Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies

The human class I alcohol dehydrogenase isozyme beta 2 beta 2 was used as the antigen to raise monoclonal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was isozyme specific and all of them exhibited a similar affinity against all isozymes of the human class I ADH. Five out of the seven monoclonal antibodies had no effect on beta 2 beta 2 activity. Antibody G3 acted as a non-competitive inhibitor with a KI of 3 micrograms/ml at pH 7.5. Increasing pH was effective in reducing the level of inhibition. On the other hand, antibody 1D4 exhibited a pH-dependent activation of ADH activity. In the presence of this antibody, the pH optimum of beta 2 beta 2 was shifted from 9 to 8.5 and total activity was increased by 70% at this optimal pH. Kinetic analysis indicates that 1D4 probably acts as a non-competitive activator and may exert its action by interacting with the coenzyme binding site.     

Territorialit?t und Brutbiologie der FeldlercheAlauda arvensis in einer intensiv genutzten Agrarlandschaft

Zusammenfassung In einer intensiv landwirtschaftlich genutzten Fläche des schweizerischen Mittellandes wurden 1983–1987 vier Feldlerchenaggregationen mit maximal 39 Brutpaaren untersucht. Die Tradition (Ortstreue) bestimmte in erster Linie die Revierwahl und das Verteilungsmuster. Die Reviere waren mit ha sehr groß; ihre Größe korrelierte negativ mit der Anzahl Parzellen pro Fläche und der Kulturendiversität. Die Nutzung des Ressourcenangebots (Nistplatz, Nahrung, Schutz) war abhängig von den Fortbewegungsmöglichkeiten. In großparzellig strukturierten Flächen mit geringer Kulturendiversität kam es häufig zu Revierverschiebungen und Reviervergrößerungen. Die Feldlerche versucht, im Laufe der Brutperiode den Getreideanteil im Revier zu verkleinern und Kulturen wie Rüben, Kartoffeln und auch Mais zu aquirieren. Die Nistplatzwahl zeigte eine deutliche Präferenz für alle nicht zu dicht stehenden, grasartigen Kulturen (Weizen, Hafer, Fettwiese). Eine Vegetationshöhe von 15–25 cm und eine Bodenbedeckung von 20–50 % bieten optimale Bedingungen für den Nestbau. 51 % aller Nester wurden in der Fettwiese angelegt, 20 % im Winterweizen und 18 % im Mais. Je nach Bewirtschaftungsart brüteten die im Mittel 2,4–2,8mal pro Brutsaison. Die Schlüpfrate von 220 Gelegen betrug 0,58. 44 % der geschlüpften Jungvögel verließen das Nest. Der Bruterfolg (nestverlassende Junge/gelegte Eier) lag bei max. 25 %. Dem Fütterungsverhalten der Altvögel nach zu schließen, wurden maximal 50 % der nestverlassenden Jungen flügge. Durchschnittlich war mit 0,9 flüggen Jungen pro Brutpaar und Jahr zu rechnen. Brutverluste waren kulturspezifisch und daher in erster Linie vom Neststandort abhängig.

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Territoriality and breeding biology of Skylark (Alauda arvensis) in an intensively farmed area in Switzerland

Summary In 1983–1987 four plots (total surface 307 ha) with up to 39 breeding pairs were studied in an area of intensive cultivation in the Swiss Lowlands (390 m asl). Distribution pattern and selection of territories are determined by a strong site tenacity of the birds. The size of territories averaged 3,3±0,9 hectares. It correlates negatively with the number of different crops per unit area and the diversity of cultures.The mobility within the vegetation determined which types of farmland ressources may be used for nesting, feeding and cover. Shifts of territories and attempts to enlarge them were frequent in areas of large plots with low crop diversity. During the season, Skylarks tried to reduce the proportion of cereal fields within their territory and to gain plots planted with sugar-beet, potatoes and corn.Their choice of nesting sites showed a strong preference for less dense grass crops such as wheat, oat or meadows. Optimal conditions for nest sites were in fields with a vegetation height of 15–25 cm and a ground coverage of 20–50 %. 51 % of 220 nests were constructed in heavily manured meadows, 20 % in winter wheat fields and 18 % in corn fields. Females bred on average 2,4–2,8 times per breeding season, depending on the type of cultivation (agriculture, husbandry). Hatching rate was 58 % out of 220 clutches, and 44 % of the hatched young left the nest. Maximum breeding success (i. e. young birds leaving the nest/number of eggs laid) was thus maximal 25 %. Observations of parental feeding behaviour suggest that a maximum of 50 % of the young larks which leave the nests survive to the age of fledging. It is estimated that 0,9 fledglings are raised per breeding pairs and year. Losses of broods vary according to farming practises in the different crops and therefore are largely depending on nesting sites.

