Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal-immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL-6) secretion in the spleen. The α₂-adrenergic agonist p -aminoclonidine (10⁻⁷ M ) inhibited IL-6 secretion (control vs. p -aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α₁-adrenergic agonist methoxamine (10⁻⁸ M ) also inhibited IL-6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β-endorphin (10⁻¹⁰ M ), methionine-enkephalin (10⁻⁹ M ), and leucine-enkephalin (10⁻⁹ M ) inhibited IL-6 secretion as well ( p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL-6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α-adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL-6 secretion was attenuated by phentolamine (10⁻⁷ M ; p = 0.0345), by naloxone (10⁻⁶ M ; p = 0.0046), by cyprodime (10⁻⁸ M ; p = 0.0014), and by the combination of cyprodime (10⁻⁷ M ) plus phentolamine (10⁻⁸ M ; p < 0.0001). We conclude from the complementary studies that the inhibition of IL-6 secretion induced by electrical pulses was mostly mediated by α-adrenergic and μ-opioidergic endogenous transmitters.     

Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413

 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10^-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10^-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997     

Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β₁-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β₁-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.     

Fracture Healing Is Delayed in Immunodeficient NOD/scid??IL2Rγ?cnull Mice

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγ_c^null mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγ_c^null mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγ_c^null mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγ_c^null mice. Our results suggest that—under aseptic, uncomplicated conditions—the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγ_c^null mice.     

Serum-Cholesterin,ABO-Blutgruppen und Hämoglobintyp

Zusammenfassung Serum-Cholesterin, ABO-Blutgruppen (N=715), Glucose-6-PhosphatDehydrogenase (G-6-PD, Farbstoff-Reduktionstest, N=611) und der Hämoglobintyp (osmotische Resistenz und Cellulose-Acetat-Elektrophorese, N=469) wurden bei anscheinend gesunden, 20 Jahre alten Männern aus 12 Distrikten der Provinz Chiang Mai in Nordthailand bestimmt. Das Körpergewicht hatte keinen Einfluß auf die Cholesterinkonzentration. Probanden der Blutgruppe A hatten signifikant höhere Cholesterinwerte als die der Gruppen 0 und B. Gruppe B hatte höhere Werte als Gruppe 0, aber die Differenz war nur schwach signifikant. Der mittlere Cholesterinwert der Probanden mit -Thalassaemia minor war signifikant niedriger als der der Gruppen mit normalem Hämoglobin und mit -Thalassaemie oder abnormalem Hämoglobin. Zwischen den drei letzteren Gruppen bestand kein signifikanter Unterschied. Diese Befunde bestätigen für eine tropische Bevölkerung mit an Fetten armer Ernährung die Beziehung zwischen -Thalassämie (Fessas et al., 1963; Mayo et al., 1969, Griechenland) und ABO-Blutgruppen (Mayo et al., 1969; Oliver et al., 1969; Langman et al., 1969; Beckman u. Olivecrona, 1970) einerseits und der Serum-Cholesterin-Konzentration.

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Serum-cholesterol, AB0 blood-groups and haemoglobin typeGenetic influences on the serum-cholesterol level

Summary Serum-cholesterol, ABO blood-groups (N=715), glucose-6-phosphate dehydrogenase (G-6-PD, dye decolorization test, N=611) and haemoglobin type (osmotic fragility, cellulose acetate electrophoresis, N=469) were determined in apparently health, 20 years old males from 12 districts of the province of Chiang Mai in northern Thailand. Body weight and G-6-PD deficiency did not seem to influence the serum-cholesterol level. Probands with blood-group A had significantly higher cholesterol concentrations than groups 0 and B. The difference between groups 0 and B, the latter having somewhat higher levels, was only weakly significant. Cholesterol levels were significantly lower in probands with -thalassaemia minor when compared with a normal control group. The difference between the control group and the probands with -thalassaemia and abnormal haemoglobins (mainly HbE trait) was not significant. These findings confirm for a tropical rural population with a diet low in fat the association between -thalassaemia and low cholesterol concentrations previously reported from Greece (Fessas et al., 1963; Mayo et al., 1969) and the association between blood-group A and high cholesterol levels found in several European populations (Mayo et al., 1969; Oliver et al., 1969; Langman et al., 1969; Beckman and Olivecrona, 1970).

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Beurlaubt von der Universitäts-Kinderklinik Bonn.

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Eingerichtet und unterstützt von der Stiftung Volkswagenwerk.     

Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.