N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells

Recombinant DNA technology enables the use of plants as the host for the production of pharmaceutical proteins, such as antibodies. The glycosylation of recombinant proteins plays physiological and biological roles. However, because glycosylation in plants is different from that in human cells, the development of glycoengineering is required. In plant cells, glycan structures are shown to correlate with the localization of the recombinant protein produced. In this study, the vacuolar sorting signal (VSS) of sporamin was fused to the heavy (H) and light (L) chains of a mouse monoclonal antibody (mAb), and the mAb was produced in suspension-cultured tobacco BY2 cells. The sugar chain structures were determined by high-performance liquid chromatography, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Typical plant glycans with α1,3-fucosylation and/or β1,2-xylosylation derived from mAb with the VSS-fused H-chain (mIgG1000) and mAb with the VSS-fused H- and L-chain (mIgG1010) occupied the large amount of the total N-glycans, 72.1% and 85.0%, respectively, such as those derived from mAb without VSS (mIgG0000), 74.6% (Fujiyama et al., J. Biosci. Bioeng., 101, 212-218, 2006). In contrast, the typical plant glycan structure Man?FucXylGlcNAc? particularly in vacuoles accounted for 37.8% of the total sugar chains derived from mIgG1000 and 58.5% of those derived from mIgG1010 compared with 24.3% of those derived from mIgG0000. These results suggest that the sporamin signal peptide fused to mAb acts as a VSS and leads to the increase in the amount of Man?FucXylGlcNAc?, which is the main N-glycan structure in vacuoles.     

Efficient production of an antibody Fab fragment using the baculovirus–insect cell system

The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (> 600 μg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus–insect cell system may allow efficient production of antibody Fab fragments.     

Dietary N‐Glycans from Bovine Lactoferrin and TLR Modulation

1 Scope

Bovine lactoferrin (bLF) is an ingredient of food supplements and infant formulas given its antimicrobial and antiviral properties. We modified bLF enzymatically to alter its N‐glycosylation and to isolate the glycan chains. The aims of this study include (1) to evaluate whether such derivates induce responses via pattern recognition receptors namely Toll‐like receptors (TLRs) and (2) to relate those responses to their different glycosylation profiles.

2 Methods and results

The unmodified and modified bLF fractions are incubated with reporter cell lines expressing pattern recognition receptors. Afterwards, we screen for TLRs and analyze for nuclear factor kappa—light‐chain enhancer of activated B cells (NF‐κB) activation. Activation of reporter cell lines show that signaling is highly dependent on TLRs. The activation pattern of bLF is reduced with the desialylated form and increased with the demannosylated form. In reporter cells for TLR, bLF activate TLR‐4 and inhibit TLR‐3. The isolated glycans from bLF inhibit TLR‐8. TLR‐2, TLR‐5, TLR‐7, and TLR‐9 are not significantly altered.

3 Conclusion

The profile of glycosylation is key for the biological activity of bLF. By understanding how this affects the human defense responses, the bLF glycan profile can be modified to enhance its immunomodulatory effects when used as a dietary ingredient.     

Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ～70%; where P indicates phosphorylation from single to triple phosphorylation (1–3P), and G indicates glycosylation from single to triple glycosylation (1–3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ～12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ～0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.     

Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system

Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI = 5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1 × 10⁶ cells/ml, and the highest rAAV yield (1.22 × 10⁸ IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13 × 10⁸ IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40 × 10⁸ IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.     

I2/I−-mediated fluorescence quenching of an Ag+-doped gold nanocluster-based immunoassay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is a type of hazardous bacteria in the field of food safety. A sensitive and effective method is urgently needed to detect it, avoiding enormous harm for the human health. In this study, we synthesized stable Ag⁺-doped gold nanoclusters (Ag-AuNC) with a fluorescence intensity 4.8 times stronger than that of AuNC. It was further demonstrated that Ag⁰ existing in the AuNC core and a fraction of Ag⁺ anchored on the AuNC shell eliminated the surface defects and improved the luminescent properties of AuNC. A combination of I₂ and I^? was used to quench fluorescence-enhanced Ag-AuNC, which was first applied in ELISA for detecting E. coli O157:H7 to improve the sensitivity. In the presence of E. coli O157:H7, the biotinylated anti-E. coli O157:H7 mAb and streptavidin-alkaline phosphatase would be immobilized and catalyze l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to produce ascorbic acid. After addition of KIO₃, I₂/I^? were generated. The I₂ could trigger oxidative etching of Ag-AuNC and I^? could combine with Ag⁺ to decrease the Ag⁺ concentration of Ag-AuNC, which resulted in fluorescence quenching of Ag-AuNC. Under optimal conditions, the linear range of I₂/I^?-mediated fluorescence quenching of Ag-AuNC-based immunoassay for detecting E. coli O157:H7 was 3.3 × 10³ to 10⁶ cfu/mL, with a detection limit of 9.2 × 10² cfu/mL, 10.7-fold lower than that of the traditional ELISA. The proposed immunoassay exhibits excellent sensitivity, specificity, recovery, and accuracy, which is useful for quantitative detection of E. coli O157:H7 in food safety.     

Hydrolysed collagen from Lates calcarifer skin: its acute toxicity and impact on cell proliferation and collagen production of fibroblasts

Acute toxicity in Wistar rat and the impact of hydrolysed collagen (HC) from seabass skin on in vitro cell proliferation and collagen production were studied using L929 fibroblasts. HC was rich in glycine (326 residue/1000 residue) and imino acids (196 residue/1000 residue). MALDI mass spectrum of HC showed several low MW peptides with MW range of 1050–1330 Da as the major components. Based on acute oral cytotoxicity test in Wistar rat, HC was considered as safe with LD₅₀ value higher than 5000 mg kg^?1 body. HC could promote L929 cell growth, especially when used in combination with vitamin C (VitC) at a ratio of 2:1. HC/VitC (2:1) mixture also exhibited the higher enhancement effect on collagen production of L929 cells, compared with HC or VitC alone. Thus, HC could be a promising candidate for biological applications, especially in combination with VitC, as nutraceuticals for skin care.     

Determination of Ochratoxin A in Cereals and Feeds by Ultra-performance Liquid Chromatography Coupled to Tandem Mass Spectrometry with Immunoaffinity Column Clean-up

This paper describes the preparation of reusable immunoaffinity columns and the development of an ultra-performance liquid chromatography tandem mass spectrometry method combined with immunoaffinity column clean-up (IAC-UPLC-MS/MS) for the determination of ochratoxin A (OTA) in cereals and feeds. The monoclonal antibody (mAb) was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1 (κ-light chain) isotype. A competitive indirect enzyme-linked immunosorbent assay was used to characterize the mAb. The concentrations causing 50 % inhibition of binding of mAb to OTA-ovalbumin by free OTA, ochratoxin B, and ochratoxin C were 1.29, 4.78, and 0.94 ng mL^?1, respectively. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N >10) ranging from 0.5 to 1.0 μg kg^?1 and a limit of detection (LOD, S/N >3) ranging from 0.2 to 0.3 μg kg^?1 in cereal and feed samples. The IAC-UPLC-MS/MS method offers a good LOQ and LOD for OTA in cereal and feed samples. The accuracy and precision at this level fall within the EU regulatory limit. This methodology has been validated in four different matrices (millet, maize, soybean, and swine finisher diet) with highly satisfactory results and applied to the analysis of samples collected from the markets.     

A high-concentrate diet provokes inflammation,endoplasmic reticulum stress,and apoptosis in mammary tissue of dairy cows through the upregulation of STIM1/ORAI1

High-concentrate feeding can induce subacute ruminal acidosis, which leads to mammary tissue injury in dairy cows. Therefore, the purpose of this research was to evaluate the effect of high-concentrate feeding on STIM1 (stromal interaction molecule 1)/ORAI1 (Orai calcium release-activated calcium modulator 1)-mediated inflammation, endoplasmic reticulum stress (ERS), and apoptosis in the mammary tissue of dairy cows. A total of 12 healthy mid-lactating Holstein cows of similar weight were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 6:4) and a low-concentrate (LC) group (concentrate:forage = 4:6). The trial lasted for 3 wk. After the feeding experiment, rumen fluid, lacteal vein blood, and mammary tissue samples were collected. The results showed that the HC diet significantly increased blood lipopolysaccharide levels, decreased ruminal pH, and upregulated the concentrations of Ca²⁺ and proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and the enzyme activities of caspase-3, caspase-9, PKC, and IKK. The upregulation of STIM1, ORAI1, PKCα, IKKβ, phosphorylated-IκBα, phosphorylated-p65, TNF-α, and IL-1α proteins in the HC group indicated activation of the STIM1/ORAI1-mediated inflammatory signaling pathway compared with that in the LC group. The HC diet also induced ERS by increasing the mRNA and protein abundances of GRP78, CHOP, PERK, ATF6, and IRE1α in the mammary tissue. Compared with the LC group, the mRNA expression levels and protein abundances of caspase-3, cleaved caspase-3, caspase-9, and BAX were markedly increased in the HC group. However, the mRNA and protein expression levels of Bcl-2 were significantly decreased in the HC group. Therefore, this study demonstrated that the HC diet can activate the store-operated calcium entry channel by upregulating the expression of STIM1 and ORAI1 and induce inflammation, ERS, and apoptosis in the mammary tissue of dairy cows.     

