Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe ( SPINE) consortium have developed and implemented high- throughput ( HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast ( Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for coexpression, selenomethionine labelling ( in all three eukaryotic systems) and control of glycosylation ( for secreted proteins in mammalian cells) are assessed.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHbeta knockout (FSHbetaKO) females where disruption of the gene encoding FSHbeta results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin alpha subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the betaA and betaB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the RA and betaB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin betaA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin alpha and betaB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk

The aim of the present study was to find out the best growing conditions for exopolysaccharide (EPS) producing bifidobacteria, which improve their functionality in yoghurt-like products. Two Bifidobacterium strains were used in this study, Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205. In the first part of the study the effect of casein hydrolysate, lactalbumin hydrolysate, whey protein concentrate and whey protein isolate, added at 1.5% w/v in skim milk, was evaluated in terms of cell growth and EPS production; skim milk supplemented with yeast extract served as the control. Among the various nitrogen sources, casein hydrolysate (CH) showed the highest cell growth and EPS production for both strains after 18 h incubation and therefore it was selected for subsequent work. Based on fermentation experiments using different levels of CH (from 0.5 to 2.5% w/v) it was deduced that 1.5% (w/v) CH resulted in the highest EPS production, yielding 102 and 285 mg L− 1 for B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486, respectively. The influence of temperature on growth and EPS production of both strains was further evaluated at 25, 30, 37 and 42 °C for up to 48 h in milk supplemented with 1.5% (w/v) CH. The temperature had a significant effect on growth, acidification and EPS production. The maximum growth and EPS production were recorded at 37 °C for both strains, whereas no EPS production was observed at 25 °C. Lower EPS production for both strains were observed at 42 °C, which is the common temperature used in yoghurt manufacturing compared to that at 37 °C. The results showed that the culture conditions have a clear effect on the growth, acidification and EPS production, and more specifically, that skim milk supplemented with 1.5% (w/v) CH could be used as a substrate for the growth of EPS-producing bifidobacteria, at 37 °C for 24 h, resulting in the production of a low fat yoghurt-like product with improved functionality.

Enhanced aglycone production of fermented soybean products by Bacillus species

This study evaluated the effect of starter culture and fermentation period on the isoflavone content of protein-rich soybeans variety TG145. Initially, soybeans were washed, soaked in water for 16 h and autoclaved at 121°C for 40min. Three different bacterial starter cultures (~104 CFU/g) namely Bacillus subtilis BEST195, B. subtilis Asaichiban and B. subtilis TN51 were then added and the fermentation was allowed to proceed at 42°C for 24 h (natto-style) and 72 h (thua nao-style). The quantities of six major isoflavones (daidzin, genistin, glycitin, daidzein, genistein, and glycitein) were then determined in these fermented soybean products using reverse phase HPLC technique. Generally, our results clearly showed that the content of total isoflavones in the fermented products prepared by Bacillus starter cultures greatly increased ranging from 43 to 99% compared to that of the unfermented autoclaved soybeans. In addition, a dramatic increase of aglycones was also observed (> 400%) in the soybean products fermented by Bacillus subtilis strain TN51. This present study suggests a promising use of Bacillus starter cultures in improving isoflavone compounds especially the aglycones which would benefit for novel functional food development.

Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture

Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2x108, 1.1x109 and 3.5x1010 at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2 mg at d18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus shown to be a flexible and powerful tool for manufacturing recombinant proteins.

Estimation of the stoichiometry of volatile fatty acid production in the rumen of lactating cows

The purpose of this study was to improve the prediction of the quantity and type of Volatile Fatty Acids (VFA) produced from fermented substrate in the rumen of lactating cows. A model was formulated that describes the conversion of substrate (soluble carbohydrates, starch, hemi-cellulose, cellulose, and protein) into VFA (acetate, propionate, butyrate, and other VFA). Inputs to the model were observed rates of true rumen digestion of substrates, whereas outputs were observed molar proportions of VFA in rumen fluid. A literature survey generated data of 182 diets (96 roughage and 86 concentrate diets). Coefficient values that define the conversion of a specific substrate into VFA were estimated meta-analytically by regression of the model against observed VFA molar proportions using non-linear regression techniques. Coefficient estimates significantly differed for acetate and propionate production in particular, between different types of substrate and between roughage and concentrate diets. Deviations of fitted from observed VFA molar proportions could be attributed to random error for 100%. In addition to regression against observed data, simulation studies were performed to investigate the potential of the estimation method. Fitted coefficient estimates from simulated data sets appeared accurate, as well as fitted rates of VFA production, although the model accounted for only a small fraction (maximally 45%) of the variation in VFA molar proportions. The simulation results showed that the latter result was merely a consequence of the statistical analysis chosen and should not be interpreted as an indication of inaccuracy of coefficient estimates. Deviations between fitted and observed values corresponded to those obtained in simulations. (c) 2005 Elsevier Ltd. All rights reserved.

A safety evaluation of genetically modified feedstuffs for livestock production; the fate of transgenic DNA and proteins

dTwo genetic constructs used to confer improved agronomic characteristics, namely herbicide tolerance (HT) in maize and soyabean and insect resistance (Bt) in maize, are considered in respect of feeding to farm livestock, animal performance and the nutritional value and safety of animal products. A review of nucleic acid (DNA) and protein digestion in farm livestock concludes that the frequency of intact transgenic DNA and proteins of GM and non-GM crops being absorbed is minimal/non existent, although there is some evidence of the presence of short fragments of rubisco DNA of non-GM soya in animal tissues. It has been established that feed processing (especially heat) prior to feeding causes significant disruption of plant DNA. Studies with ruminant and non-ruminant farm livestock offered GM feeds demonstrated that animal performance and product composition are unaffected and that there is no evidence of transgenic DNA or proteins of current GM in the products of animals consuming such feeds. On this evidence, current HT and Bt constructs represent no threat to the health of animals, or humans consuming the products of such animals. However as new GM constructs become available it will be necessary to subject these to rigorous evaluation.

Simulation of milk production by dairy cows fed sugarcane top-based diets with locally available supplements under Indian conditions

A model of sugarcane digestion was applied to indicate the suitability of various locally available supplements for enhancing milk production of Indian crossbred dairy cattle. Milk production was calculated according to simulated energy, lipogenic, glucogenic and aminogenic substrate availability. The model identified the most limiting substrate for milk production from different sugarcane-based diets. For sugarcane tops/urea fed alone, milk production was most limited by amino acid followed by long chain fatty acid availability. Among the protein-rich oil cake supplements at 100, 200 and 300 g supplement/kg total DM, cottonseed oil cake proved superior with a milk yield of 5.5, 7.3 and 8.3 kg/day, respectively. This was followed by mustard oil cake with 5.1, 6.5 and 7.6 kg/day, respectively. In the case of a protein-rich supplement (fish meal), milk yield was limited to 6.6 kg/day due to a shortage of long chain fatty acids. However, at 300 g of supplementation, energy became limiting, with a milk yield of 6.7 kg/day. Supplementation with rice bran and rice polishings at 100, 200 and 300 g restricted milk yield to 4.3, 4.9 and 5.5 and 4.5, 5.3 and 6.1 kg/day, respectively, and amino acids became the factor limiting milk production. The diet comprising basal sugarcane tops supplemented by leguminous fodder, dry fodder (e.g. rice or wheat straw) and concentrates at levels of 100, 200 and 300 g supplements/kg total diet DM proved to be the most balanced with a milk yield of 5.1, 6.7 and 9.0 kg/day, respectively.

