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See detailInvestigation of pregnancy-associated glycoproteins (PAGs) by means of an enzymoimunoassay (ELISA) sandwich kit for pregnancy monitoring in sheep
El Amiri, Bouchra; Delahaut, Philippe; Colemans, Yves et al

in Chentouf, M; López-Francos, A; Bengoumi, M (Eds.) et al Options méditerranéennes : Series A (2014)

The aim of the present study was to investigate the use of a PAGs ELISA-Sandwich kit (Ret. code E.G.7. CER. Marloie, Belgium) for both early pregnancy diagnosis (in Sardi sheep) and the pregnancy follow ... [more ▼]

The aim of the present study was to investigate the use of a PAGs ELISA-Sandwich kit (Ret. code E.G.7. CER. Marloie, Belgium) for both early pregnancy diagnosis (in Sardi sheep) and the pregnancy follow up (in Boujaâd and Boujaâd x D'man sheep). ln Sardi breed, plasma samples were obtained from pregnant ewes (n = 17) from day 18 to 30 of gestation at 2 days interval. ln Boujaâd (n = 8) and Boujaâd x D'man (n = 20) the blood samples were weekly collected from the first week of gestation till the 4th week after lambing. The PAG concentrations were determined by a sandwich-ELISA based on purified bovine PAG (boPAG-67 kDa) as a standard, the antiserum raised against caprine PAG (caPAG-55+62 kDa) as a capture antibody (1/40000) and antiserum raised against purified PAG from buffalo (AS 859) as detection antibody (1/32 000). The Avidin-HRP and TMB were used to reveal reactions. Ewes were assumed to be pregnant when PAG concentrations were higher than 0.8 ng/ml. Results showed that in Sardi sheep it is possible to detect ail ewes as pregnant as early as on 24 days of gestation. ln Boujaâd and Boujaâd x D'man the PAG concentrations reached the higher values just before lambing. This value is higher in ewes carrying more than one lamb than in those carrying a single lamb. To conclude, the present study shows that the ELISA kit used for early pregnancy diagnosis in sheep as weil as for pregnancy fellow up could be a good alternative to the radioimmunoassay RIA in countries where this last technique is hard to set up. [less ▲]

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See detailIdentification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora ULg; Barbato, Olimpia et al

in Acta Veterinaria Scandinavica (2014), 56(4), 1-11

Background: This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from ... [more ▼]

Background: This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDSPAGE and N-terminal microsequencing. Results: Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues. Conclusion: Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family. [less ▲]

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See detailCharacteristics of Pregnancy-Associated glycoprotein (PAG) like proteins in red deer
Okuyama, MW; Melo de Sousa, Noelita ULg; Beckers, Jean-François ULg

Poster (2013, June 19)

The Pregnancy-Associated glycoproteins (PAGs) constitute an asparatic proteinase family and are divided into 2 groups; bovine PAG-1 (boPAG-1) group and boPAG-2 group. PAGs have been detected in many ... [more ▼]

The Pregnancy-Associated glycoproteins (PAGs) constitute an asparatic proteinase family and are divided into 2 groups; bovine PAG-1 (boPAG-1) group and boPAG-2 group. PAGs have been detected in many domestic ruminant species and their biochemical characteristics were reported. However, information in wild ruminant, especially in deer species is scarce. The aim of this study is to investigate the characteristics of placental protein extracted in red deer (cervus elaphus); placental proteins which belong to each type of PAG group were identified in purification steps. Fresh frozen placenta (fetal cotyledon (FC): 2,247 g, maternal cotyledon (MC): 2,255 g respectively) was used as following purification step; protein extraction, acid precipitation (pH 4.5), Ammonium sulfate precipitation (SA0-40%, SA40-80%) and anion exchange chromatography (DEAE-cellulose 0M, 0,02M, 0,04M, 0,08M, 0,16M and 0,32M NaCl). In every step, Immunoreactivity against anti-PAG antisera was checked by Radioimmunoassay with using anti-PAG-1 antiserum (AS706) and anti-boPAG-2 antiserum (AS438). After Ammonium sulfate precipitation, much higher concentrations were gained in SA40-80% step (AS706: 5.35 mg/FCkg and 6.73 mg/MCkg, AS438: 24.79 mg/FCkg and 84.6 mg/MCkg) than SA0-40% step (AS706: 0.64 mg/FCkg and 0.79 mg/MCkg, AS438: 1.3 mg/FCkg and 9.34 mg/MCkg) in both part of cotyledon. After DEAE chromatography, the highest concentrations of protein against AS438 were gained in 0.08M NaCl step (5.49 mg/FCkg and 17.8 mg/MCkg) and concentrations of protein against AS706 were almost same in 0.08M NaCl (1.01 mg/FCkg and 1.35 mg/MCkg) and 0.16M NaCl (0.93 mg/FCkg and 1.34 mg/MCkg). In many ruminants such as cattle, sheep and goat, PAGs are contained largely in fetal cotyledon and most amounts of PAGs are belonging to PAG-1 group. On the other hand, in red deer, much larger amount of PAGs was obtained from maternal cotyledon. And stronger immunoreactivity with anti-boPAG-2 antisera was detected than with anti-PAG-1 antisera. This characteristic is similar to the results which were reported in pig and dromedary. Therefore, these results suggest that PAGs in red deer have different characteristics from other ruminant species. [less ▲]

