Growth and tissue accretion rates of swine expressing an insulin-like growth factor I transgene

The goal of this research was to determine whether directing expression of an insulin-like growth factor I (IGF-I) transgene specifically to striated muscle would alter the growth characteristics in swine. Transgenic pigs were produced with a fusion gene composed of avian skeletal alpha-actin regulatory sequences and a cDNA encoding human IGF-I. Six founder transgenic pigs were mated to nontransgenic pigs to produce 11 litters of G1 transgenic and sibling control progeny. Birth weight, weaning weight, and proportion of pig survival did not differ between transgenic and control pigs. The ADG of pigs as they grew incrementally from 20 to 60 kg, 60 to 90 kg, and 90 to 120 kg, respectively, did not significantly differ between transgenic and control pigs. Efficiency of feed utilization (gain:feed) was also similar for transgenic and control pigs. Plasma IGF-I and porcine growth hormone (pGH) concentrations were determined at 60, 90, and 120 kg body weight. Plasma IGF-I concentrations were 19% higher in transgenic gilts than control gilts and 11.1% higher in transgenic boars than control boars (P=0.0005). Plasma IGF-I concentrations for boars were also higher than for gilts (P=0.0001). At 60, 90, and 120 kg body weight each pig was scanned by dual energy X-ray absorptiometry (DXA) to derive comparative estimates of carcass fat, lean, bone content of the live animal. Control pigs had more fat and less lean tissue than transgenic pigs at each of the scanning periods and the difference became more pronounced as the pigs grew heavier (P<0.005 at each weight). Transgenic pigs also had a slightly lower percentage of bone than control pigs (P<0.05 at each weight). While daily rates of lean tissue accretion did not differ for transgenic and control pigs, daily rates of fat accretion were lower in transgenic pigs than in control pigs (P<0.05). Based on these results we conclude that expression of IGF-I in the skeletal muscles gradually altered body composition as pigs became older but did not have a major affect on growth performance.     

A substrate variant as a high-affinity,reversible inhibitor: insight from the X-ray structure of cilastatin bound to membrane dipeptidase

An analysis of the X-ray structure of cilastatin bound to membrane dipeptidase, together with docking studies, is presented here to reveal how a simple amide may act as a high-affinity, reversible, amidase inhibitor. Cilastatin binds as a normal substrate and is orientated in a perfect near-attack conformer for formation of a tetrahedral intermediate with the zinc-bound water/hydroxide. This intermediate is fated, however, only to revert to its starting components as scission of the amide bond is prevented by the precise fit of cilastatin within the active site. The cilastatin alkyl end groups that are tightly buttressed against amino acid residues on opposite sides of the active site, are aligned along the C-N reaction coordinate axis thereby preventing collapse of the intermediate via rupture of the C-N bond. Such a feature could have more general applicability in the explicit design of substrate variants as selective, tight-binding, and reversible inhibitors.     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

Linkage disequilibrium patterns across a recombination gradient in African Drosophila melanogaster

Previous multilocus surveys of nucleotide polymorphism have documented a genome-wide excess of intralocus linkage disequilibrium (LD) in Drosophila melanogaster and D. simulans relative to expectations based on estimated mutation and recombination rates and observed levels of diversity. These studies examined patterns of variation from predominantly non-African populations that are thought to have recently expanded their ranges from central Africa. Here, we analyze polymorphism data from a Zimbabwean population of D. melanogaster, which is likely to be closer to the standard population model assumptions of a large population with constant size. Unlike previous studies, we find that levels of LD are roughly compatible with expectations based on estimated rates of crossing over. Further, a detailed examination of genes in different recombination environments suggests that markers near the telomere of the X chromosome show considerably less linkage disequilibrium than predicted by rates of crossing over, suggesting appreciable levels of exchange due to gene conversion. Assuming that these populations are near mutation-drift equilibrium, our results are most consistent with a model that posits heterogeneity in levels of exchange due to gene conversion across the X chromosome, with gene conversion being a minor determinant of LD levels in regions of high crossing over. Alternatively, if levels of exchange due to gene conversion are not negligible in regions of high crossing over, our results suggest a marked departure from mutation-drift equilibrium (i.e., toward an excess of LD) in this Zimbabwean population. Our results also have implications for the dynamics of weakly selected mutations in regions of reduced crossing over.     

Testing models of selection and demography in Drosophila simulans

We analyze patterns of nucleotide variability at 15 X-linked loci and 14 autosomal loci from a North American population of Drosophila simulans. We show that there is significantly more linkage disequilibrium on the X chromosome than on chromosome arm 3R and much more linkage disequilibrium on both chromosomes than expected from estimates of recombination rates, mutation rates, and levels of diversity. To explore what types of evolutionary models might explain this observation, we examine a model of recurrent, nonoverlapping selective sweeps and a model of a recent drastic bottleneck (e.g., founder event) in the demographic history of North American populations of D. simulans. The simple sweep model is not consistent with the observed patterns of linkage disequilibrium nor with the observed frequencies of segregating mutations. Under a restricted range of parameter values, a simple bottleneck model is consistent with multiple facets of the data. While our results do not exclude some influence of selection on X vs. autosome variability levels, they suggest that demography alone may account for patterns of linkage disequilibrium and the frequency spectrum of segregating mutations in this population of D. simulans.     

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.     

Renal pathology resulting from PGHS-2 gene ablation in DBA/B6 mice

Kidneys of prostaglandin H synthase-2 (PGHS-2) null mice fail to develop normally, leading to renal insufficiency. We have found that in a mixed DBA/B6 background, the lack of a functional PGHS-2 gene causes less severe renal pathology than was reported previously for PGHS-2 null mice in a B6 genetic background. The increase in blood urea nitrogen in the DBA/B6 strain of PGHS-2 null mice was significantly lower than reported for B6 PGHS-2 null mice (200% versus 270%). Cystic changes in DBA/B6 PGHS-2 null mice were also less severe. The DBA/B6 PGHS-2 null adult mice did not die from renal failure, unlike their B6/PGHS-2 counterparts that showed excessive neonatal and adult deaths. Therefore, DBA/B6 PGHS-2 null may be highly suitable to study the functional consequences of the lack of PGHS-2 in the kidney due to their less severe pathology and greater survival.