A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.     

Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Assessment of Optimal Selected Prognostic Factors

The identification and assessment of prognostic factors is one of the major tasks in clinical research. The assessment of one single prognostic factor can be done by recently established methods for using optimal cutpoints. Here, we suggest a method to consider an optimal selected prognostic factor from a set of prognostic factors of interest. This can be viewed as a variable selection method and is the underlying decision problem at each node of various tree building algorithms. We propose to use maximally selected statistics where the selection is defined over the set of prognostic factors and over all cutpoints in each prognostic factor. We demonstrate that it is feasible to compute the approximate null distribution. We illustrate the new variable selection test with data of the German Breast Cancer Study Group and of a small study on patients with diffuse large B‐cell lymphoma. Using the null distribution for a p‐value adjusted regression trees algorithm, we adjust for the number of variables analysed at each node as well. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Plant regeneration from protoplasts of the monocotyledonous Haworthia magnifica v. Poelln.

Calli were initiated from flower buds, gynoecia and inflorescence segments of Haworthia magnifica v. Poelln. and subcultured on solid medium. Two liquid culture steps were necessary to prepare the calli for the isolation of protoplasts capable of sustained cell divisions. Plants were regenerated from protoplast-derived calli. The influence of both the osmolality of the culture media and exudates on the viability of protoplasts and protoplast-derived cell colonies is briefly discussed.     

No adverse prognostic influence of hepatitis B virus infection in acute childhood lymphoblastic leukemia

In the years 1980-1985 72 children with acute lymphoblastic leukemia were diagnosed and treated by intensive combination chemotherapy (BFM protocols 79, 81, 83). Of these children 33 acquired a Hepatitis B-virus-carrier state with 1983 as the peak year of incidence. Both groups of patients, the infected and the uninfected ones, were comparable as to prognostic factors. All except 8 patients are off chemotherapy after a total duration of treatment of 1 1/2 or 2 years. Probability for event-free survival (life table analysis, maximum observation time 82 months, minimum 12 months) is equal (0.77 vs. 0.75) in both groups. With 3 exceptions, all HBV-infected patients still carry the HBs-antigen in the serum; 22 of the 30 living patients in the infected group developed anti-HBc.     

Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa

Summary Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.     

Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies

Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.     

Die Bestandsentwicklung von Kleinv?geln in Mitteleuropa: Analyse von Fangzahlen

Zusammenfassung Im Mettnau-Reit-Illmitz-Programm (MRI-Programm) der Vogelwarte Radolfzell wurden von 1974–1983 auf drei Fangstationen in Süddeutschland, Norddeutschland und Ostösterreich (Abb. 1) 184 939 Vögel von 37 Kleinvogelarten gefangen. Die Fänge erfolgten jährlich während der gesamten Wegzugperiode von Ende Juni bis Anfang November an Durchzüglern in Japannetzen unter strikter und umfassender Standardisierung aller Fang- und Arbeitsmethoden. Die Fangzahlen (Tab. 1, Abb. 2–6) werden in fünf Regressionsanalysen (Tab. 2–9) auf systematische Änderungen untersucht.Von insgesamt 496 errechenbaren Koeffizienten waren 317 oder 63,9% negativ und nur 179 oder 38,1 % positiv. Bei 34 der 37 untersuchten Arten ließen sich signifikante Trends errechnen. Sie sind für 20 oder 54 % dieser Arten ausschließlich oder überwiegend negativ. 14 Arten zeigten mindestens auf zwei Stationen negative Trends. Nur für insgesamt 10 Arten ließen sich überwiegend positive Trends errechnen. Faßt man negative Trends und Tendenzen (Vorzeichen) zusammen, so ergibt sich für 26 oder 70 % der untersuchten Arten ein negatives Bild. Dieses stark negative Gewicht ist im statistischen Sinne keine zufällige Erscheinung. Da die Bestandszunahmen die Abnahmen nicht aufwiegen konnten, zeigten auch die jährlichen Gesamtfangzahlen aller drei Stationen negative Trends. Die mittlere jährliche Abnahme betrug auf den Stationen Mettnau, Reit und Illmitz etwa 1,6 %. Die Tendenzen und Trends der einzelnen Arten stimmen auf den drei Stationen weitgehend überein. Sie lassen für ihr Zustandekommen auf weitgehend gleichförmige Ursachen bei den durch Mitteleuropa wandernden Populationen schließen.Die Fangzahlen und Literaturdaten zeigen, daß beträchtliche Teile unserer Kleinvogelwelt von Rückgangserscheinungen betroffen sind, wie wir sie von vielen Großvogelarten seit langem kennen. Bei einer Reihe von Arten sind die Abnahmen gravierend und die jährlichen Fangzahlen inzwischen so niedrig, daß sich bei ihnen mit den in den 70er Jahren konzipierten Fanganlagen eine Reihe von Fragestellungen nicht mehr untersuchen lassen. Wir können der weiteren Entwicklung der Bestände unserer derzeit noch artenreichen Kleinvogelwelt — wie der vieler Großvögel — nur mit größter Sorge entgegensehen. Die Ursachen der Rückgänge sind weitgehend unbekannt, und demzufolge mangelt es fast vollständig an geeigneten Gegenmaßnahmen. Bisherige Schutzmaßnahmen haben die Rückgänge nicht aufhalten können. Künftiger Vogelschutz wird sich folglich weitergehender Maßnahmen bedienen müssen.

