Purifications of Rat Adrenal Dopamine-β-Hydroxylase: Immunological Analysis of Its Soluble and Membrane-Bound Forms with the Use of an Antibody Raised Against the Soluble Form

Soluble and membrane-bound dopamine-beta-hydroxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A-Sepharose chromatography, gel filtration, and ion-exchange high-performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 75,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A-Sepharose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000-molecular-weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher-molecular-weight protein could be the 75,000 protein before covalent modifications and cleavage.     

Fertilizer nitrogen budget of Na15NO3 and (15NH4)2SO4 split-applied to winter wheat in microplots on a loam soil

Labelled fertilizer N applied to winter wheat as Na¹⁵NO₃ and (¹⁵NH₄)₂SO₄ at a total N dressing of 100kg ha⁻¹ was used in a microplot balance study to investigate the fate of each split fraction at three growth stages: end of tillering, heading and beginning of flowering. Results indicated that while the percentage utilization of the applied N by the grain and total crop increased considerably from the first to the third split application, these values diminished steadily in the straw. Grain recovery values for the first, second and third split applications were 34.2%, 51.5% and 55.7% for the NO₃ and 32.3%, 48.4% and 52.5% for the NH₄ carrier, respectively. The corresponding recovery values for the whole plant were 54.6%, 67.8% and 69.9% for the NO₃ and 51.7%, 63.5% and 66.1% for the NH₄ carrier. A greater proportion of the fertilizer N applied at the end of tillering stage was found in the vegetative plant components as compared with the grain. The reverse occurred for the N applied at the heading and at the beginning of the flowering stages. The residual fertilizer N found in the soil amounted to 18.0%, 10.4% and 11.6% of the applied NO₃−N and to 22.5%, 12.7% and 15.2% of the applied NH₄−N for the respective split applications. No differences were found for each split application between the two carriers as far as the unaccounted fertilizer N was concerned. The losses were 26.6%, 22.3% and 18.6% of the applied N for the three split applications, respectively. The application of fertilizer N did not lead to any increase in soil N uptake by the crop.     

ANTHEPROT: a package for protein sequence analysis using a microcomputer

A simple microcomputer package is described to make the theoreticalanalysis of protein sequences. Several methods designed to comparetwo sequences, to model proteolytic reactions and to predictthe secondary structure, the hydro-phobic/hydrophilic regionsand the potential antigenic sites of proteins have been includedin an Apple II microcomputer software. The package comprises21 programs as well as the secondary structure database of Kabschand Sander (1983). Received on November 24, 1987; accepted on March 8, 1988     

Implications of power law exponent in synergy and inhibition of olfactory mixtures

An index of olfactory quantitative interaction has been definedby Laffort and Dravnieks (1982). This index is obtained by combiningthe experimental effectiveness of mixtures with the power lawexponent of the components. When its value equals 1, this meansthat the ‘apparent’ inhibitions and synergies canbe expressed only through the power law exponents of the components.This, of course, holds true when a substance is added to itself.Values less than or greater than 1 are interpreted as ‘true’inhibition and synergy respectively. In a slightly modifieddefinition, this index [called (Gamma)] has been applied totwo sets of data, (i) By using an appropriate computer program,theoretical iso-intensity curves for binary mixtures were generatedby given values of this index, the variables being the concentrations.The diversity of curves is comparable to that observed in certainpsychophysical studies (Köster, 1969). (ii) EAG responsesof honey bees to 18 binary mixtures of three reciprocal concentrationseach, have been recorded. Absence of true interaction was observedin two-thirds of cases and the results concerning the last thirdare discussed. These two sets of results illustrate the integrativestrength of the index. The present work was also presentedin a condensed form in French by Laffort et al. (1984).     

