The effect of dietary supplementation of algae rich in docosahexaenoic acid on boar fertility.

The objective of this study was to assess the effects of dietary supplementation of a commercial algal product rich in docosahexaenoic acid (DHA) on boar fertility as assessed in vitro and in vivo. Boars were fed one of three experimental diets for 19 weeks: (i) Control (Ctl) diet (n = 31), (ii) Ctl diet plus 75g All-G-Rich per day (n = 31) or (iii) Ctl diet plus 150g All-G-Rich per day (n = 30). Parameters assessed were (i) raw semen quality; volume, sperm concentration, total motility and morphology (ii) liquid semen quality; progressive motility, viability, hypotonic resistance and acrosomal integrity (iii) frozen-thawed semen quality; motility, thermal stress, viability, membrane fluidity and mitochondrial activity (iv) sperm and seminal plasma (SP) fatty acid composition (FAC) (v) total antioxidant capacity (TAC) of SP and (vi) farrowing rates and litter sizes of sows (n = 1158) inseminated with liquid semen. Boars consuming 75g All-G-Rich had a larger semen volume (P < 0.05) and a higher total sperm number (P < 0.01) than the Ctl treatment, however, there was no effect of treatment on any other semen quality parameter (P > 0.05). There was no effect of dietary treatment on the FAC and TAC of SP or on farrowing rate and litter size (P > 0.05). There was an effect of dietary treatment on the FAC of sperm, represented by an 1.72 and 1.60 fold increase in the DHA content for 75 and 150g treatments, respectively, compared to the Ctl treatment. In conclusion, a significant increase in semen volume and total sperm number in boars supplemented 75g All-G-Rich daily, resulted in an increase in production of 3 to 4 more doses per ejaculate, thus, indicating that the feeding regime described within this study has the potential for increasing the output of boar studs.

Effect of the interaction of seaweed extracts containing laminarin and fucoidan with zinc oxide on the growth performance, digestibility and faecal characteristics of growing piglets.

Seaweed extracts (SWE) rich in laminarin and fucoidan have shown promise as a supplement for weaned piglets. However, successful application in pig nutrition depends on their bioactivity in the presence of additives such as ZnO. In the present study, a 2 × 2 factorial experiment was carried out to investigate the effect of the interaction between SWE and ZnO on the growth performance, digestibility and faecal characteristics of 192 weaned piglets (6·5 kg). The piglets were penned in groups of 4 (n 12 pens). The study consisted of two phases after weaning: a starter diet period from the day of weaning (0 d) to 21 d and a transition diet period from 21 to 40 d. The dietary treatments were as follows: (1) control diet; (2) control diet+ZnO; (3) control diet+SWE; (4) control diet+ZnO+SWE. Diets containing ZnO improved the faecal consistency of the piglets throughout the experimental period (0-40 d). An effect of the interaction between ZnO and SWE on several variable was observed. The diet containing only SWE or ZnO improved the feed conversion efficiency of the piglets during the transition diet period; however, this effect was not observed when the diet containing both ZnO and SWE was fed. The diet containing only SWE increased the N and organic matter digestibility of the piglets; however, this effect was not observed in the presence of ZnO. An interaction between ZnO and SWE was observed, whereby the faecal counts of Escherichia coli were decreased when piglets were fed the diet containing only SWE, but not when fed the diet containing both SWE and ZnO. In summary, SWE and ZnO improve growth performance when given alone, but not when given in combination. The biological effect of SWE on selected digestibility and faecal characteristics was markedly different when compared with that of ZnO.

The effect of seaweed extract as an alternative to zinc oxide diets on growth performance, nutrient digestibility, and fecal score of weaned piglets.

This study investigated if supplementing the diet with seaweed extracts (SWE) containing laminarin and fucoidan would promote growth performance, nutrient digestibility, and fecal consistency in newly weaned piglets during 2 growth phases as compared with ZnO. The experiment was designed as a 2 × 2 factorial with 2 levels of SWE (0 or 300 mg/kg laminarin + 240 mg/kg fucoidan) and 2 levels of ZnO [0 or added (3.1 g/kg for the starter diet and 2.5 g/kg for the transition diet)]. Dietary treatments were (i) basal diet, (ii) basal diet + ZnO, (iii) basal diet + SWE, and (iv) basal diet + ZnO + SWE. Newly weaned 6.5-kg pigs (n = 12; 4 pigs per pen) were offered supplements in a starter diet from weaning (day 0) to day 21 and in a transition diet from day 22 to day 40. There was an interaction (P = 0.005) between SWE and ZnO on G:F whereby pigs supplemented with SWE and ZnO individually had improved G:F when compared with the combination diet. There was an interaction between SWE and ZnO interaction on digestibility of DM (P < 0.01), N (P < 0.01), and NDF (P < 0.01). Pigs offered the SWE diets alone had a higher digestibility of DM, N, and NDF compared with pigs offered the basal diet. In summary, SWE induced a comparable growth performance pattern as obtained with ZnO inclusion. However, this was negated when supplements were offered in combination. Improvements in growth performance of pigs consuming SWE alone may reflect improvements in nutrient digestibility.

