Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral-bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral-bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral-bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.     

Metabolic responses of wheat roots to alkaline stress

Aims The aim of this study was to investigate the effects of alkaline stress on primary, secondary metabolites and metabolic pathways in the roots of wheat (Triticum aestivum). The results were used to evaluate the physiological adaptive mechanisms by which wheat tolerated alkali stress.Methods A pot experiment was carried out in the greenhouse. For each plastic pot, five wheat seeds were planted. After germination, seedlings were allowed to grow under controlled water and nutrient conditions for two months, then seedlings were exposed to alkaline stress (NaHCO₃-Na₂CO₃) for 12 days. The relative growth rate (RGR), absolute water content (AWC), metal elements, free cations and metabolites were measured.Important findings The alkaline stress caused the reduction of RGR and AWC. Alkaline stress caused a rapid increase of Na content with the concurrent decrease in K and Cl content, resulting in inhibited metal element accumulation and an ionic imbalance. In the present study, alkaline stress strongly enhanced Ca accumulation in wheat roots, suggesting that an increased Ca concentration can immediately trigger the salt overly sensitive (SOS)-Na exclusion system and reduce Na-associated injuries. Also, 70 metabolites, including organic acids, amino acids, sugars/polyols and others, behaved differently in the alkaline stress treatments according to a GC-MS analysis. The metabolic profiles of wheat were closely associated with alkaline-stress conditions. Alkaline stress caused the accumulation of organic acids, accompanied by the depletion of sugars/polyols and amino acids. Organic acids could play a central role in the regulation of intracellular pH by accumulating vacuoles to neutralize excess cations. Glycolysis and amino acid synthesis in roots were inhibited under salt stress while prolonged alkaline stress led to a progressive tricarboxylic acid (TCA) cycle. The severe negative effects of alkaline stress on sugar synthesis and storage may reflect the toxic levels of Na⁺ accumulating in plant cells in a high-pH environment, implying that the reactive oxygen species detoxification capacity was diminished by the high pH. A lack of NO₃^- in wheat roots can decrease synthase enzyme activities, limiting the synthesis of amino acids. Under salt stress, the TCA cycle and organic acid accumulation increased, but glycolysis and amino acid synthesis were inhibited in roots. Thus, energy levels and high concentrations of organic acids may be the key adaptive mechanisms by which wheat seedlings maintain their intracellular ion balance under alkaline stress.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

The influence of medium components on Cu(2+)-dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase.

The extent of in vitro Cu(2+)-dependent oxidation of low-density lipoproteins (LDL) has been reported to vary widely depending upon reaction conditions. In this study, the effect of proteins and amino acids on Cu(2+)-induced LDL oxidation was examined. Treatment of LDL with 5 microM CuSO4 for 18 h in either phosphate-buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TBARS) and by increased lipoprotein electronegativity. In PBS, oxidation was entirely blocked by histidine and the tripeptide, gly-his-lys (GHK). Oxidation was also prevented by bovine serum albumin, but superoxide dismutase (SOD) provided only 20% protection. Both proteins bound similar amounts of Cu2+, but albumin appeared to be a more effective peroxyl radical trap as evidenced by its ability to prevent LDL oxidation induced by 2,2'-azo-bis(2-amidinopropane hydrochloride). In F-10 medium, SOD had marked inhibitory effects, in contrast to PBS. The addition of disulfides to PBS markedly enhanced the ability of SOD to inhibit oxidation. These results indicate that medium components which affect Cu2+ availability influence LDL oxidation and suggest that albumin is ideally suited as a plasma antioxidant to prevent oxidative modification of LDL. Furthermore, in certain instances, the inhibitory effects of SOD may be attributable to effects such as Cu2+ binding rather than dismutation of superoxide.     

Cu2(+)-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein.

We previously reported that the expression of an epitope of apolipoprotein B (apoB), mapped to the C-terminus and defined by antibody Bsol7, increased during Cu2(+)-mediated oxidation of isolated low density lipoprotein (LDL). We describe now the properties of Bsol7 as a marker of LDL oxidation in whole plasma in relation to other effects of oxidative treatment of plasma, such as the distribution of apoA-I and cholesteryl ester transfer protein (CETP). In dialyzed plasma, no LDL oxidation was detected at Cu2+ concentrations (5 microM) sufficient for extensive oxidation of isolated LDL. At a higher Cu2+ concentration (50 microM), an increased expression of the Bsol7 epitope was observed; at 250 microM Cu2+, other evidence of LDL oxidation was found. The pattern of LDL response to Cu2+ observed in dialyzed plasma could be reproduced by adding 3% bovine serum albumin to isolated LDL. We demonstrate that the effect of albumin most likely results from its ability to bind copper ions. Incubation of plasma with increasing concentrations of Cu2+ resulted first in the disappearance of alpha 2-migrating HDL, the usual carrier of CEPT; free CETP and high molecular weight apoA-I-containing particles were also generated during oxidation. Addition of oxidized, but not native, LDL to plasma resulted in a transfer to LDL of some of the CETP initially associated with apoA-I. In conclusion, the increased immunoreactivity of the Bsol7 epitope was the most sensitive parameter of LDL oxidation, but other parameters, such as the presence of alpha 2-HDL and CETP-lipoprotein associations were even more sensitive evidence of lipoprotein oxidation.     

The role of amino acid metabolism during abiotic stress release

Plant responses to abiotic stress include various modifications in amino acid metabolism. By using a hydroponic culture system, we systematically investigate modification in amino acid profiles and the proteome of Arabidopsis thaliana leaves during initial recovery from low water potential or high salinity. Both treatments elicited oxidative stress leading to a biphasic stress response during recovery. Degradation of highly abundant proteins such as subunits of photosystems and ribosomes contributed to an accumulation of free amino acids. Catabolic pathways for several low abundant amino acids were induced indicating their usage as an alternative respiratory substrate to compensate for the decreased photosynthesis. Our results demonstrate that rapid detoxification of potentially detrimental amino acids such as Lys is a priority during the initial stress recovery period. The content of Pro, which acts as a compatible osmolyte during stress, was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments. The production of amino acid derived secondary metabolites was up‐regulated specifically during the recovery period, and our dataset also indicates increased synthesis rates of the precursor amino acids. Overall, our results support a tight relationship between amino acid metabolism and stress responses.     

Effects of emulsified policosanols with different chain lengths on cholesterol metabolism in heterozygous LDL receptor-deficient mice

Policosanol is a mixture of long-chain primary aliphatic saturated alcohols. Previous studies in humans and animals have shown that these compounds improved lipoprotein profiles. However, more-recent placebo-controlled studies could not confirm these promising effects. Octacosanol (C28), the main component of sugarcane-derived policosanol, is assumed to be the bioactive component. This has, however, never been tested in an in vivo study that compared individual policosanol components side by side. Here we present that neither the individual policosanol components (C24, C26, C28, or C30) nor the natural policosanol mixture (all 30 mg/100 g diet) lowered serum cholesterol concentrations in LDL receptor knock-out (LDLr(+/-)) mice. Moreover, there was no effect on gene expression profiles of LDLr, ABCA1, HMG-CoA synthase 1, and apolipoprotein A-I (apoA-I) in hepatic and small intestinal tissue of female LDLr(+/-) mice after the 7 week intervention period. Finally, none of the individual policosanols or their respective long-chain fatty acids or aldehydes affected de novo apoA-I protein production in vitro in HepG2 and CaCo-2 cells. Therefore, we conclude that the evaluated individual policosanols, as well as the natural policosanol mixture, have no potential for reducing coronary heart disease risk through effects on serum lipoprotein concentrations.     

Low density lipoprotein degradation by secretory granules of rat mast cells. Sequential degradation of apolipoprotein B by granule chymase and carboxypeptidase A

The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B component of low density lipoproteins (LDL) Kokkonen, J. O., and Kovanen, P. T. (1985) J. Biol. Chem. 260, 14756-14763). The granules are known to contain two neutral proteases with complementary specificities: a chymotrypsin-like endopeptidase called chymase, and an exopeptidase, the granule carboxypeptidase A. The role of this enzyme pair in the proteolytic degradation of LDL was studied with the aid of specific enzyme inhibitors. Incubation of LDL with intact granules (both enzymes active) led to the formation of numerous low molecular weight peptides and the liberation of free amino acids, most of which (95%) were aromatic (Phe, Tyr, Trp) or branched-chain aliphatic (Leu, Ile, Val). Selective inhibition of granule carboxypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of peptides uninhibited. On the other hand, selective inhibition of granule chymase (leaving carboxypeptidase A active) totally abolished the proteolytic degradation of LDL. The results are consistent with a model according to which the proteolytic degradation of LDL by mast cell granules results from coordinated action of the two granule-bound enzymes, whereby the chymase first cleaves peptides from the apolipoprotein B of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed.     

In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection.     

Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Effects of Cadmium Exposure on Growth and Metabolic Profile of Bermudagrass [Cynodon dactylon (L.) Pers.]

Metabolic responses to cadmium (Cd) may be associated with variations in Cd tolerance in plants. The objectives of this study were to examine changes in metabolic profiles in bermudagrass in response to Cd stress and to identify predominant metabolites associated with differential Cd tolerance using gas chromatography-mass spectrometry. Two genotypes of bermudagrass with contrasting Cd tolerance were exposed to 0 and 1.5 mM CdSO₄ for 14 days in hydroponics. Physiological responses to Cd were evaluated by determining turf quality, growth rate, chlorophyll content and normalized relative transpiration. All these parameters exhibited higher tolerance in WB242 than in WB144. Cd treated WB144 transported more Cd to the shoot than in WB242. The metabolite analysis of leaf polar extracts revealed 39 Cd responsive metabolites in both genotypes, mainly consisting of amino acids, organic acids, sugars, fatty acids and others. A difference in the metabolic profiles was observed between the two bermudagrass genotypes exposed to Cd stress. Seven amino acids (norvaline, glycine, proline, serine, threonine, glutamic acid and gulonic acid), four organic acids (glyceric acid, oxoglutaric acid, citric acid and malic acid,) and three sugars (xylulose, galactose and talose) accumulated more in WB242 than WB144. However, compared to the control, WB144 accumulated higher quantities of sugars than WB242 in the Cd regime. The differential accumulation of these metabolites could be associated with the differential Cd tolerance in bermudagrass.     

