Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time ±4 cm pan-and-tilt action. Robot-driven ±60° pivots of an integrated ±185° rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[¹⁸F] fluoro-2-deoxy-ᴅ-glucose (¹⁸F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less¹. In this article, we describe new radiation plans for the treatment of moving lung tumors.     

Experimentelle Beeinflußbarkeit des Vitellogenin-Titers bei der Bienenkönigin (Apis mellifica)

Ohne Zusammenfassung

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Experimental manipulation of the vitellogenin titer in the honeybee queen (Apis mellifica)

     

Neutrophilic Hypersegmentation Without Macrocytic Anemia

In five months of examining some 25,000 blood smears of hospital and clinic patients, 200 patients were found whose blood had hypersegmented neutrophiles without macrocytosis of the erythrocytes. Sixty-five of these patients subsequently received adequate laboratory work-up. Seven proved to have a bacterial inhibitor in their serum which interfered with the microbiological assay of folic acid. Of these patients, six had normal B-12 levels. Of the remaining 58, 45 had a deficiency of folate, of vitamin B-12 or of both. In 13, no such deficiency could be established. Of these, seven proved to be uremic. It is concluded that the additional effort required to search carefully for hypersegmentation, even in the absence of erythroid macrocytosis, is thoroughly justified, and that a large percentage of those patients in whose blood hypersegmented neutrophiles are found will prove to have important deficiencies of folate or B-12, or will prove to be uremic.     

Strukturelle Chromosomenstörungen bei Intelligenzminderung

Anomalies of chromosome number and structure are among frequent causes of intellectual disability (ID) and psychomotor developmental delay. The great heterogeneity of ID is reflected in the diversity of the possible aberration types and causative chromosomal regions. In this context, conventional cytogenetics using light microscopy detect—amongst others—structural aberrations of sizes above 5–10 megabases (Mb), and also in the form of small mosaics, and locates them within the genome. Clinically suspected microdeletion and microduplication syndromes of much smaller aberration sizes can be detected by fluorescence in situ hybridization. Chromosomal microarrays (CMAs) can identify submicroscopic microdeletions and -duplications in the entire genome owing to their much superior resolution, which can reach significantly less than 0.1 Mb; however, CMAs cannot give evidence about the location of the duplications and usually barely detect low-grade mosaics of less than 20%. Because of their varying abilities, conventional cytogenetics and CMAs complement each other and detect causative chromosomal aberrations in approximately 15% each of patients with ID, including Down syndrome. Together with modern sequencing techniques, they constitute an important element of the etiological analysis of ID in human genetics. Typical chromosome aberration types are discussed with examples and are categorized in an overview of the present-day situation.     

Quantitative Untersuchungen zum Dotterprotein-Haushalt der Honigbiene (Apis mellifica)

Zusammenfassung Die löslichen Hämolymph-, Ovarund Eiproteine der Honigbiene wurden elektrophoretisch auf Celluloseacetat-Streif en und Polyacrylamid-Gelen aufgetrennt. Die dominierende Hämolymph-Fraktion (60–80%) ist ein Weibchen-spezifisches Protein. Korrespondierende Fraktionen sind im Ovar und in ungefurchten Eiern zu finden. Aufgrund verschiedener Ergebnisse wird es als sehr wahrscheinlich angesehen, daß diese Fraktionen das Dotter-Material (= Vitellogenin) reprentieren. Bei Apis besteht das Vitellogenin nur auseiner Protein-Bande ohne Kohlenhydrat-Komponenten.Der Titer des Dotter-Proteins in der Hämolymphe hängt vom physiologischen Zustand der weiblichen Bienen ah. Bei der Arbeiterin ist nur während der Ammen-Tätigkeit Vitellogenin in der Hämolymphe zu finden, bei der Königin dagegen permanent und zu allen Jahreszeiten. Bemerkenswerterweise ist bei begatteten und eierlegenden Königinnen die Konzentration des Dotter-Materials in der Hämolymphe niedriger als bei unbegatteten und adulten, nicht-eierlegenden.Die Proteinsynthese-Raten wurdenin vivo nach Injektion eines¹⁴C-Aminosäuren-Gemisches bestimmt. Zur Messung der Radioaktivität der einzelnen Pherogramm-Banden wurde ein Methan-Durchflußzähler verwendet. Bei intensiver Legetätigkeit einer Königin liegt die Rate der Vitellogenin-Synthese dreimal höher als die der nicht-weibehen-spezifischen Blutproteine. Ungefähr 85% der vom Fettkörper abgegebenen Proteine sind Dotter-Material. Im Gegensatz zu anderen Insekten synthetisieren auch nicht-eierlegende Königinnen Vitellogenin; in diesem Falle liegt die Syntheserate jedoch nicht höher als die der übrigen Serum-Proteine. Der Verbleib des Dotter-Materials bei solchen Königinnen ist unbekannt. Im Ovar verläuft die Synthese der Euplasma-Proteine ungefähr mit der gleichen Rate wie die der nicht-weibchen-spezifischen Hämolymph-Fraktionen. Im Vergleich mit allen anderen Proteinen tritt die Markierung des Ovar-Dotters verspätet auf. Daraus kann geschlossen werden, daß das Dotter-Material nicht innerhalb des Ovars synthetisiert wird. Dieser Schluß wird außerdem durch den Befund gestützt, daß der Anstieg der spezifischen Aktivität der Dotter-Proteine im Ovar erst zu einem Zeitpunkt erfolgt, in dem in der Hämolymphe keine freien¹⁴C-Aminosäuren mehr verfügbar sind. Auf das Markierungs-Maximum der Dotter-Proteine folgt ein relativ rascher Abfall der spezifischen Aktivität. Aus den Werten kann die Vitellogenese-Dauer eines einzelnen Follikels mit 2 Tagen bestimmt werden. Bis zur Eiablage werden anschließend noch 2 weitere Tage benötigt, in denen die Glykogen-Synthese und Chorion-Bildung ablaufen.

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Quantitative investigations on vitellogenic protein metabolism in the honey bee (Apis mellifica)

Summary The soluble proteins of the haemolymph, ovary and eggs of the honey bee were separated by electrophoresis on cellulose acetate foils and on polyacrylamide gels. The predominant haemolymph fraction (60–80%) is a female-specific protein. Corresponding fractions are found in the ovary and in uncleaved eggs. According to the results, it is highly probable that these fractions represent the yolk material or vitellogenin. InApis, this vitellogenin is represented by only one protein band which carries no carbohydrate components.The haemolymph titre of vitellogenic protein depends on the physiological state of the female bees. In the worker bees, haemolymph yolk proteins are detectable only during the nurse age, whereas in queens they can be found throughout the year. It may be noted that in the mated and egg producing queens the concentration of vitellogenin in the haemolymph is lower than in virgin queens and in adult non-laying queens.The rate ofin vivo protein synthesis was determined by injecting a mixture of¹⁴C-amino acids and measuring the radioactivity in pherogram bands using a methane flow counter. In the actively laying queen the rate of vitellogenin synthesis is 3 times higher than that of the non-sex-specific blood proteins. Approximately 85% of the labeled proteins which are released from the fat body are yolk material. In contrast to other insects, non-laying queens were also found to synthesize vitellogenin; however, the rate did not exceed that of the other serum proteins. The fate of yolk material in such females is unknown. In the ovary, synthesis of the euplasmic proteins occurs at a rate similar to that of the nonspecific haemolyph fractions. The appearance of radioactivity in the ovarian yolk is delayed compared with all other proteins. This indicates that these proteins are labeled byde novo synthesis, whereas the yolk is not built up within the ovary. This conclusion is further supported by the fact that the increase in specific radioactivity of ovarian yolk proteins occurs at a time when free¹⁴C-amino acids are no longer available in the haemolymph. Following the maximum intensity of labelling of yolk proteins the specific activity declines. From the data it can be estimated that the period of vitellogenesis for a single follicle is 2 days; 2 additional days of maturation, during which glycogen synthesis and chorion formation occur, are required before oviposition.

