DNA-based analysis of protein variants reveals different genetic variability of the paralogous equine ß-lactoglobulin genes LGB1 and LGB2

The genetic variability of milk protein genes may influence the nutritive value or processing and functional properties of the milk. While numerous protein variants are known in ruminants, knowledge about milk protein variability in horses is still limited. Mare's milk is, however, produced for human consumption in many countries. Beta-lactoglobulin belonging to the protein family of lipocalins, which are known as common food- and airborne allergens, is a major whey protein. It is absent from human milk and thus a key agent in provoking cow's milk protein allergy. Mare's milk is, however, usually better tolerated by most affected people. Several functions of β-lactoglobulin have been discussed, but its ultimate physiological role remains unclear. In the current study, the open reading frames of the two equine β-lactoglobulin paralogues LGB1 and LGB2 were re-sequenced in 249 horses belonging to 14 different breeds in order to predict the existence of protein variants at the DNA-level. Thereby, only a single signal peptide variant of LGB1, but 10 different putative protein variants of LGB2 were identified. In horses, both genes are expressed and in such this is a striking previously unknown difference in genetic variability between the two genes. It can be assumed that LGB1 is the ancestral paralogue, which has an essential function causing a high selection pressure. As horses have very low milk fat content this unknown function might well be related to vitamin-uptake. Further studies are, however, needed, to elucidate the properties of the different gene products.

Experiences with a voluntary surveillance system for early detection of equine diseases in Switzerland

Clinical observations made by practitioners and reported using web- and mobile-based technologies may benefit disease surveillance by improving the timeliness of outbreak detection. Equinella is a voluntary electronic reporting and information system established for the early detection of infectious equine diseases in Switzerland. Sentinel veterinary practitioners have been able to report cases of non-notifiable diseases and clinical symptoms to an internet-based platform since November 2013. Telephone interviews were carried out during the first year to understand the motivating and constraining factors affecting voluntary reporting and the use of mobile devices in a sentinel network. We found that non-monetary incentives attract sentinel practitioners; however, insufficient understanding of the reporting system and of its relevance, as well as concerns over the electronic dissemination of health data were identified as potential challenges to sustainable reporting. Many practitioners are not yet aware of the advantages of mobile-based surveillance and may require some time to become accustomed to novel reporting methods. Finally, our study highlights the need for continued information feedback loops within voluntary sentinel networks.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(−)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.

Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of α-eleostearic (18:3Δ9cis,11trans,13trans) and α-parinaric (18:4Δ9cis,11trans,13trans,15cis) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of α-eleostearic and α-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Δ12-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of α-eleostearic and α-parinaric acids, neither of which is present in untransformed soybean embryos. α-Eleostearic and α-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Δ12-oleic acid desaturase-type enzymes that we have termed “conjugase.”

Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α2ββ′), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ70 was bound to the core enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71–75], the cut sites from different probe-modified σ70 proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).

Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

4-Hydroxylation of estrogens as marker of human mammary tumors.

Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2-hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development.

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.