The impact of founder events and introductions on genetic variation in the muskox <Emphasis Type="Italic">Ovibos moschatus</Emphasis> in Sweden

The muskox Ovibos moschatus (Zimmerman 1780) is a specialised arctic mammal with a highly fragmented circumpolar distribution, with native populations in Canada and east Greenland and introduced populations in west Greenland, Alaska, Siberia and Eurasia. In 1971, five O. moschatus individuals from an introduced population in Norway migrated to Sweden. After a peak population of 36 individuals in the mid-1980s, the Swedish population now numbers seven individuals, making it vulnerable to both demographic and genetic stochasticity (i.e. inbreeding). Here, we analyse genetic variation among native and introduced populations of O. moschatus to evaluate the genetic effect of sequential founder events in this species. Our results show that genetic variation among native and introduced O. moschatus populations do not conform entirely to the expectations from sequential founder events, most likely because of random processes associated with introduction. In the Swedish population, a calf resulting from the mating of a wild cow and a captive Greenlandic bull contributes significantly to the current genetic variation. Thus, even a single outbreeding event may, at least momentarily, increase the genetic variation and potentially prevent inbreeding depression. Our results should aid the long-term preservation of O. moschatus in Sweden and Europe.     

Electrostatic contributions to the kinetics and thermodynamics of protein assembly

The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.     

Zn2+ binding to human calbindin D(28k) and the role of histidine residues

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.     

Seven new species of Pavonia (Malvaceae) from the Horn of Africa

Pavonia friisii , sp. nov., from south-eastern Ethiopia and south-central and southern Somalia, P. nigrescens , sp. nov., from south-central and southern Somalia, P. matteiana , sp. nov., from south-central Somalia, P. longipilosa , sp. nov., from eastern Ethiopia, P. rotundifolia , sp. nov., from eastern Ethiopia and northern and central Somalia, P. marginata , sp. nov., from central Somalia, and P. paucibracteata , sp. nov., from central Somalia, are described and illustrated.     

<Emphasis Type="Italic">Vernonia bayensis</Emphasis> (Asteraceae/Compositae), a new species from Somalia and a key to the Somali species of <Emphasis Type="Italic">Vernonia</Emphasis>

The new species Vernonia bayensis Thulin & Beentje from southern Somalia is described and illustrated, and a new key to the Somali species of the genus is presented.     

Calliandra (Leguminosae) in continental Africa

The mainly neotropical genus Calliandra Benth. (Leguminosae–Mimosoideae–Ingeae) is reported for the first time as native in continental Africa. There are two species known, C. gilbertii Thulin & Hunde sp. nov. in E Kenya and Somalia and C. redacta (J. H. Ross) Thulin & Hunde comb. nov. in South Africa near the Namibia border. On pollen morphological grounds they are supposed to be most related to certain neotropical species but the pollen does not agree entirely with any previously known group of the genus. The distribution of C gilbertii and C. redacta stresses the phytogeographical link between the arid zones of northern and southern Africa.