α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.     

Use of [Cit312,314] filaggrin (306–324) analogue for the diagnosis of rheumatoid arthritis. Conformational study by circular dichroism and fourier transformed infrared spectroscopy

Antifilaggrin autoantibodies (AFA) have described to be the most specific markers of rheumatoid arthritis (RA) and epitopes containing citrulline within the sequence of filaggrin identified as major antigenic sites recognised by AFA. In this paper we confirm that citrulline is an essential constituent of filaggrin related antigenic determinants recognised by RA specific autoantibodies, thus having an important role for the development of filaggrin peptides based serological tests. Moreover, we describe our findings on the comparative conformational analysis of filaggrin (306–324) peptide sequence and an analogue in which two arginines were simultaneously substituted by citrulline carried out by Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR). Results might contribute to the understanding of the structural features that may be important to explain the enhanced binding characteristics of citrullinated peptides to the autoantibodies in rheumatoid arthritis.     

Population and community-level compositional patterns shape the realized niche of the rare arctic-alpine species Carex lachenalii Schkuhr

The ability for vegetative growth and development of generative organs often reflects an adaptation to the environment and may be a suitable proxy for understanding population dynamics of rare relict species. An example of such a plant is Carex lachenalii Schkuhr, an arctic-alpine species, in the temperate zone of Europe only occurring in isolated localities of high-elevation mountain ranges. We aimed to assess whether there were relationships between flower production and clonal growth of C. lachenalii, both at the tuft and plot level, and how co-occurring vegetation could modify this relationship. In the study we focused on population-level traits of C. lachenalii, vegetation traits and components of functional diversity. At the tuft level we found that the proportion of flowering ramets of C. lachenalii decreased with increasing diameter of the tuft. At the plot level, in snowbed vegetation C. lachenalii produced more flowering ramets. We suggest this is due to higher environmental stress, expressed by high importance of habitat filtering (low functional dispersion) in shaping species composition of co-occurring vegetation. In granite grasslands and milder environment (expressed by higher functional dispersion), C. lachenalii produced more vegetative ramets, which we suggest is a result of a more competitive environment. While in snowbeds investment in flowering ramets could promote successful persistence of C. lachenalii, survival of subpopulations occurring in the highly competitive conditions of granite grasslands may be uncertain due to potentially weak adaptation to competition with graminoids and dwarf shrubs.     

Heritability of running economy: a study made on twin brothers

Running economy (RE), defined as the steady-state of oxygen uptake (V˙O₂) for a given running velocity, is a factor of sports performance the genetic component of which has seldom been reported to date. We studied this component using a heritability index (HI) in a group of 32 male twins, 8 monozygotic (MZ) and 8 dizygotic (DZ) pairs, all sportsmen with similar perinatal and environmental backgrounds. Zygocity was determined by the identity of erythrocytic antigenic, protein and enzymatic polymorphism, and human leucocyte antigen serologic types between co-twins. The subjects exercised twice on a treadmill, once until exhaustion and again at submaximal intensities. Pulmonary gas exchange was measured continuously using an automatic analyser system during both tests. Blood samples were obtained during the recovery period to determine lactate concentrations. No significant differences were observed between MZ and DZ, in respect of RE at any speed or in maximal V˙O₂ relative to body mass. Nevertheless, significant HI (P < 0.05) was found in maximal lactate concentrations (HI = 0.75) and in respiratory equivalent for oxygen at two speeds, 7 km · h⁻¹ (HI = 0.71) and 8 km · h⁻¹ (HI = 0.79), differences which probably suggest that there are differences in RE. In conclusion, we did not detect a genetic component in RE or in maximal oxygen uptake, but a genetic component for markers of anaerobic metabolism was present. Accepted: 5 November 1997     

Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases and determining how this affects chronological life span (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity, and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS. Extension of CLS by caloric restriction also required respiration above a similar threshold during exponential growth and completely alleviated the need for respiration in the stationary phase. Finally, we show that supplementation of media with 1% trehalose, a storage carbohydrate, restores wild-type CLS to respiratory-null cells. We conclude that mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction by increasing stress resistance, an important component of which is the optimal accumulation and mobilization of nutrient stores.     

Potentially handicapped but otherwise functional: Malformations in prey capture tools show no impacts on octopus life

Larval mortality is a keystone ecological factor for many benthic octopus since it mostly occurs before their settlement in the sea bottom as benthic juveniles. The literature had revealed that records of adult animals with morphological abnormalities (teratologies) are fewer in species with complex life cycle than in those with direct development. This is a direct consequence of the morphological, physiological, and development challenges that the transition from the larval to the adult morphology represents. During a routine fishing sample, we found an immature female horned octopus with additional buccal structures in two suckers of its ventral arms, likely rendering these suckers as inefficient. Based on the literature about the natural history of octopus, we provide evidence that these abnormalities were present at the moment of hatch. We evaluated the impact of the teratologies by comparing the shape of the buccal beaks and the trophic niche of the individual with five normal conspecifics. Although the beaks showed a different shape than normal individuals, the trophic niche was similar. Surprisingly, the teratological condition of the individual likely had no severe impacts on its life, even though it likely represents a handicap for its survival during its planktonic life. We also comment on other previous records from the literature of teratological adult octopus to highlight the amazing adaptive capacity of octopus to deal with challenging morphologies.