Effects of corn silage derived from a genetically modified variety containing two Transgenes on feed intake, milk production, and composition, and the absence of detectable Transgenic deoxyribonucleic acid in milk in holstein dairy cows

The objectives were to compare the chemical composition, nutritive value, feed intake, milk production and composition, and presence in milk of transgenic DNA and the encoded protein Cry1Ab when corn silages containing 2 transgenes (2GM: herbicide tolerance: mepsps and insect resistance: cry1Ab) were fed as part of a standard total mixed ration (TMR) compared with a near isogenic corn silage ( C) to 8 multiparous lactating Holstein dairy cows in a single reversal design study. Cows were fed a TMR ration ad libitum and milked twice daily. Diets contained [ dry matter (DM) basis] 45% corn silage, 10% alfalfa hay, and 45% concentrate (1.66 Mcal of net energy for lactation/kg of DM, 15.8% crude protein, 35% neutral detergent fiber, and 4.1% fat). Each period was 28-d long. During the last 4 d of each period, feed intake and milk production data were recorded and milk samples taken for compositional analysis, including the presence of transgenic DNA and Cry1Ab protein. There was no significant difference in the chemical composition between C and 2GM silages, and both were within the expected range (37.6% DM, 1.51 Mcal of net energy for lactation/kg, 8.6% crude protein, 40% neutral detergent fiber, 19.6% acid detergent fiber, pH 3.76, and 62% in vitro DM digestibility). Cows fed the 2GM silage produced milk with slightly higher protein (3.09 vs. 3.00%), lactose ( 4.83 vs. 4.72%) and solids-not-fat (8.60 vs. 8.40%) compared with C. However, the yield (kg/d) of milk (36.5), 3.5% fat-corrected milk (34.4), fat (1.151), protein (1.106), lactose (1.738), and solids-not-fat ( 3.094), somatic cell count (log(10): 2.11), change in body weight (+ 7.8 kg), and condition score (+ 0.09) were not affected by type of silage, indicating no overall production difference. All milk samples were negative for the presence of transgenic DNA from either trait or the Cry1Ab protein. Results indicate that the 2GM silage modified with 2 transgenes did not affect nutrient composition of the silages and had no effect on animal performance and milk composition. No transgenic DNA and Cry1Ab protein were detected in milk.

Comparison of energy evaluation systems and a mechanistic model for milk production by dairy cattle offered fresh grass-based diets

Grass-based diets are of increasing social-economic importance in dairy cattle farming, but their low supply of glucogenic nutrients may limit the production of milk. Current evaluation systems that assess the energy supply and requirements are based on metabolisable energy (ME) or net energy (NE). These systems do not consider the characteristics of the energy delivering nutrients. In contrast, mechanistic models take into account the site of digestion, the type of nutrient absorbed and the type of nutrient required for production of milk constituents, and may therefore give a better prediction of supply and requirement of nutrients. The objective of the present study is to compare the ability of three energy evaluation systems, viz. the Dutch NE system, the agricultural and food research council (AFRC) ME system, and the feed into milk (FIM) ME system, and of a mechanistic model based on Dijkstra et al. [Simulation of digestion in cattle fed sugar cane: prediction of nutrient supply for milk production with locally available supplements. J. Agric. Sci., Cambridge 127, 247-60] and Mills et al. [A mechanistic model of whole-tract digestion and methanogenesis in the lactating dairy cow: model development, evaluation and application. J. Anim. Sci. 79, 1584-97] to predict the feed value of grass-based diets for milk production. The dataset for evaluation consists of 41 treatments of grass-based diets (at least 0.75 g ryegrass/g diet on DM basis). For each model, the predicted energy or nutrient supply, based on observed intake, was compared with predicted requirement based on observed performance. Assessment of the error of energy or nutrient supply relative to requirement is made by calculation of mean square prediction error (MSPE) and by concordance correlation coefficient (CCC). All energy evaluation systems predicted energy requirement to be lower (6-11%) than energy supply. The root MSPE (expressed as a proportion of the supply) was lowest for the mechanistic model (0.061), followed by the Dutch NE system (0.082), FIM ME system (0.097) and AFRCME system(0.118). For the energy evaluation systems, the error due to overall bias of prediction dominated the MSPE, whereas for the mechanistic model, proportionally 0.76 of MSPE was due to random variation. CCC analysis confirmed the higher accuracy and precision of the mechanistic model compared with energy evaluation systems. The error of prediction was positively related to grass protein content for the Dutch NE system, and was also positively related to grass DMI level for all models. In conclusion, current energy evaluation systems overestimate energy supply relative to energy requirement on grass-based diets for dairy cattle. The mechanistic model predicted glucogenic nutrients to limit performance of dairy cattle on grass-based diets, and proved to be more accurate and precise than the energy systems. The mechanistic model could be improved by allowing glucose maintenance and utilization requirements parameters to be variable. (C) 2007 Elsevier B.V. All rights reserved.

Genetic parameters and evaluations from single- and multiple-trait analysis of dairy cow fertility and milk production

Genetic parameters and breeding values for dairy cow fertility were estimated from 62 443 lactation records. Two-trait analysis of fertility and milk yield was investigated as a method to estimate fertility breeding values when culling or selection based on milk yield in early lactation determines presence or absence of fertility observations in later lactations. Fertility traits were calving interval, intervals from calving to first service, calving to conception and first to last service, conception success to first service and number of services per conception. Milk production traits were 305-day milk, fat and protein yield. For fertility traits, range of estimates of heritability (h(2)) was 0.012 to 0.028 and of permanent environmental variance (c(2)) was 0.016 to 0.032. Genetic correlations (r(g)) among fertility traits were generally high ( > 0.70). Genetic correlations of fertility with milk production traits were unfavourable (range -0.11 to 0.46). Single and two-trait analyses of fertility were compared using the same data set. The estimates of h(2) and c(2) were similar for two types of analyses. However, there were differences between estimated breeding values and rankings for the same trait from single versus multi-trait analyses. The range for rank correlation was 0.69-0.83 for all animals in the pedigree and 0.89-0.96 for sires with more than 25 daughters. As single-trait method is biased due to selection on milk yield, a multi-trait evaluation of fertility with milk yield is recommended. (C) 2002 Elsevier Science B.V. All rights reserved.

Effect of energy and protein supplementation on phosphorus utilization in lactating dairy cows

Two experiments were undertaken in which grass silage was used in conjunction with a series of different concentrate types designed to examine the effect of carbohydrate source, protein level and degradability on total dietary phosphorus (P) utilization with emphasis on P pollution. Twelve Holstein-Friesian dairy cows in early to mid-lactation were used in an incomplete changeover design with four periods consisting of 4 weeks each. Phosphorus intake ranged from 54 to 80 g/day and faecal P represented the principal route by which ingested P was disposed of by cows, with insignificant amounts being voided in urine. A positive linear relationship between faecal P and P intake was established. In Experiment 1, P utilization was affected by dietary carbohydrate type, with an associated output of 3.3 g faecal P/g milk P produced for all treatments except those utilizing low degradable starch and low protein supplements, where a mean value of 2.8 g faecal P/g milk P was observed. In Experiment 2, where two protein levels and three protein degradabilities were examined, the efficiency of P utilization for milk P production was not affected by either level or degradability of crude protein (CP) but a significant reduction in faecal P excretion due to lower protein and P intake was observed. In general, P utilization in Experiment 2 was substantially improved compared to the Experiment 1, with an associated output of 1.8 g faecal P/g milk P produced. The improved utilization of P in Experiment 2 could be due to lower P content of the diets offered and higher dry matter (DM) intake. For dairy cows weighing 600 kg, consuming 17-18 kg DM/day and producing about 25 kg milk, P excretion in faeces and hence P pollution to the environment might be minimized without compromising lactational performance by formulating diets to supply about 68 g P/day, which is close to recent published recommended requirements for P.