The effect of protease and nonstarch polysaccharide enzymes on manure odor and ammonia emissions from finisher pigs.

A 2 × 2 factorial experiment was conducted to investigate the effect of dietary protease (0 and 200 mg/kg) and xylanase (0 and 200mg/kg) in reducing manure odor and NH(3) from finisher pigs. Sixteen pigs were assigned to 1 of 4 dietary treatments, (i) basal diet, (ii) basal diet + xylanase, (iii) basal diet + protease, or (iv) basal diet + xylanase + protease, for 24 d. The manure samples from pigs offered diets containing protease showed increased (P < 0.05) molar proportions of isobutyric acid, isovaleric acid, valeric acid, and branched-chain fatty acids in contrast to pigs offered diets without protease supplementation. The pigs offered diets with xylanase inclusion had reduced (P < 0.05) manure odor emissions compared to pigs offered diets without xylanase supplementation [598 vs. 1306 European odor units (OuE)/m(3)]. Pigs offered protease-supplemented diets alone had significantly higher NH(3) emissions compared to basal fed pigs. However, NH(3) emissions were reduced when protease was combined with xylanase. In summary, this study indicates that protease supplementation increased protein-derived VFA in manure and also increased manure NH(3) emissions when offered singularly. Consumption of diets containing xylanase reduced manure odor emissions.

The effect of protease and xylanase enzymes on growth performance and nutrient digestibility in finisher pigs.

Two 2 × 2 factorial experiments were conducted to investigate the interaction between xylanase (0 vs. 200 mg/kg) and protease (0 vs. 200 mg/kg) enzyme supplementation on growth performance (Exp. 1) and coefficient of ileal and total tract apparent digestibility in grower-finisher pigs (Exp. 2). One hundred and twenty-eight individual fed pigs (BW = 34.2 kg; n = 32) were assigned to 1 of 4 dietary treatments: basal diet (T1), T1 + xylanase enzyme (T2), T1 + protease enzyme (T3), or T1 + xylanase + protease enzymes (T4). The pigs offered diets containing protease enzymes had reduced daily gain (0.795 vs. 0.840 kg/d; P < 0.05) and final body weight (96.4 vs. 99.1 kg; P < 0.05) compared to pigs offered diets without protease enzymes. Pigs offered xylanase-supplemented diets had reduced daily gain (0.787 vs. 0.848 kg/d; P < 0.05) compared to pigs offered diets without xylanase enzymes. In Exp. 2, the nutrient digestibility experiment consisted of 24 intact male pigs (n = 6; BW = 78 kg), offered identical diets to that offered in Exp. 1. Following the fecal collections, the pigs were slaughtered and digesta samples were taken from the ileum in order to measure apparent ileal N and GE digestibilities. Pigs offered diets supplemented with protease had increased coefficients of ileal digestibility of N compared to pigs offered diets without protease supplementation (0.583 vs. 0.449; P < 0.05). There was a xylanase × protease interaction (P < 0.05) on the apparent ileal digestibility of GE. Pigs offered diets containing protease only had increased apparent ileal digestibility of GE compared to basal fed pigs; however, the ileal digestibility of GE decreased when protease was combined with xylanase. Neither xylanase nor protease enzymes had any effect on total tract digestibility of GE or N. In conclusion, xylanase and protease enzyme supplementation had no positive effects on grower-finisher pig performance.

Novel use of accelerator mass spectrometry for the quantification of low levels of systemic therapeutic recombinant protein.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

Emergence of tics in children with attention deficit hyperactivity disorder treated with stimulant medications.

The emergence of tics in children treated with stimulant medication for attention deficit hyperactivity disorder (ADHD) was investigated. A retrospective chart review of the medical records of 555 subjects was performed to examine the emergence of tics in relation to treatment with a stimulant medication, dosage, duration of treatment, and age of subjects. A total of 7.8% of the subjects treated with stimulants developed tics: 8.3% of subjects treated with methylphenidate, 6.3% with dextroamphetamine, and 7.7% with pemoline. The subjects who developed tics were significantly younger than those who did not. Subjects treated with higher doses of stimulant medication were not more likely to develop tics. While the emergence of tics was common, these subjects may have developed tics irrespective of stimulant medication. Controversy remains as to the long-term risk of tics in relation to stimulant medication and to appropriate practice should tics emerge during the course of stimulant medication treatment.