Borna disease virus infection perturbs energy metabolites and amino acids in cultured human oligodendroglia cells

Background

Borna disease virus is a neurotropic, non-cytolytic virus that has been widely employed in neuroscientific research. Previous studies have revealed that metabolic perturbations are associated with Borna disease viral infection. However, the pathophysiological mechanism underlying its mode of action remains unclear.

Methodology

Human oligodendroglia cells infected with the human strain Borna disease virus Hu-H1 and non-infected matched control cells were cultured in vitro. At day 14 post-infection, a proton nuclear magnetic resonance-based metabonomic approach was used to differentiate the metabonomic profiles of 28 independent intracellular samples from Borna disease virus-infected cells (n = 14) and matched control cells (n = 14). Partial least squares discriminant analysis was performed to demonstrate that the whole metabonomic patterns enabled discrimination between the two groups, and further statistical testing was applied to determine which individual metabolites displayed significant differences between the two groups.

Findings

Metabonomic profiling revealed perturbations in 23 metabolites, 19 of which were deemed individually significant: nine energy metabolites (α-glucose, acetate, choline, creatine, formate, myo-inositol, nicotinamide adenine dinucleotide, pyruvate, succinate) and ten amino acids (aspartate, glutamate, glutamine, glycine, histidine, isoleucine, phenylalanine, threonine, tyrosine, valine). Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between the two groups.

Conclusion

Borna disease viral infection perturbs the metabonomic profiles of several metabolites in human oligodendroglia cells cultured in vitro. The findings suggest that Borna disease virus manipulates the host cell’s metabolic network to support viral replication and proliferation.     

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.     

Isolation and identification of bovine nasopharyngeal mucosal epithelial cells and establishment of cell models of acute infection by foot-and-mouth disease virus

Foot-and-mouth disease (FMD) commonly occurs via the respiratory tract, and bovine nasopharyngeal mucosal epithelial cells are the primary infection cells in cattle. The aim of the present study was to isolate and culture epithelial cells from the bovine nasopharyngeal mucosa in vitro using a mechanical separation method. The cells were expanded, established in continuous cell culture, and used for immunofluorescence cytochemistry and establishment of infection models. We detected pan-cytokeratin markers of bovine nasopharyngeal mucosal epithelial cells by immunofluorescence. Bovine nasopharyngeal mucosal epithelial cells were then infected with foot-and-mouth disease virus (FMDV) serum type O. RT-PCR demonstrated the successful establishment of acute FMDV infection in the cell models. This infection model provides the basis for clarification of the interaction between FMDV and host bovine nasopharyngeal mucosal epithelial cells in vitro.     

Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti- receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.     

An immunochemical marker of low density lipoprotein oxidation

Using monoclonal antibodies against apolipoprotein B (apoB) we studied changes in apoB immunoreactivity during copper ion-mediated oxidation of human low density lipoprotein (LDL). The radioimmunoassay experiments demonstrated the decrease of immunoreactivity of three different epitopes of apoB located in different parts of the protein; at the same time the immunoreactivity of another epitope, previously mapped to the C-terminal 20 amino acids of apoB increased markedly during the first 6 h of LDL oxidation and diminished gradually upon prolonged incubation with copper ions. The fate of LDL during oxidation was also monitored using electrophoretic techniques combined with immunodetection. These experiments showed a rapid fragmentation and disappearance of immunoreactive apoB. They also indicated that the diminishing LDL immunoreactivity detectable during oxidation is associated with apoB fragments still attached to the lipid core. The changes in apoB immunoreactivity during Cu2+ treatment of LDL are similar to those observed upon LDL aging. Therefore, it appears that the enhancement of immunoreactivity of the C-terminus of apoB is a general phenomenon associated with various kinds of oxidative modifications of LDL.     

Human respiratory syncytial virus vaccine antigen produced in plants.

Human respiratory syncytial virus (RSV) is the primary cause of respiratory infection in infants worldwide. Currently there is no available vaccine, although studies in animal models have demonstrated protective immunity induced by an epitope of the RSV G-protein representing amino acids 174-187. Two peptides containing amino acids 174-187 of the G-protein of the human RSV A2 strain (NF1-RSV/172-187 and NF2-RSV/170-191) were separately engineered as translational fusions with the alfalfa mosaic virus coat protein and individually expressed in Nicotiana tabacum cv. Samsun NN plants through virus infection. RSV G-protein peptides were expressed in infected plant tissues at significant levels within 2 wk of inoculation and purified as part of recombinant alfalfa mosaic virions. BALB/c mice immunized intraperitoneally with three doses of the purified recombinant viruses showed high levels of serum antibody specific for RSV G-protein and were protected against infection with RSV Long strain.