Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14

We have assigned ¹H, ¹⁵N and ¹³C resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.     

Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique

1. An apparatus was developed for the simultaneous measurement of steady-state values of respiration rate and oxidation level of respiratory pigments at low oxygen tensions. An open reaction system is utilized. The liquid sample is in contact with a gas mixture whose oxygen tension can be increased linearly with time at a rate so slow that the system is always practically at a steady state.

2. Assuming Michaelis-Menten kinetics in the respiration, theoretical curves for oxygen tension in the liquid and oxidation level of the terminal oxidase during a linear increase of the oxygen tension in the gas were calculated.

3. Measurements were performed on rat liver mitochondria. Steady-state curves for oxygen tension in the liquid and oxidation level of the terminal oxidase, cytochrome a₃, obtained with coupled mitochondria resembled the theoretical curves. For uncoupled mitochondria the cytochrome a₃ curve was signmoidal, deviating strongly from the theoretical curve.

4. The apparent K_m for oxygen uptake of coupled mitochondria in the presence of pyruvate and malate, in the absence of phosphate was found to be 0.5 μM. In the case of uncoupled mitochondria the oxygen tension in the liquid could not be measured with sufficient accuracy to allow comparison with Michaelis-Menten kinetics. The apparent K_m for oxygen uptake was less than 0.05 μM.     

Microsatellites reveal clonal structure of populations of the thelytokous ant platythyrea punctata (F. Smith) (Hymenoptera; formicidae)

Parthenogenesis is often thought to constitute an evolutionary dead end as compared with sexual reproduction because genetic recombination is limited or nonexistent in parthenogenetic populations. Yet there are many species to demonstrate that parthenogenesis can initially be extremely successful under certain environmental conditions. In this study we used microsatellite markers to investigate the genetic structure of four natural populations of the neotropical thelytokous parthenogenetic ant Platythyrea punctata. Ten dinucleotide microsatellites were isolated from a partial genomic library of P. punctata. Five of these were found to be polymorphic. In a subsequent analysis of 314 workers taken from 51 colonies, we detected low intraspecific levels of variation at all loci, expressed both in the number of alleles detected and heterozygosities observed. Surprisingly, we found almost no differentiation within populations. Populations rather had a clonal structure, with all individuals from all colonies usually sharing the same genotype. Only in one colony from Puerto Rico did some workers have an additional genotype. This low level of genotypic diversity probably reflects the predominance of thelytoky in P. punctata, together with genetic bottlenecks and founder effects. Cross-species amplification of all 10 loci in 29 ant species comprising four different subfamilies yielded positive amplification products in only a limited number of species.     

Suppression of growth hormone by oral glucose in the evaluation of tall stature

Excess secretion of growth hormone is a rare diagnosis in children or adolescents with tall stature. An oral glucose tolerance test (OGT) with determination of growth hormone is generally recommended to exclude this disorder. In order to test the validity of this approach in pediatric subjects, OGT tests were performed in 126 tall subjects (age: 12.4 +/- 1.8 years; height: 3.1 +/- 0.8 SDS). Nonsuppression was present in 39 subjects, however, anthropometric analysis and follow-up excluded the diagnosis of eosinophilic pituitary adenoma in all patients. The lowest GH concentration was reached 90 min after ingestion of oral glucose, GH rose above baseline at 180 min. Plasma concentrations of glucose and insulin did not differ between suppressors and nonsuppressors. In conclusion, absent suppression of growth hormone by oral glucose is common in tall children and adolescents. The test is therefore not recommended as a general screening for excess growth hormone. Prolonging the test beyond 120 min does not increase the diagnostic value.     

Modulation of red cell glycolysis: interactions between vertebrate hemoglobins and cytoplasmic domains of band 3 red cell membrane proteins

Several vital functions/physical characteristics of erythrocytes (including glycolysis, the pentose phosphate pathway, ion fluxes, and cellular deformability) display dependence on the state of hemoglobin oxygenation. The molecular mechanism proposed involves an interaction between deoxyhemoglobin and the cytoplasmic domain of the anion-exchange protein, band 3 (cdB3). Given that band 3 also binds to membrane proteins 4.1 and 4.2, several kinases, hemichromes, and integral membrane proteins, and at least three glycolytic enzymes, it has been suggested that the cdB3-deoxyhemoglobin interaction might modulate the pathways mediated by these associated proteins in an O(2)-dependent manner. We have investigated this mechanism by synthesizing 10-mer peptides corresponding to the NH(2)-terminal fragments of various vertebrate cdB3s, determining their effects on the oxygenation reactions of hemoglobins from the same and different species and examining binding of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase to the erythrocytic membrane of mouse erythrocytes. The cdB3 interaction is strongly dependent on pH and the number of negative and positive charges of the peptide and at the effector binding site, respectively. It lowers the O(2) association equilibrium constant of the deoxygenated (Tense) state of the hemoglobin and is inhibited by magnesium ions, which neutralize cdB3's charge and by 2,3-diphosphoglycerate, which competes for the cdB3-binding site. The interaction is stronger in humans (whose erythrocytes derive energy predominantly from glycolysis and exhibit higher buffering capacity) than in birds and ectothermic vertebrates (whose erythrocytes metabolize aerobically and are poorly buffered) and is insignificant in fish, suggesting that its role in the regulation of red cell glycolysis increased with phylogenetic development in vertebrates.     

Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.     

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.     

Adhesins and invasins of pathogenic bacteria: a structural view

Adhesion and invasion of pathogenic bacteria represent the important initial step of infection. Pathogens utilize surface-located adhesins/invasins for specific interaction with host cell receptors. The three-dimensional structures of a number of adhesins/invasins show that many are elongated molecules containing domains commonly found in eukaryotic proteins. Similar folds are employed repeatedly to target different receptors.