A common frame of reference for learned and communicated vectors in honeybee navigation

Humans draw maps when communicating about places or verbally describe routes between locations. Honeybees communicate places by encoding distance and direction in their waggle dances. Controversy exists not only about the structure of spatial memory but also about the efficiency of dance communication. Some of these uncertainties were resolved by studies in which recruits' flights were monitored using harmonic radar. We asked whether the two sources of vector information--the previously learned flight vector to a food source and the communicated vector--are represented in a common frame of spatial reference. We found that recruits redirect their outbound flights and perform novel shortcut flights between the communicated and learned locations in both directions. Guidance by beacons at the respective locations or by the panorama of the horizon was excluded. These findings indicate a spatial reference based on either large-scale vector integration or a common geocentric map-like spatial memory. Both models predict a memory structure that stores the spatial layout in such a way that decisions are made according to estimated distances and directions. The models differ with respect to the role of landmarks and the time of learning of spatial relations.     

Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC–MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.     

Real-time changes of the local vasoactive peptide systems (angiotensin,endothelin) in the bovine corpus luteum after induced luteal regression

There is evidence that angiotensin II (Ang II) and endothelin-1 (ET-1) may interact in an additive or synergistic way during luteal regression. The aim of the study was to investigate real time changes in luteal tissue of angiotensin and endothelin system members in mRNA expression, tissue concentrations, tissue localization, and ACE (angiotensin converting enzyme) antagonist application after prostaglandin F(2alpha) (PG) induced (days 8-12) luteal regression in cow. Corpora lutea (CL) were collected by transvaginal ovaryectomy before and 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PG injection. ACE mRNA expression (RT-PCR) increased continuously and peaked at 12, 24 hr; ECE-1 (endothelin converting enzyme) peaked at 12 hr, and both peptides in tissue (Ang II and ET-1) increased significantly and peaked at 24 hr. The expression of receptors for Ang II (AT1R and AT2R) did not change in contrast to ET receptors (ETR-A and ETR-B), which were up-regulated. Localization in tissue revealed very weak staining for Ang II and ET-1 before PG application followed by a clear increase of staining predominantly in large luteal cells, but also in endothelial cells. In two experiments, the attempt was made to block ACE by the antagonist captopril with two different doses. In both experiments with captopril, progesterone levels were not significantly different from controls. Ang II alone seems to be not essential for functional luteolysis in bovine system. In conclusion, the results suggest that both Ang II and ET-1 are in parallel up-regulated during luteal regression and may act as vasoconstrictors during functional luteolysis, but also as apoptosis inducer during functional/structural luteolysis.     

The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase

Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall-related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.     

Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB)2

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL₂ bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)₂ complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)₂ complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)₂ crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)₂(ChlL₂)₂ complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.     

Evidence of luminal phosphatidylcholine secretion in rat ileum

BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.     

Combined use of proteomic analysis and enzyme activity assays for metabolic pathway analysis of glycerol fermentation by Klebsiella pneumoniae

The fed-batch fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae displayed an unusual dynamic behavior that can be clearly divided into four distinct phases according to cell growth and CO(2) evolution rate. Metabolism changed significantly during the different phases as reflected by the varied specific rates of substrate consumption and product formation. An assay of activities of the three initial enzymes of glycerol metabolism, namely glycerol dehydratase (GDHt), glycerol dehydrogenase (GDH), and 1,3-propanediol-oxidoreductase (PDOR), showed apparently different patterns of expression. To understand the culture dynamics and patterns of enzyme formation at a more systemic level we analyzed the expression patterns of intracellular proteins of K. pneumoniae from different phases of the fed-batch fermentation using two-dimensional gel electrophoresis (2DE). Two new enzymes, namely a phosphoenolpyruvate-dependent dihydroxyacetone kinase (DHAK II) and a hypothetical oxidoreductase (HOR), which are directly related to glycerol metabolism and 1,3-propanediol formation, were identified among the highly expressed proteins. The changes in expression of these new enzymes and several other proteins identified from the 2DE analysis helped to understand not only the dynamic behavior of the fed-batch fermentation reported in this work but also some previously insufficiently understood phenomena related to this fermentation process. In particular, we demonstrated the combined use of proteomic analysis and enzyme activity assay data for metabolic pathway analysis and for a better identification of targets for bioprocess improvement.     

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.     

Ambruticin S production in air-lift and stirred-tank bioreactors

Summary Using the method of equi-inocular synchronized comparative fermentation (EISCF) the cultivation of Sorangium cellulosum So ce 10 and production of the polyketide antibiotic ambruticin S was compared in stirred-tank and air-lift reactors of different geometry. This method requires that inocula originate from the same pre-culture and cultivation parameters are synchronized to similar values. Similar ambruticin yields were obtained from both reactor systems provided that the concentration of dissolved oxygen (DO) was maintained above a certain value (ca. 40%). For cultivation of S. cellulosum it is the DO level rather than the oxygen transfer rate which presents the proper criterion for scale-up and comparative reactor studies. Offprint requests to: W.-D. Deckwer