Chemical modification of a variant of human MIP-1alpha; implications for dimer structure.

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.

Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES. Characterization of active disaggregated chemokine variants.

Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES. Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Techniques for development of safety-related software for surgical robots.

Regulatory bodies require evidence that software controlling potentially hazardous devices is developed to good manufacturing practices. Effective techniques used in other industries assume long timescales and high staffing levels and can be unsuitable for use without adaptation in developing electronic healthcare devices. This paper discusses a set of techniques used in practice to develop software for a particular innovative medical product, an endoscopic camera manipulator. These techniques include identification of potential hazards and tracing their mitigating factors through the project lifecycle.

A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development.

Analytical ultracentrifugation (AUC) has reemerged as a powerful technique for protein characterisation. We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1 alpha (MIP-1 alpha) as a protein therapeutic. MIP-1 alpha has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers. Using AUC as a screening technique, we have produced a biologically active variant of MIP-1 alpha, BB-10010, which has a reduced tendency to aggregate in physiological buffers. The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC). Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association. This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies.

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Fluorescence spectroscopy.

This chapter has introduced the theory of fluorescence, and briefly shown that it is a technique with many wide and varied applications in biochemistry. It has the overall advantage of being able to carry out measurements on small amounts of sample in solution, often revealing important information concerning the structure and dynamics of these systems.

Characterization of the solution properties and conformation of pneumolysin, the membrane-damaging toxin of Streptococcus pneumoniae.

Pneumolysin is a membrane-damaging toxin produced by Streptococcus pneumoniae. In order to understand fully the mode of action of this toxin, it is necessary to have an appreciation of the size, self-association behaviour and solution conformation of pneumolysin. A combination of analytical ultracentrifugation methodologies has shown that pneumolysin lacks self-association behaviour in solution and has provided a weight-average M(r) (M omega) of 52,000 +/- 2000, which was in agreement with that derived from the amino acid sequence. By determining a sedimentation coefficient (S20,w0) of 3.35 +/- 0.10 S, it was possible to suggest a model for the gross solution conformation of pneumolysin monomers. Spectroscopic methods provide additional secondary and tertiary structure information.

Characterization of the quaternary structure and conformational properties of the human stem cell inhibitor protein LD78 in solution.

The human LD78 protein (sometimes referred to as human macrophage inflammatory protein-1 alpha) has been shown to protect multipotential hemopoietic stem cells from the effects of cytotoxic agents. Administration of the recombinant stem cell inhibitor molecule LD78 as an adjunct to chemotherapy has potential clinical benefit in reducing or preventing the neutropenia associated with this treatment. At physiological ionic strength, the 8-kDa LD78 molecule exists as soluble, heterogeneous, multimeric complexes of mass ranging from 100 to > 250 kDa. The hydrodynamic and structural properties of LD78 have been determined in various buffer solutions using analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy. The results demonstrate that defined, homogeneous monomer and tetramer forms of LD78 can be prepared which display distinct conformational properties. The combined use of hydrodynamic and spectroscopic analysis provides an insight into the pathway and molecular mechanics of LD78 self-association.

Kinetics of folding of the all-beta sheet protein interleukin-1 beta.

The folding of the all-beta sheet protein, interleukin-1 beta, was studied with nuclear magnetic resonance (NMR) spectroscopy, circular dichroism, and fluorescence. Ninety percent of the beta structure present in the native protein, as monitored by far-ultraviolet circular dichroism, was attained within 25 milliseconds, correlating with the first kinetic phase determined by tryptophan and 1-anilinonaphthalene-8-sulfonate fluorescence. In contrast, formation of stable native secondary structure, as measured by quenched-flow deuterium-hydrogen exchange experiments, began after only 1 second. Results from the NMR experiments indicated the formation of at least two intermediates with half-lives of 0.7 to 1.5 and 15 to 25 seconds. The final stabilization of the secondary structure, however, occurs on a time scale much greater than 25 seconds. These results differ from previous results on mixed alpha helix-beta sheet proteins in which both the alpha helices and beta sheets were stabilized very rapidly (less than 10 to 20 milliseconds).

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.

Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus.

3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity. It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein. As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature. Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature. These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins. They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity.