Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.     

Recombinant avidin and avidin-fusion proteins

Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.     

Structure-function analysis of enoyl thioester reductase involved in mitochondrial maintenance

Candida tropicalis enoyl thioester reductase Etr1p and the Saccharomyces cerevisiae homologue Mrf1p catalyse the NADPH-dependent reduction of trans-2-enoyl thioesters in mitochondrial fatty acid synthesis (FAS). Unlike prokaryotic enoyl thioester reductases (ETRs), which belong to the short-chain dehydrogenases/reductases (SDR), Etr1p and Mrf1p represent structurally distinguishable ETRs that belong to the medium-chain dehydrogenases/reductases (MDR) superfamily, indicating independent origin of two separate classes of ETRs. The crystal structures of Etr1p, the Etr1p-NADPH complex and the Etr1Y79Np mutant were refined to 1.70A, 2.25A and 2.60A resolution, respectively. The native fold of Etr1p was maintained in Etr1Y79Np, but the mutant had only 0.1% of Etr1p catalytic activity remaining and failed to rescue the respiratory deficient phenotype of the mrf1Delta strain. Mutagenesis of Tyr73 in Mrf1p, corresponding to Tyr79 in Etr1p, produced similar results. Our data indicate that the mitochondrial reductase activity is indispensable for respiratory function in yeast, emphasizing the significance of Mrf1p (Etr1p) and mitochondrial FAS for the integrity of the respiratory competent organelle.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

Structure of bradavidin-C-terminal residues act as intrinsic ligands

Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ～25 μM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.     

Spatial and Temporal Analysis of Contact Rates in Female White-Tailed Deer

Abstract: White-tailed deer (Odocoileus virginianus) are important game mammals and potential reservoirs of diseases of domestic livestock; thus, diseases of deer are of great concern to wildlife managers. Contact, either direct or indirect, is necessary for disease transmission, but we know little about the ecological contexts that promote intrasexual contact among deer. Using pair-wise direct contacts estimated from Global Positioning System collar locations and joint utilization distributions (JUDs), we assessed habitats in which contacts occur to test whether direct contact rates among female white-tailed deer in different social groups differs among land-cover types. We also tested whether contact rates differed among seasons, lunar phases, and times of day. We obtained locations from 27 female deer for periods of 0.5–17 months during 2002–2006. We designated any simultaneous pair of locations for 2 deer <25 m apart as a direct contact. For each season, we used compositional analysis to compare land-cover types where 2 deer had contact to available land-cover weighted by their JUD. We used mixed-model logistic regression to test for effects of season, lunar phase, and time of day on contact rates. Contact rates during the gestation season were greater than expected from random use in forest and grassland cover, whereas contact rates during the fawning period were greater in agricultural fields than in other land-cover types. Contact rates were greatest during the rut and lowest in summer. Diel patterns of contact rates varied with season, and contact rates were elevated during full moon compared to other lunar periods. Both spatial and temporal analyses suggest that contact between female deer in different social groups occurs mainly during feeding, which highlights the potential impact of food distribution and habitat on contact rates among deer. By using methods to associate contacts and land-cover, we have created beneficial tools for more elaborate and detailed studies of disease transmission. Our methods can offer information necessary to develop spatially realistic models of disease transmission in deer.