Real-space processing of helical filaments in SPARX

We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G₂/M and cells with lower G₀/G₁ DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.     

Heterogeneity of the Glyoxylate-Condensing Enzymes.

Wegener, Warner S. (Albert Einstein Medical Center, Philadelphia, Pa.), Henry C. Reeves, and Samuel J. Ajl. Heterogeneity of the glyoxylate-condensing enzymes. J. Bacteriol. 90:594-598. 1965.-Evidence is presented that the enzymatic condensations of glyoxylate with acetyl-CoA (malate synthase), propionyl-CoA (alpha-hydroxyglutarate synthase), butyryl-CoA (beta-ethylmalate synthase), and valeryl-CoA (beta-n-propylmalate synthase) are catalyzed by different enzymes. The possibility that these activities resulted from a single enzyme possessing a broad fatty acid acyl-CoA substrate specificity was ruled out. The latter was suggested by the fact that cells grown on a number of short-chain fatty acids exhibited all the above activities. The conclusion that these reactions are catalyzed by different enzymes is based on the following considerations: (i) the enzymes can be differentially inactivated by heat; (ii) under various growth conditions, where all the condensing enzymes are present, their respective activities do not show a constant ratio, as would be expected if they were catalyzed by a single enzyme; and (iii) under appropriate growth conditions, one or more of these enzymes has been shown to be present to the exclusion of others.     

Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free conditions and incubated with 4 μM verapamil and [U-¹³C₅] glutamine. Specific extracellular metabolite uptake/production rates together with mass isotopomer fractions in alanine and glutamate were implemented into a metabolic network model to calculate metabolic flux distributions in the central metabolism. Verapamil decreased specific glucose consumption rate and glycolytic activity by 60%. Although the HL-1 cells show Warburg effect with high lactate production, verapamil treated cells completely stopped lactate production after 24 h while maintaining growth comparable to the untreated cells. Calculated fluxes in TCA cycle reactions as well as NADH/FADH₂ production rates were similar in both treated and untreated cells. This was confirmed by measurement of cell respiration. Reduction of lactate production seems to be the consequence of decreased glucose uptake due to verapamil. In case of tumors, this may have two fold effects; firstly depriving cancer cells of substrate for anaerobic glycolysis on which their growth is dependent; secondly changing pH of the tumor environment, as lactate secretion keeps the pH acidic and facilitates tumor growth. The results shown in this study may partly explain recent observations in which verapamil has been proposed to be a potential anticancer agent. Moreover, in biotechnological production using cell lines, verapamil may be used to reduce glucose uptake and lactate secretion thereby increasing protein production without introduction of genetic modifications and application of more complicated fed-batch processes.     

Zur Kompartimentierung der Synthese von Mono- und Sesqui-Terpenen des ätherischen Öls beiPoncirus trifoliata

Zusammenfassung In der Frucht vonPoncirus trifoliata liegen in der Außenschale Drüsenzellkomplexe, die ein monoterpenreiches ätherisches Öl mit geringem Anteil an Sesquiterpenen und O-haltigen Substanzen produzieren. Ähnlich aussehende Exkretzellkomplexe aus den Saftschläuchen enthalten hauptsächlich Sesquiterpenkohlenwasserstoffe (STKW) und O-haltige Komponenten und sehr wenig Monoterpenkohlenwasserstoffe (MTKW). Im Schalenöl konnten nach gaschromatographischer Trennung mit Hilfe der Massenspektrometrie 19 Komponenten identifiziert werden, im Saftschlauchöl 25.Elektronenmikroskopische Aufnahmen der jüngsten Drüsenzellen beider Drüsenkomplexe lassen erkennen, daß beide Terpenklassen wahrscheinlich hauptsächlich bzw. ausschließlich plastidär entstehen.Exogen angebotenes¹⁴CO₂ wird zunächst überwiegend in die MTKW eingebaut, erst später nimmt die Markierung der STKW und O-haltigen Komponenten stark zu. Über den Ferntransportweg angebotenes¹⁴C-Leucin führt anfangs zu einer starken Markierung der STKW und O-haltigen Komponenten, erst später verschiebt sich der Einbau etwas mehr in Richtung MTKW. Als Hauptursache für den differenten Einbau wird das Vorhandensein zweier Typen von Drüsenzellkomplexen mit unterschiedlichen Syntheseleistungen angesehen.Die aus dem¹⁴CO₂ in der Außenrinde gebildeten Assimilate werden zuerst in das MTKW-reiche Öl der Schalenexkretbehälter eingebaut. Die überwiegend STKW erzeugenden Saftschlauchbehälter werden erst später beliefert. Beim Leucinangebot über die Fruchtstiele scheint es gerade umgekehrt zu verlaufen. Die aufeinanderfolgenden Maxima der Ölproduktion in den beiden Drüsenzellkomplex-Typen und die Änderung des Komponentenspektrums ihres ätherischen Öls im Verlauf der Vegetationsperiode tragen ebenfalls zu einem je nach Jahreszeit unterschiedlichen Einbau in die MTKW und STKW bei.

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Compartmentation of mono- and sesqui-terpene biosynthesis of the essential oil inPoncirus trifoliata

Summary The fruit ofPoncirus trifoliata shows glandular cell complexes in the exocarp, which produce a volatile oil rich in monoterpenes but poor in sesquiterpenes and oxigenated compounds. The juice vesicles of the endocarp possess similar cell complexes mainly containing sesquiterpenes and oxigenated compounds, whereas monoterpenes only occur in small amounts. By the use of combined gas chromatography-mass spectrometry 19 components of the rind oil and 15 compounds of the endocarp oil could be identified.As demonstrated by electron microscopy the terpenes most probably are synthesized predominantly, if not exclusively in plastids. As shown by gasradiochromatography radioactive precursors (¹⁴CO₂ and¹⁴C-leucine) are incorporated into mono- and sesqui-terpenes to a different extent.This is due to two gland types producing essential oils of different composition with regard to their mono- and sesqui-terpene percentage. In fruit development the exocarp glands differentiate earlier than the endocarp glands do. The activity of exogenously applied¹⁴CO₂ first reaches the peripheral glands and later on appears in the interior glands. Depending upon the growth season, labelled leucine transported by the conducting tissues from lower plant parts leads to a high specific activity of the sesqui-terpenes and oxigenated compounds. It could be argued that in this instance the glands of the pulp are better provided with precursors than the exocarp glands. The successive maxima of essential oil production in both glandular complexes, and the changes in the concentration of individual oil constituents during the ontogeny of the fruit also contribute to different incorporation ratios of radioactive precursors into mono- and sesqui-terpenes.

     

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.     

Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load

Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α₁-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.