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The development of songbird populations in central Europe: Analysis of trapping data

Summary In the Mettnau-Reit-Illmitz-Program (MRI-Program) of the Vogelwarte Radolfzell 184 939 birds of 37 songbird species were caught in the period 1974–1983 on three trapping stations in S. and N. Germany and E. Austria (Fig. 1). Trapping of passage migrants in mist nets occurred annually from the end of June to the beginning of November, the entire autumn migratory period. All trapping and working methods were strictly and comprehensively standardized. The trapping figures (Tab. 1, Fig. 2–6) were analyzed with respect to systematic alterations to gain information about changes in the population levels of species migrating through central Europe. The investigation is based above all on five regression analyses (Tab. 2–9).The most important individual results are: out of a total of 496 coefficients which could be calculated 317 or 63.9 % were negative and only 179 or 38.1 % positive. For 34 of the 37 investigated species significant trends could be calculated. In 20 or 54 % of these species they were (exclusively or predominately) negative. 14 species showed negative trends at least at two stations. Mainly positive trends could be found in only 10 species. Taking negative trends and tendencies (negative signs) together, 26 or 70 % of the species were included. This strongly negative bias is not accidental in a statistical sense. Since the increases could not balance the declines, the annual trapping totals of all three stations also showed negative trends. The mean annual decline in the stations Mettnau, Reit and Illmitz was about 1.6 %. Trends and tendencies of the species show a high degree of agreement for the three stations. This suggests that in the species migrating through central Europe rather uniform factors control the population levels. From the most recent literature results, it appears that most of the species with declining trapping figures in the MRI-Program demonstrate decreases elsewhere.The trapping figures of the MRI-Program and data from the literature together clearly indicate that considerable parts of our small birds are affected by declines as have been known in many large bird species for long time. In a number of species the decreases have been quite dramatic and their annual trapping totals are now so low that a number of problems can no longer be studied. We should be alarmed when considering this negative development among our small birds as we must be with respect to many large bird species. The reasons for the declines are almost unkown and thus there is lack of appropriate counter-measures. Hitherto existing efforts for conservation which are briefly discussed have not been able to prevent the decline. Consequently, bird conservation in the future has to be based on more effective methods. Proposals, briefly raised, will be discussed in a forthcoming paper.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und gefördert mit Hilfe von Forschungsmitteln des Landes Niedersachsen. 15. Mitteilung aus dem MRI-Programm