast to CID. The ECD technique is therefore expected to support the understanding of strain-specific Fe(III) transport processes of PVDs.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

Effects of immobilizing agents and procedures on viability of cultured celery (Apium graveolens) cells

Summary In an attempt to develop concurrent permeabilization/immobilization systems for the production of secondary plant metabolites, the effects of chitosan, alginate, carrageenan gel and carrageenan/chitosan copolymers as immobilizing agents and immobilization procedures on viability of culturedApium graveolens cells have been examined. Chitosan immobilization, ascorbic and succinic acid resulted in low viability of plant cells but use of carrageenan/chitosan copolymers enabled maintenance of viable cell lines providing the potential for concurrent immobilization/permeabilization of cells and elicitation of secondary metabolites by chitosan.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: II. RELATIONSHIP BETWEEN GROWTH RATE, CARBOHYDRATE CONCENTRATION AND 14C-PARTITIONING WITHIN TUBERS

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. The growth rates of individualtubers were closely reflected by their ¹⁴C-content 20 h after¹⁴CO₂ had been applied to the aerial parts of the shoot for4 h. The ¹⁴C-content of the tuber (sink strength) was significantlycorrelated to the ¹⁴C-concentration of the tuber tissue (¹⁴Cg^–1 fr. wt.=sink activity). The sink activity, which differedbetween individual tubers by up to a factor of 10, was alsoclosely related to the conversion rates of ¹⁴C into the starchand the remainder as well as to the ¹⁴C-content in the ethanolsoluble fraction. This indicates the simultaneous use of photosynthatefor growth and storage in the growing tubers. No preferenceof photosynthate utilization for either of these processes couldbe detected in relation to the sink activity of the tubers.Tubers with high sink activity imported ¹⁴C-labelled photosynthateat higher rates although their tissue contained higher concentrationsof reducing sugars and sucrose than the tissue of tubers withlow sink activity. Despite the close relationship between sinkactivity and the rate of starch synthesis (¹⁴C-conversion intostarch), no significant correlation was found between sink activityand the actual starch concentration of the tissue. The applicationof zeatin riboside directly onto individual tubers increasedtheir growth rates in comparison to non-treated tubers of thesame plant. The results indicate the importance of both growthand storage processes for the regulation of sink activity inyoung potato tubers. Key words: Potato tuber, ¹⁴C-photosynthate partitioning, zeatin riboside application     

Sex steroid and glucocorticoid receptors in the bursa of Fabricius of obese strain chickens spontaneously developing autoimmune thyroiditis

The female sex develops autoimmune disease far more often than the male. This is claimed to be due to differences in peripheral sex steroid levels. We have examined in the bursa of Fabricius of Obese strain (OS) chickens, which spontaneously develop autoimmune thyroiditis, as well as in their healthy counterparts androgen(AR)-, estrogen(ER)-, progestin(PR)- and glucocorticoid(GR)-receptors in an attempt to elucidate possible further pathomechanisms, namely at the target site of steroid hormones. The characterization (affinity, specificity, association- and dissociation-rate, sedimentation behaviour) of all four types of receptors revealed no difference between sex or strain. Furthermore, the ontogeny study of the receptor capacity and affinity from the 7th embryonic day (i.e. before lymphocyte settlement) until bursa involution, again showed no difference between OS and healthy chickens of either sex. Thus, it can be concluded that the principal sex dependency of the susceptibility to autoimmune disease results predominantly from differences in sex steroid levels per se, although alterations in mechanisms beyond the cytosolic receptor level can presently not be excluded.     

Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

Group II intron self-splicing. Alternative reaction conditions yield novel products

Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I. One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products. A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified. Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.     

Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures

Treatment of suspension-cultured parsley (Petroselinum crispum) cells with fungal elicitor triggers rapid, transient and sequential phosphorylation of a number of proteins, as shown by electrophoretic analysis on two-dimensional gels. This response is rapidly reversed by removal of the elicitor from the medium and appears to be specific. It is not observed in cells exposed to other environmental stress factors, such as heat shock, UV irradiation or treatment with mercuric chloride. Pronase digestion of the elicitor has the same negative effect on protein phosphorylation as its previously demonstrated effect on the activation of some pathogen defense-related genes, suggesting a link between these two phenomena. Some of the changes in protein phosphorylation are among the earliest known events following elicitation. The phosphorylation of a neutral 45-kDa protein, which is found in both the microsomal and cytoplasmic fractions, can be observed as early as 1 min after the onset of elicitor treatment. The phosphorylation of a 26-kDa nuclear protein also starts increasing very early. The changes in protein phosphorylation in response to the elicitor are dependent on the presence of Ca2+ in the medium. Our data are compatible with the hypothesis that protein phosphorylation is involved in the signal transduction processes following elicitor recognition by parsley cells.