New evidence fortrans-species evolution of theH-2 class I polymorphism

A serological survey using alloantisera specific for the H-2 class I antigens in Japanese wild mice,Mus musculus molossinus, revealed a high frequency of the H-2K^f antigen. This antigen has also been found in European wild mice,M. m. domesticus andM. m. musculus. In this survey, the H-2K^f antigen was characterized through the use of ten newly isolated monoclonal antibodies raised against cells of a Japanese wild mouse, and by Southern blot analysis using anH-2K locus-specific probe which hybridizes with the 3′ end of the gene. The serologically identified H-2K^f antigens revealed several minor variations in reactivities to the monoclonal antibodies. However, all the antigens examined could be clearly separated into two types with respect to the restriction fragment length polymorphism (RFLP) pattern. The first type, found together with a single, characteristic RFLP pattern, was always associated with the presence of reactivity to one particular monoclonal antibody, MS54. The second type, found to represent different RFLP patterns, is associated with the absence of reactivity to MS54. This concordance between the presence of an antigenic determinant and a particular RFLP was observed not only withinMus musculus subspecies but also in a different species:M. spretus, carrying the same antigenic determinant, gave an identical RFLP to that of the other MS54-positiveMus musculus subspecies. The data suggest that the antigenic determinant specific for MS54 is an ancient polymorphic structure which has survived the long period of diversification ofMus species (approximately 2–3 million years) without alteration, and is associated with a stable DNA structure at the 3′ end of theH-2K gene.     

Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.     

Self-pollination,style length development and seed set in self-compatible Asteraceae: evidence from Senecio vulgaris L.

Background: Variation in style length has been reported in Senecio vulgaris and has been associated with outcrossing rate.

Aims: To determine if (i) long styles lack germinated pollen on stigmas left to self-pollinate, (ii) successful self-pollination causes styles to stop elongating and shrink in length and (iii) seed set increases with the amount of pollen deposited on stigmas.

Methods: Determined germinated self-pollen on stigmas of long and short styles after auto-self-pollination; scored style length over 48 h in self-pollinated and non-pollinated florets; recorded seed set after placing different amounts of pollen on stigmas.

Results: Most long-styled florets had zero or low amounts of germinated pollen on stigmas in contrast to most short-styled florets. Styles initially elongated to the same length in self-pollinated and non-pollinated florets, then shrank in length in self-pollinated florets while continuing to elongate in non-pollinated florets. Seed set increased with number of pollen grains deposited on stigmas.

Conclusions: Successful self-pollen deposition and/or germination on stigmas of S. vulgaris are indicated by presence of short styles, whereas the opposite is indicated by presence of long styles in florets left to self-pollinate. Self-pollination causes styles to shrink after initially elongating. Seed set is dependent on the amount of pollen deposited on stigmas.     

Conserving a variety of ancient forest patches maintains historic arthropod diversity

Forests are naturally extensive tracts. However, in South Africa natural fires over many millennia have reduced forested areas into small remnants spread throughout a grassland matrix. Small patches, especially distant patches, are generally considered to be adverse for forest specialists, owing to decreased forest interior and increased edge. Here we test this assumption by determining the impact of forest interpatch distance and patch size on epigaeic arthropod diversity in this globally rare vegetation type. Forty patches were selected: ten large (100–435 m diameter) that are distant (500–645 m) from other patches, ten large that are close to other patches (38–97 m), ten small (30–42 m) that are distant, and ten small-close patches. Each patch had two plots: edge and interior. Arthropods were sampled using pitfall traps, Berlese-Tullgren funnels and active searches. Interiors and edges had similar species richness and composition, excluding spiders, which were richer in interiors. Patch size significantly influenced species richness of predatory beetles and arthropod assemblages, excluding spiders. Effect of the interaction between patch size and interpatch distance on species richness and composition varied among taxa. Furthermore, large patches supported similar assemblages regardless of interpatch distance. Arthropod response, particularly ants to patch size and interpatch distance, was partly shaped by the matrix type. The percentage of surrounding grassland had little effect on arthropod diversity. We can conclude that large and close patches are important for arthropod conservation. Nevertheless, it is also important to conserve a variety of patch sizes at various distances to maximize overall arthropod composition.