     

The effect of chronic fatty acid treatment on lipolysis in 3T3-L1 adipocytes.

Various saturated and unsaturated fatty acids were included in the culture medium to test their effects on lipolysis in 3T3-L1 adipocytes. Following prolonged incubation, only oleate was found to exert enhancing effect on basal and isoproterenol-stimulated lipolysis. The effect of oleate was concentration-dependent and was accompanied with increased intracellular cAMP content. Furthermore, the lipolytic response induced by isobutyl-methylxanthine, forskolin or dibutyryl cAMP was also increased in adipocytes treated with oleate. Thus, it appears that in addition to an increased cAMP accumulation, a step distal to cAMP production in the cells may be involved in inducing enhanced lipolysis in 3T3-L1 adipocytes by prolonged exposure to oleate.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

Asymmetric distribution of phosphatidylethanolamine in the membrane of vesicular stomatitis virus.

The membrane-impermeable reagent trinitrobenzenesulfonate has been shown to react only with the surface components of vesicular stomatitis virus (VSV) membranes. When the amount of phosphatidylethanolamine (PE) available to modification by trinitrobenzenesulfonate in intact virions was determined, it was found that 36% of the total membrane PE was converted to the trinitrophenyl derivative. The same proportion of the total membrane PE was reactive after removal of the surface glycoprotein by trypsin digestion, but disruption of the virus membrane by sonication rendered all of the PE reactive. These results indicate that PE is asymmetrically distributed in the VSV membrane; 36% is present in the outer lipid leaflet, whereas 64% is found on the inner layer.     

A dynamic mass balance model for phosphorus fluxes and concentrations in coastal areas

This paper presents a general, process-based mass balance model (CoastMab) for total phosphorus (TP) in defined coastal areas (at the ecosystem scale). The model is based on ordinary differential equations and calculates inflow, outflow and internal fluxes on a monthly basis. It consists of four compartments: surface water, deep water, erosion/transportation areas for fine sediments and accumulation areas for fine sediments. The separation between surface water and deep water is not done based on water temperature, but on sedimentological criteria instead (from the theoretical wave base). There are algorithms for all major internal TP fluxes (sedimentation, resuspension, diffusion, mixing and burial). Validations were performed using data from 21 different Baltic coastal areas. The results show that the model predicts monthly TP in water and chlorophyll a very well (generally within the uncertainty bands of the empirical data). The model has also been put through sensitivity tests, which show that the most important factor regulating the predictions of the model is generally the TP concentration in the sea beyond the coast. The model is simple to apply, since all driving variables may be accessed from maps or monitoring programs. The driving variables include coastal area, section area (between the defined coastal area and the adjacent sea), mean and maximum depths, latitude (used to predict water temperatures, stratification and mixing), salinity and TP concentration in the sea. Many of the model structures are general and could be used for areas other than those included in this study, e.g., for open coasts, estuaries or tidal coasts, as well as for other substances than phosphorus.     

Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.     

Residents’ support for wolf and bear conservation: the moderating influence of knowledge

This article examines the combined influence of cognitions (i.e., impact beliefs) and affect (i.e., feelings) on normative beliefs (i.e., support for management options) about wolves and brown bears. Data were obtained from stratified random face-to-face interviews (n = 1,611). The survey was conducted in the Abruzzo Lazio and Molise National Park (central Italy), where people have a long history of coexistence with large carnivores. Knowledge was hypothesized to moderate the relationships of beliefs and feelings on support for management actions. Path analyses supported the role of affect in mediating perceived impact beliefs and support for the protection of large carnivores. Knowledge moderated these relationships in the case of wolves but not brown bears. Residents of the national park had more knowledge about bears than wolves, which might partly explain both the stronger effect that knowledge had on the affective component and its lack of a moderating effect on the bear model. Overall, our findings show the positive attitude of residents toward large carnivores and support the idea of affect being more important than cognition in predicting normative beliefs.