Maternal Choline Intake Programs Hypothalamic Energy Regulation and Later‐Life Phenotype of Male Wistar Rat Offspring

Scope: High‐folic‐acid diets during pregnancy result in obesity in the offspring, associated with altered DNA‐methylation of hypothalamic food intake neurons. Like folic acid, the methyl‐donor choline modulates foetal brain development, but its long‐term programing effects on energy regulation remain undefined. This study aims to describe the effect of choline intake during pregnancy on offspring phenotype and hypothalamic energy‐regulatory mechanisms. Methods and results: Wistar rat dams are fed an AIN‐93G diet with recommended choline (RC, 1 g kg^?1 diet), low choline (LC, 0.5‐fold), or high choline (HC, 2.5‐fold) during pregnancy. Male pups are terminated at birth and 17 weeks post‐weaning. Brain 1‐carbon metabolites, body weight, food intake, energy expenditure, plasma hormones, and protein expression of hypothalamic neuropeptides are measured. HC pups have higher expression of the orexigenic neuropeptide‐Y neurons at birth, consistent with higher cumulative food intake and body weight gain post‐weaning compared to RC and LC offspring. LC pups have lower leptin receptor expression at birth and lower energy expenditure and activity during adulthood. Conclusion: Choline content of diets that are consumed by rats during pregnancy affects the later‐life phenotype of offspring, associated with altered in utero programing of hypothalamic food intake regulation.     

Detection of histamine-producing bacteria using polymerase chain reaction techniques and DNA probes

The polymerase chain reaction technique was used to enable the detection of DNA specific amplicons, corresponding to gene fragments coding for histidine decarboxylase present in the histidine decarboxylating bacteria frequently found in canned fish. The level of histamine in several fish product samples was quantified by HPLC and the DNA of the samples containing histamine was isolated and amplified with hdcA histidine-decarboxylase gene-specific primers. The primer sets used, CL1/CL2, JV16HC/JV17HC and CL1/JV17HC, amplified 150 bp, 370 bp and 500 bp products respectively. Non-expected fragments (100 bp, 150 bp and 250 bp) were also amplified by JV16HC/JV17HC. Some amplified fragments were sequenced automatically, presenting high homologies with the Clostridium perfringens hdcA gene. A DNA probe from a C. perfringens pure strain was hybridized with the DNA fragments amplified from the contaminated samples. The hybridization blots were then detected by colorimetry, revealing that the samples were contaminated by C. perfringens.     

Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC₅₀ value of 8.9 ng mL^?1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg^?1 in muscle matrix and from 25.2 to 55.4 μg kg^?1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r?>?0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.     

Contraceptive responses of mice immunized with purified recombinant mouse zona pellucida subunit 3 (mZP3) proteins

Mouse zona pellucida subunit 3 (mZP3) was tested for efficacy as an immunocontraceptive antigen by comparing the fertility of mice immunized with recombinant mZP3 proteins. Recombinant protein was expressed using either the vaccinia virus T7 mammalian (vmZP3 protein) or baculovirus insect cell (bmZP3 protein)-expression systems. Female BALB/c or wild mice were immunized by i.p. injection using Freund's complete adjuvant and boosted three times with affinity purified recombinant proteins in Freund's incomplete adjuvant. Most mice developed antibodies that crossreacted to the respective mZP3 antigens by ELISA or western blot. In BALB/c mice immunized with vmZP3, fertility and mean litter size were reduced transiently to 25% and 10%, respectively, of those of control mice. However, immunization with bmZP3 did not affect either the fertility or mean litter sizes in BALB/c or wild mice immunized with bmZP3. The results demonstrate that reduction in fertility can be achieved in female BALB/c mice immunized using Freund's adjuvants and recombinant mZP3 protein produced in a mammalian, but not an insect, cell-expression system. Arguments are presented for the likely role of glycosylation of the mZP3 antigen in inducing contraceptive immune responses.     

Autocrine insulin-like growth factor II inhibits beta-casein mRNA expression in a mammary cell line

A hallmark of mammary cell differentiation is the induction of beta-casein mRNA expression. A mouse mammary epithelial cell line (COMMA-1D) was treated with insulin, hydrocortisone (HC), and prolactin (Prl) at concentrations (50, 500, and 20 ng/ml, respectively) that resulted in less than half-maximal beta-casein mRNA expression. The cells secreted insulin-like growth factor (IGF)-II (106 pg/ml per 24 h) in the condition media under these conditions. Replacement of insulin with rhIGF-II (150 ng/ml) resulted in significantly less beta-casein mRNA expression. Long-Arg IGF-I (50 ng/ml) was similar to insulin in terms of its ability to induce differentiation, but its activity differed from that of insulin in that it also induced cell proliferation. When the two receptor-specific IGF-II analogs, Arg54,55IGF-II and Leu27IGF-II, were used in studies, only at high concentrations (150 ng/ml) was either analog capable of stimulating any beta-casein mRNA expression. When autocrine IGF-II was immuno-neutralized or bound by the addition of rhIGF binding protein-3 (IGFBP-3)beta-casein mRNA expression was enhanced seven-fold and three-fold, respectively. Exogenous application of IGF-II to counteract the IGF-II mAb stimulation resulted in increased cellular growth and reduced differentiation. We conclude that autocrine IGF-II inhibits mammary cell differentiation and that the blockage of autocrine IGF-II benefits mammary cell differentiation.     

Genomics and proteomics of deleted ovine CSN1S11I

Ovine α_s1-casein (CSN1S1) allele I (CSN1S1¹I) was characterized at the molecular genetic and protein level. Sequencing of CSN1S1 cDNA and mature protein showed the absence of exon 7 from CSN1S1¹I in comparison with the C″ genetic variant of the C phenotype. This allelic aberration is correlated with a sequence difference in 5′-splice donor sequence of intron 7 (g.656T > A), leading to upstream skipping of exon 7. Consequently mRNA sequence of ovine CSN1S1¹I is 24 bp shorter than complete coding sequence leading to an abbreviation of eight amino acids in the mature protein, resulting in a lower degree of phosphorylation in comparison with CSN1S1¹C″. However CSN1S1¹I was expressed at a quantitative level similar to that for the C″ reference variant. Using amplified created restriction site polymerase chain reaction, a DNA-based test for identification of CSN1S1¹I was developed. Altogether six nucleotide substitutions were identified within intron 6 and intron 7 of CSN1S1 variants, forming three different haplotypes.     

Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs

The extraction efficiency of aflatoxin B₁ (AFB₁) in cattle feed containing nine adsorbents (ADSs) was investigated using two organic/aqueous solvents composed of methanol/water (80/20 v/v; MeOH) and acetone/water (85/15 v/v; AC). Samples were obtained including a highly AFB₁-contaminated (HC) and a low-level AFB₁-contaminated (LC) feedstuff (15.33 and 7.57 µg kg^–1, respectively), nine ADSs (four clay minerals; one yeast cell wall-based product; one activated carbon and three commercial ADS products) at two different levels of inclusion (10 and 20 g kg^?1). After solvent extraction and immunoaffinity column clean-up, all samples were analysed for AFB₁ by high-performance liquid chromatography (HPLC) with fluorescence detection. For each contamination level (HC and LC), the data obtained were analysed using a factorial arrangement in a completely randomized design. Means were compared with the correspondent controls using the Dunnett's test. No statistical difference was found in AFB₁ levels of feedstuffs not containing ADSs when extracted with AC or MeOH, even if numerically higher values were obtained with AC. A dose-dependent effect (p < 0.01) of ADSs inclusion was observed on AFB₁ recoveries that were lower when the higher ADS level (20 g kg^?1) was included in the HC and LC feedstuffs. Higher AFB₁ recoveries were obtained using AC compared with MeOH, both in HC (75.0% versus 12.0%, respectively) and in LC (84.0% versus 22.8%, respectively) ADSs containing feedstuffs. However, when the activated carbon and the sodium bentonite were included in feeds, lower AFB₁ concentrations with respect to control values (p < 0.001 and <0.05, respectively) were obtained also using AC. The data obtained in this study indicate that routine use of the MeOH solvent for AFB₁ analysis of unknown feedstuffs, can produce misleading results if they contain an ADS.     

Development of an Anti‐Insect Sachet Using a Polyvinyl Alcohol−Cinnamon Oil Polymer Strip Against Plodia interpunctella

Plodia interpunctella is a major storage pest that penetrates into food packaging and causes serious economic losses, as well as posing health risks. The goal of this study was to develop effective anti‐insect polymer strips against P. interpunctella by using plant essential oil (EO) and polyvinyl alcohol (PVA). The EO of cinnamon (Cinnamomum zeylanicum, CO) bark was used as an insect repellent, and fumigant mortality and the repellent activity of CO were measured to evaluate subsistent anti‐insect properties through newly designed traps. Repellent activity was also examined with several foods to simulate the storage environment. The mortality rate with CO after fumigation for 120 h was 63%. In the repellent assay, CO‐treated strips, but not control strips, effectively repelled P. interpunctella in both “with foods” and “without foods” groups. A PVA–CO strip sachet (PCO sachet) was developed to control the volatility of CO, and the PCO sachet demonstrated robust repellent activity. The loading contents of CO at the center and edges of strips were 39.41% and 39.59%, respectively, and through the results of FT‐IR, it inferred that CO was physically diffused in the PVA polymer matrix, not forming chemical bonds. In a release test using a gas chromatography, the PCO sachet showed remarkable controlled release of CO. These results demonstrate that the anti‐insect effects of CO can be maintained throughout the distribution and storage periods of foods using PCO sachets.     

Production of a horse-specific monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA

A stable hybridoma cell line (DD3) has been produced secreting a monoclonal antibody specific for horse muscle proteins. The DD3 monoclonal antibody (mAb) did not show significant cross-reactivity when tested against beef, chicken, pig and soya proteins or bovine caseins, gelatine and bovine serum albumin. The DD3 mAb was used in an indirect ELISA format for the detection of defined amounts of horse meat (10–500 g kg-¹) in beef meat mixtures. Immunorecognition of monoclonal antibodies adsorbed to horse meat adsorbed onto the ELISA plate was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of the substrate gave clear optical density differences when assaying mixtures of minced beef containing different amounts of horse meat.     

Emissions of regulated pollutants from a spark ignition engine. Influence of fuel and air/fuel equivalence ratio

A spark ignition engine is used to determine the influence of fuel composition and air/fuel equivalence ratio on the exhaust emissions of regulated pollutants. Two specific fuel matrices are used: the first contains eight hydrocarbons and the second contains four oxygenated compounds. A specific experimental design is used for these tests. Fuel aromatics increase the exhaust CO, HC, and NOx at stoichiometry, lean and rich conditions. Lambda is more important than fuel composition in the case of CO and HC. At stoichiometry, the addition of oxygenated compounds can decrease exhaust CO, HC, and NOx up to 30%, 50%, and 60%, respectively. Under these conditions, the addition of 5% of 2-propanol is the most effective for the reduction of CO, the addition of 20% of ethanol forthe reduction of HC, and this of 5% of methyl tributyl ester (MTBE) for the NOx. The addition of oxygenated compounds can decrease CO by 30% at lean conditions, while no decrease is observed at rich ones; HC and NOx can decrease up to 30% and 80%, respectively, under lean conditions and 50% under rich ones. At all lambda tested, exhaust NOx increases with the addition of 20% of 2-propanol.     

Production of recombinant protein by baculovirus-infected insect cells in immobilized culture using porous biomass support particles

Immobilization of insect cells using porous biomass support particles (BSPs) and production of a recombinant protein by the immobilized cells after infection with a baculovirus were investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin BSPs (2 x 2 x 2 mm cubes) with matrices of 60 mum mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5 x 10(7) cells/cm3-BSP in cultures with regular replacement of the culture medium, as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After infection with a recombinant baculovirus carrying the beta-galactosidase gene, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5 x 10(7) cells/cm3-BSP were infected with the baculovirus, efficient beta-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion.