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.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

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.

Differential effects of thyroid hormone manipulation and ß adrenoceptor agonist administration on uncoupling protein mRNA abundance in adipose tissue and thermoregulation in neonatal pigs

We have shown that there is significant disparity in the expression of uncoupling proteins (UCP) 2 and 3 between modern-commercial and ancient-Meishan porcine genotypes, commercial pigs also have higher plasma triiodothyronine (T(3)) in on the first day of life. T(3) and the sympathetic nervous system are both known to regulate UCPs in rodents and humans; their role in regulating these proteins in the pig is unknown. This study examined whether thyroid hormone manipulation or administration of a selective beta3 adrenoceptor agonist (ZD) influenced plasma hormones, colonic temperature and UCP expression in adipose tissue of two breeds of pig. To mimic the differences observed in thyroid hormone status, piglets from Meishan and commercial litters were randomly assigned to control (1 ml/kg water), T(3) (10 mg/kg) (Meishan only), methimazole (a commonly used antithyroid drug) (50 mg/kg) (commercial only) or ZD (10 mg/kg) oral administration for the first 4 days of postnatal life. Adipose tissue UCP2/3 mRNA abundance was measured on day 4 using PCR. T(3) administration raised plasma T(3) concentrations and increased colonic temperature on day 4. UCP3 mRNA abundance was higher in Meishan, than commercial piglets (p = 0.042) and was downregulated following T(3) administration (p = 0.014). Irrespective of genotype, ZD increased UCP2 mRNA abundance (Meishan p = 0.05, commercial p = 0.03). Expression of neither UCP2 nor 3 was related to colonic temperature, regardless of treatment. In conclusion, we have demonstrated a dissociation between thyroid hormones and the sympathetic nervous system in the regulation of UCPs in porcine adipose tissue. We have also suggested that expression of adipose tissue UCP2 and 3 are not related to body temperature in piglets.

Detection of transgenic DNA and protein, in rumen fluid, duodenal digesta, milk, blood and faeces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Limit-feeding a high-energy diet to meet energy requirements in the dry period alters plasma metabolite concentrations but does not affect intake or milk production in early lactation

Limit-feeding dry cows a high-energy diet may enable adequate energy intake to be sustained as parturition approaches, thus reducing the extent of negative energy balance after parturition. Our objective was to evaluate the effect of dry period feeding strategy on plasma concentrations of hormones and metabolites that reflect energy status. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition, paired by expected calving date, parity, and previous lactation milk yield, and randomly assigned to 1 of 2 dry-period diets formulated to meet nutrient requirements at ad libitum or limited intakes. All cows were fed the same diet for ad libitum intake after parturition. Prepartum dry matter intake (DMI) for limit-fed cows was 9.4 kg/d vs. 13.7 kg/d for cows fed ad libitum. During the dry period, limit-fed cows consumed enough feed to meet calculated energy requirements, and ad libitum-fed cows were in positive calculated net energy for lactation (NEL) balance (0.02 vs. 6.37 Mcal/d, respectively). After parturition, milk yield, milk protein concentration, DMI, body condition score, and body weight were not affected by the prepartum treatments. Cows limit fed during the dry period had a less-negative calculated energy balance during wk 1 postpartum. Milk fat concentration and yield were greater for the ad libitum treatment during wk 1 but were lower in wk 2 and 3 postpartum. Plasma insulin and glucose concentrations decreased after calving. Plasma insulin concentration was greater in ad libitum-fed cows on d -2 relative to calving, but did not differ by dietary treatment at other times. Plasma glucose concentrations were lower before and after parturition for cows limit-fed during the dry period. Plasma nonesterified fatty acid concentrations peaked after parturition on d 1 and 4 for the limit-fed and ad libitum treatments, respectively, and were greater for limit-fed cows on d -18, -9, -5, and -2. Plasma tumor necrosis factor-alpha concentrations did not differ by treatment in either the pre- or postpartum period, but tended to decrease after parturition. Apart from a reduction in body energy loss in the first week after calving, limit feeding a higher NEL diet during the dry period had little effect on intake and milk production during the first month of lactation.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Assembly rules for protein networks derived from phylogenetic-statistical analysis of whole genomes

Background: We report an analysis of a protein network of functionally linked proteins, identified from a phylogenetic statistical analysis of complete eukaryotic genomes. Phylogenetic methods identify pairs of proteins that co-evolve on a phylogenetic tree, and have been shown to have a high probability of correctly identifying known functional links. Results: The eukaryotic correlated evolution network we derive displays the familiar power law scaling of connectivity. We introduce the use of explicit phylogenetic methods to reconstruct the ancestral presence or absence of proteins at the interior nodes of a phylogeny of eukaryote species. We find that the connectivity distribution of proteins at the point they arise on the tree and join the network follows a power law, as does the connectivity distribution of proteins at the time they are lost from the network. Proteins resident in the network acquire connections over time, but we find no evidence that 'preferential attachment' - the phenomenon of newly acquired connections in the network being more likely to be made to proteins with large numbers of connections - influences the network structure. We derive a 'variable rate of attachment' model in which proteins vary in their propensity to form network interactions independently of how many connections they have or of the total number of connections in the network, and show how this model can produce apparent power-law scaling without preferential attachment. Conclusion: A few simple rules can explain the topological structure and evolutionary changes to protein-interaction networks: most change is concentrated in satellite proteins of low connectivity and small phenotypic effect, and proteins differ in their propensity to form attachments. Given these rules of assembly, power law scaled networks naturally emerge from simple principles of selection, yielding protein interaction networks that retain a high-degree of robustness on short time scales and evolvability on longer evolutionary time scales.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to approximate to 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Analysis of void volumes in proteins and application to stability of the p53 tumour suppressor protein

We have developed a new method for the analysis of voids in proteins (defined as empty cavities not accessible to solvent). This method combines analysis of individual discrete voids with analysis of packing quality. While these are different aspects of the same effect, they have traditionally been analysed using different approaches. The method has been applied to the calculation of total void volume and maximum void size in a non-redundant set of protein domains and has been used to examine correlations between thermal stability and void size. The tumour-suppressor protein p53 has then been compared with the non-redundant data set to determine whether its low thermal stability results from poor packing. We found that p53 has average packing, but the detrimental effects of some previously unexplained mutations to p53 observed in cancer can be explained by the creation of unusually large voids. (C) 2004 Elsevier Ltd. All rights reserved.

Cupins: the most functionally diverse protein superfamily?

The cupin superfamily of proteins, named on the basis of a conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), was originally discovered using a conserved motif found within germin and germin-like proteins from higher plants. Previous analysis of cupins had identified some 18 different functional classes that range from single-domain bacterial enzymes such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors including one linked to the nodulation response in legumes. Recent advances in comparative genomics, and the resolution of many more 3-D structures have now revealed that the largest subset of the cupin superfamily is the 2-oxyglutarate-Fe2+ dependent dioxygenases. The substrates for this subclass of enzyme are many and varied and in total amount to probably 50–100 different biochemical reactions, including several involved in plant growth and development. Although the majority of enzymatic cupins contain iron as an active site metal, other members contain either copper, zinc, cobalt, nickel or manganese ions as a cofactor, with each cofactor allowing a different type of chemistry to occur within the conserved tertiary structure. This review discusses the range of structures and functions found in this most diverse of superfamilies.

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.