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Herrn H. Fahrenhorst danke ich für Hilfe bei den Bienen-Arbeiten, Frl. B. Höltken und Frl. A. Rath für sorgfältige technische Assistenz, Frl. M. Krink für Zeichenhilfe bei den Abbildungen. Ein Teil der Untersuchungen wurde durch eine Herrn Prof. Dr. K. Bier gewährte Sachmittel-Zuwendung des Landesamtes für Forschung in Nordrhein-Westfalen gefördert     

Impact of Plant Functional Group, Plant Species, and Sampling Time on the Composition of nirK-Type Denitrifier Communities in Soil

We studied the influence of eight nonleguminous grassland plant species belonging to two functional groups (grasses and forbs) on the composition of soil denitrifier communities in experimental microcosms over two consecutive years. Denitrifier community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. The impact of experimental factors (plant functional group, plant species, sampling time, and interactions between them) on the structure of soil denitrifier communities (i.e., T-RFLP patterns) was analyzed by canonical correspondence analysis. While the functional group of a plant did not affect nirK-type denitrifier communities, plant species identity did influence their composition. This effect changed with sampling time, indicating community changes due to seasonal conditions and a development of the plants in the microcosms. Differences in total soil nitrogen and carbon, soil pH, and root biomass were observed at the end of the experiment. However, statistical analysis revealed that the plants affected the nirK-type denitrifier community composition directly, e.g., through root exudates. Assignment of abundant T-RFs to cloned nirK sequences from the soil and subsequent phylogenetic analysis indicated a dominance of yet-unknown nirK genotypes and of genes related to nirK from denitrifiers of the order Rhizobiales. In conclusion, individual species of nonleguminous plants directly influenced the composition of denitrifier communities in soil, but environmental conditions had additional significant effects.     

Human health risks of metals and metalloids in muscle tissue of Synodontis zambezensis Peters, 1852 from Flag Boshielo Dam,South Africa

Muscle tissue from 63 Synodontis zambezensis collected bimonthly in 2013 at Flag Boshielo Dam were analysed for metals and metalloids in a desktop human health risk assessment. The Hazard Quotient, based on a weekly meal of 67 g of fish muscle, exceeded the maximum acceptable level of one for lead, cobalt, cadmium, mercury, arsenic and selenium. The concentrations of these elements were higher in 2013 than those recorded in 2009 and 2012 in other fish species from Flag Boshielo Dam and these may pose a long-term health risk if consumed regularly by impoverished rural communities reliant on fish as a source of protein.     

Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Resting energy expenditure and delayed-onset muscle soreness after full-body resistance training with an eccentric concentration

The purpose of this investigation was to determine the effect of an acute bout of high-volume, full-body resistance training with an eccentric concentration on resting energy expenditure (REE) and indicators of delayed-onset muscle soreness (DOMS). Eight resistance trained (RT) and eight untrained (UT) participants (mean: age = 23.5 years; height = 180.76 cm; weight = 87.58 kg; body fat = 19.34%; lean mass = 68.71 kg) were measured on four consecutive mornings for REE and indicators of DOMS: creatine kinase (CK) and rating of perceived muscle soreness (RPMS). Delayed-onset muscle soreness was induced by performing eight exercises, eight sets, and six repetitions using a 1-second concentric and 3-second eccentric muscle action duration. A two-factor repeated-measures analysis of variance revealed that REE was significantly (p < 0.05) elevated at 24, 48, and 72 hours post compared with baseline measures for both UT and RT groups. Ratings of perceived muscle soreness were significantly elevated within groups for UT and RT at 24 and 48 hours post and for UT only at 72 hours post compared with baseline (p < 0.05). Nonparametric analyses revealed that CK was significantly increased at 24 hours post for both UT and RT and at 48 and 72 hours post for UT only compared with baseline (p < 0.05). Resting energy expenditure and indicators of DOMS were higher in UT compared with RT on all measures, but no significant differences were determined. The main finding of this investigation is that full-body resistance training with an eccentric concentration significantly increased REE up to 72 hours postexercise in UT and RT participants.