Biological Control of Sheep Parasites using Duddingtonia flagrans: Trials on Commercial Farms in Sweden

   

Trials were conducted on 3 commercial sheep farms in Sweden to assess the effect of administering spores of the nematode trapping fungus, Duddingtonia flagrans, together with supplementary feed to lactating ewes for the first 6 weeks from turn-out on pastures in spring. Also control groups of ewes, receiving only feed supplement, were established on all 3 farms. Groups were monitored by intensive parasitological investigation. The ewes and their lambs were moved in late June to saved pastures for summer grazing, the lambs receiving an anthelmintic treatment at this time. After approximately 6 weeks on summer pasture the lambs were weaned, treated a second time with anthelmintic, and returned to their original lambing pastures for finishing. Decisions as to when lambs were to be marketed were entirely at the discretion of the farmer co-operators. No difference in lamb performance was found between the two treatments on all three farms. This was attributed to the high levels of nutrition initially of the ewes limiting their post-partum rise in nematode faecal egg counts in spring, which in turn resulted in low levels of nematode infection on pastures throughout the autumn period. Additionally, pastures were of good quality for the lambs during the finishing period, so they grew at optimal rates as far as the farmers were concerned.     

Intravascular ultrasound observations of atherosclerotic lesion formation and restenosis in patients with diabetes mellitus

Coronary artery disease is more aggressive in diabetic patients than in nondiabetics; they have more diffuse disease, higher mortality rates and worse clinical outcomes after coronary interventions. Intravascular ultrasound (IVUS) produces transmural tomographic images of the coronary arteries in vivo. Recent IVUS studies have provided new insights into the mechanisms of stenosis formation and restenosis in both nondiabetic and diabetic patients. Arterial remodeling is defined as a change in arterial area. During atherogenesis, an increase in arterial area usually accompanies plaque accumulation to delay lumen compromise. Stenosis formation is related to: (a) the rate of plaque accumulation versus the rate of positive remodeling; and (b) the limits and ultimate failure of positive remodeling. However, there is a marked variability in remodeling. IVUS studies have suggested that remodeling may be impaired in some diabetic patients during atherogenesis. Following non-stent catheter-based interventions, serial (post-intervention and follow-up) IVUS studies have shown that the change in lumen area correlates better with the change in arterial area (remodeling) than with the change in plaque area (neointimal hyperplasia). In some patients, a positive remodeling response mitigates against the increase in plaque area to limit late lumen loss and restenosis. Neointimal hyperplasia is exaggerated in diabetic patients. Despite this, there is a reduced frequency of positive remodeling, potentially similar to the impaired positive remodeling in some diabetic patients during atherogenesis. Failed or inadequate arterial remodeling may contribute to the pathogenesis and natural history of atherosclerotic coronary artery disease in diabetic patients.     

Evaluation and molecular characterization of EHD1, a candidate gene for Bardet-Biedl syndrome 1 (BBS1)

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.     

Alanine-scanning mutagenesis of alpha-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine

We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.     

A rapid enzymatic procedure for production of 1-beta-D-(3H)arabinofuranosyl cytosine 5'-triphosphate.

A rapid method of sequentially phosphorylating picomole quantities of [³H]-araC to [³H]araCTP is described (ara = 1-β-d-arabinofuranosyl). The procedure utilizes a system of phosphorylating enzymes isolated from rat spleen and requires a single incubation step. The [³H]araCTP product is isolated by ion-exchange chromatography and analyzed by PEI-cellulose thin-layer chromatography. At low concentrations of [³H]araC as much as 80% can be phosphorylated to the triphosphate, and the produet may be obtained in radiochemical purity greater than 97%.     

Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin

The glycoprotein hormone, human chorionic gonadotropin (hCG), contains both N- and O-linked oligosaccharide chains linked to its beta-subunit. Using the human choriocarcinoma cell line, BeWo, we have examined the temporal relationship between N- and O-glycosylation of hCG and the subsequent processing of both types of oligosaccharide chains. The results indicate that, as observed in related cell lines, mature, completely glycosylated forms of the subunits of hCG cannot be detected intracellularly in BeWo cells during pulse-chase experiments with [35S]methionine. To more directly study the temporal relationship between N- and O-glycosylation of hCG in BeWo cells, 14C-amino acids and [3H]glucosamine (which also serves as a precursor to N-acetylgalactosamine) were used to label hCG. The results of these studies are consistent with a model for the N- and O-glycosylation of hCG in which 1) N-glycosylation of hCG occurs co-translationally or very shortly after translation, and 2) the addition of O-linked GalNAc residues to the polypeptide and the addition of peripheral GlcNAc residues to the N-linked oligosaccharide chains occur just prior to secretion, presumably in the Golgi complex.     

Generation and analysis of a protein-protein interface data set with similar chemical and spatial patterns of interactions

Protein-protein interfaces are regions between 2 polypeptide chains that are not covalently connected. Here, we have created a nonredundant interface data set generated from all 2-chain interfaces in the Protein Data Bank. This data set is unique, since it contains clusters of interfaces with similar shapes and spatial organization of chemical functional groups. The data set allows statistical investigation of similar interfaces, as well as the identification and analysis of the chemical forces that account for the protein-protein associations. Toward this goal, we have developed I2I-SiteEngine (Interface-to-Interface SiteEngine) [Data set available at http://bioinfo3d.cs.tau.ac.il/Interfaces; Web server: http://bioinfo3d.cs.tau.ac.il/I2I-SiteEngine]. The algorithm recognizes similarities between protein-protein binding surfaces. I2I-SiteEngine is independent of the sequence or the fold of the proteins that comprise the interfaces. In addition to geometry, the method takes into account both the backbone and the side-chain physicochemical properties of the interacting atom groups. Its high efficiency makes it suitable for large-scale database searches and classifications. Below, we briefly describe the I2I-SiteEngine method. We focus on the classification process and the obtained nonredundant protein-protein interface data set. In particular, we analyze the biological significance of the clusters and present examples which illustrate that given constellations of chemical groups in protein-protein binding sites may be preferred, and are observed in proteins with different structures and different functions. We expect that these would yield further information regarding the forces stabilizing protein-protein interactions.     

A possible regulatory role for the metal-binding domain of CadA, the Listeria monocytogenes Cd2+-ATPase

Using the baculovirus/Sf9 expression system, we produced CadA and DeltaMBD, a metal-binding domain, truncated CadA. Both proteins had the expected properties of P-type ATPases: ATP-induced Cd2+ accumulation, Cd2+-sensitive ATP and Pi phosphorylation and ATPase activity. DeltaMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA. MBD truncation flattened the Cd2+ dependence of the ATPase activity and increased apparent Cd2+ affinity, suggesting a positive cooperativity between MBD and membranous transport sites. We propose that occupancy of MBD by Cd2+ modulates CadA activity.     

Intravascular-ultrasound-guided percutaneous transluminal coronary angioplasty/provisional stent implantation strategy: impact on long-term clinical follow-up

Two hundred and eighty-four consecutive patients with 438 native coronary artery stenoses were enrolled prospectively in a study of intravascular ultrasound (IVUS)-guided percutaneous transluminal coronary angioplasty (PTCA) with provisional stenting: (1) aggressive lesion-site media-to-media balloon-sizing; (2) IVUS-assessment of residual lumen dimensions to identify optimal PTCA results (minimum lumen area = 65% of the average of the proximal and distal reference lumen areas or = 6.0 mm(2) and no major dissection); and (3) liberal stent crossover. Overall, 206 stenoses in 134 patients (47%) were treated with PTCA alone. Reasons for crossover were flow-limiting or lumen-compromising dissections in 28% of patients and suboptimal IVUS minimum lumen area in 72% of patients. At one year, 8% of stenoses in the PTCA group and 16% in the stent crossover group required revascularization. In approximately half of the patients treated using an IVUS-guided aggressive PTCA strategy, stent implantation could be avoided without sacrificing an increase in acute complications or worse clinical outcome.