Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

Influence of the carboxyl terminus of luteinizing hormone-releasing hormone and bradykinin on hydrolysis by brain endo-oligopeptidases

A homogeneous preparation of endo-oligopeptidase A from rabbit brain cleaves luteinizing hormone-releasing hormone (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the Tyr-Gly bond only after the removal of Gly-NH2 from the COOH-terminal position of the molecule. The influence of the carboxyl terminus on hydrolysis by brain endo-oligopeptidases was studied using bradykinin as a model substrate. The substitution of the carboxyl group of bradykinin by the amide reduces by 2.5-fold the rate of Phe-Ser bond hydrolysis by endo-oligopeptidase A but has no effect on the rate of hydrolysis of the Pro-Phe bond by endo-oligopeptidase B. On the other hand, the deletion of Phe-Arg from the COOH-terminal portion of bradykinin makes the peptide resistant to hydrolysis by endo-oligopeptidase A whereas it increases by 5-fold the rate of hydrolysis of the Pro-Gly bond by endo-oligopeptidase B.     

Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Signs of stabilisation and stable coexistence

Many empirical studies motivated by an interest in stable coexistence have quantified negative density dependence, negative frequency dependence, or negative plant–soil feedback, but the links between these empirical results and ecological theory are not straightforward. Here, we relate these analyses to theoretical conditions for stabilisation and stable coexistence in classical competition models. By stabilisation, we mean an excess of intraspecific competition relative to interspecific competition that inherently slows or even prevents competitive exclusion. We show that most, though not all, tests demonstrating negative density dependence, negative frequency dependence, and negative plant–soil feedback constitute sufficient conditions for stabilisation of two‐species interactions if applied to data for per capita population growth rates of pairs of species, but none are necessary or sufficient conditions for stable coexistence of two species. Potential inferences are even more limited when communities involve more than two species, and when performance is measured at a single life stage or vital rate. We then discuss two approaches that enable stronger tests for stable coexistence‐invasibility experiments and model parameterisation. The model parameterisation approach can be applied to typical density‐dependence, frequency‐dependence, and plant–soil feedback data sets, and generally enables better links with mechanisms and greater insights, as demonstrated by recent studies.     

Environmental Supply Chain Cooperation and Its Effect on the Circular Economy Practice‐Performance Relationship Among Chinese Manufacturers

Due to resource scarcity and environmental degradation, a new development concept emphasizing environmental concerns, called the circular economy (CE), has been enacted in legislation in China. This environmental management concept can be implemented at three levels, namely, region, industrial zone, and individual enterprise, with the objective of boosting economic development while lessening environmental and resource challenges. Environmental supply chain cooperation (ESCC), an approach that utilizes customer and supplier cooperation in environmental management, has been initiated among Chinese enterprises. Using survey data collected from 396 Chinese manufacturers, we examine the role of ESCC practices in influencing the relationship between implementing CE practices and the achievement of performance outcomes by testing the moderation and mediation effects of ESCC practices on the CE practice‐performance relationship through hierarchical regression analysis. Our data analyses indicate that ESCC practices are useful by moderation and, in some cases, essential by mediation, for Chinese manufacturers seeking to realize the performance targets desired in CE practices. The results highlight the need for Chinese manufacturers to improve supply chain coordination in their implementation of CE. On the policy side, our research findings suggest that ESCC practices are beneficial and, in some cases, necessary for the development of CE in China.     

Common and rare species respond to similar niche processes in macroinvertebrate metacommunities

Ecologists have long investigated why communities are composed of a few common species and many rare species. Most studies relate rarity to either niche differentiation among species or spatial processes. There is a parallel between these processes and the processes proposed to explain the structure of metacommunities. Based on a metacommunity perspective and on data on stream macroinvertebrates from different regions of Brazil, we answer two questions. 1) Are sets of common and rare species affected by similar niche and spatial processes? 2) How does the community composition of common and of rare species differ? The main hypothesis we test is that common species are mainly affected by environmental factors, whereas rare species are mostly influenced by dispersal limitation. We used variation partitioning to determine the proportion of variation explained by the environment and space in common and rare species matrices. Contrary to our expectations, evidence supported the idea that both common and rare species are affected mainly by environmental factors, even after controlling for the differing information content between common and rare species matrices. Moreover, the abundance of some common species is also a good predictor of variation in rare species matrices. Niche differences are unlikely to be the sole cause of patterns of rarity in these metacommunities. We suggest that sets of common and rare species react to similar major environmental gradients and that rare species also respond to processes that operate at a more fine‐grained spatial scale, particularly biotic interactions. We extend the view that species sorting is the dominant process structuring metacommunities and argue that future studies focusing on rarity would benefit from a metacommunity perspective.