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See detailIntrafollicular LH administration in dairy heifers treated with a GnRH agonist
Mala, J; Beckers, Jean-François ULg; Melo de Sousa, Noelita ULg et al

in Veterinarni Medicina (2013), 58(2), 8186

The aim of this study was to evaluate the effect of intrafollicular treatment (IFT) with different doses of luteinising hormone. Experimental heifers were treated with a single deslorelin implant to ... [more ▼]

The aim of this study was to evaluate the effect of intrafollicular treatment (IFT) with different doses of luteinising hormone. Experimental heifers were treated with a single deslorelin implant to desensitise gonadotroph cells of the pituitary gland. Thereafter, follicular development was stimulated by exogenous FSH treatment. Intrafollicular treatment with 10, 5, 1 and 0.01 μg LH was performed on one single follicle while other follicles remained untreated. Human chorionic gonadotrophine (2000 UI) was administered intravenously as a control. Ovulation and development of the corpus luteum occurred after all intrafollicular treatments with 10 and 5 μg LH. After IFT using 1 μg of LH 75% animals (3/4) ovulated. The dose of 0.01 μg was not followed by any ovulation whereas control treatments with hCG were followed by an ovulation of the majority of follicles present in the ovaries. In conclusion, IFT with different doses of LH (greater than 0.01 μg) is capable of inducing ovulation. [less ▲]

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See detailProfiles of pregnancy-associated glycoprotein (PAG) concentrations during gestation in swamp buffalo
Van Hahn, N; Viet Linh, N; Melo de Sousa, Noelita ULg et al

in Proceedings of the 10th World Buffalo Congress and 7th Asian Buffalo Congress (2013)

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See detailPregnancy-Associated Glycoproteins (PAGs) in prolific (Boujaâd x D’man) and non- prolific (Boujaâd) Moroccan sheep by means of four radio-immunoassays
El Amiri, Bouchra; Melo de Sousa, Noelita ULg; El Abbadi, N et al

in Proceedings of the 23rd International Congress of Hungarian Association for Buiatrics (2013)

The aim of the present study was: i) to investigate the use of PAGs for pregnancy follow up in Boujaâd non prolific (n=8) vs. Boujaâd x D’man a prolific sheep (n=20), ii) to study whether the boPAG tracer ... [more ▼]

The aim of the present study was: i) to investigate the use of PAGs for pregnancy follow up in Boujaâd non prolific (n=8) vs. Boujaâd x D’man a prolific sheep (n=20), ii) to study whether the boPAG tracer (daily available at the laboratory of physiology of reproduction, Liège, could be used in RIA without affecting the results, iii) to prospect the possibility of transferring the technique to a Moroccan laboratory. The blood samples were weekly collected from the first week of gestation till the 4 week after lambing. The RIA1, RIA2, RIA3 and RIA4 were all based on ovPAG standard, while the tracer and antiserum changed according to the system. The RIA1, RIA3 and RIA4 were performed in Belgium while the RIA2 was carried out in Morocco. The results showed that the highest correlation was obtained between the RIA3 and RIA4 (R2 = 0.83) while the lowest was recorded between the RIA2 and RIA4 (R2 = 0.69). The lowest concentrations derived from the RIA2. The highest concentrations were recorded before lambing (279.29; 88.21; 268.75; 152.67 ng/ml respectively for RIAs 1, 2, 3 and 4). To conclude, the profiles of ovPAG showed that it is possible to flow-up with success using the four systems. In addition, the RIA1 using boPAG as tracer could be a good alternative to the homologous one and finally, it is possible to set up routinely the RIA technique for PAG detection in Moroccan laboratories. [less ▲]

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See detailPregnancy-associated glycoproteins in sheep: breeds, techniques and achievements
El Amiri, Bouchra; Melo de Sousa, Noelita ULg; Szenci, Otto et al

in Proceedings of the 23rd International Congress of Hungarian Association for Buiatrics (2013)

The aim of the present review is to summarize the achievements on pregnancy-associated glycoproteins in sheep during the last 20 years on terms of results obtained after purifications, molecular biology ... [more ▼]

The aim of the present review is to summarize the achievements on pregnancy-associated glycoproteins in sheep during the last 20 years on terms of results obtained after purifications, molecular biology, radio-immunoassays (RIA), enzyme linked immune sorbent assay (ELISA), double radial immune-diffusion (Ouchterlony) and Western blot techniques. The analysis of documented papers that concerned these attempts showed that the animal genetic material used was composed of variable breeds from north Africa, Europe and USA (the Aragonesa, Ouled Djellal, Laucaun drairy sheep, Berrichone, Texel x Norvegian crossed ewes, Awassi x Merino, Blackheaded Moutton sheep x Blackheaded German Mutton sheep (BGM), Rhoen sheep crosses of BGM with Dorper sheep, Assaf, Chura, Merino, Texel, Suffolk, Boujaâd Moroccan breed). The RIA represents the wildspread technique used (54%) while Oucherlony and Western blot had been limited in use (4% each). From 1990 to 2000 only 27% of the achievements being available today were realized. The main data, profiles and studies were performed from 2000 to now (63%). To conclude, this review allows summarizing and updating the knowledge about the PAG in sheep. [less ▲]

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See detailSerum albumin [Fragment] from Dama dama placenta - Accession number C0HJD2
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailAlpha-2-HS-glycoprotein [Fragment] from Dama dama placenta - Accession number C0HJD1
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailPregnancy-Associated Glycoprotein D-56 kDa [Fragment] from Dama dama placenta - Accession number C0HJD0
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailPregnancy-Associated Glycoprotein C-39 kDa [Fragment] from Dama dama placenta - Accession number C0HJC9
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailPregnancy-Associated Glycoprotein B-56 kDa [Fragment] from Dama dama placenta - Accession number C0HJC8
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailPregnancy-Associated Glycoprotein A-62 kDa [Fragment] from Dama dama placenta - Accession number C0HJC7
Bériot, Mathilde ULg; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia et al

E-print/Working paper (2013)

N-terminal microsequence obtained after purification and characterization of placental proteins in Dama dama (fallow deer). Proteins were submitted to SwissProt databank.

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See detailPlasma concentrations of pregnancy-associated glycoproteins measured using anti-bovine PAG-2 antibodies on Day 120 of gestation predict abortion in dairy cows naturally infected with Neospora caninum.
Garcia-ispierto, I; Almeria, S; Serrano, B et al

in Reproduction in Domestic Animals (2013), 48

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy-associated glycoprotein-2 (PAG-2) concentrations throughout pregnancy and (ii ... [more ▼]

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy-associated glycoprotein-2 (PAG-2) concentrations throughout pregnancy and (ii) whether plasma PAG-2 concentrations could predict abortion in N. caninum-infected cows. The study was performed on a commercial Holstein-Friesian dairy herd in northeastern Spain and the final data included those recorded in 53 non-aborting and 19 aborting animals. Blood samples were collected immediately before pregnancy diagnosis (on Days 40, 90, 120, 150, 180 and 210 post-insemination) in non-aborting cows or until the time of abortion detection in aborting cows. General lineal models (GLM) repeated measures ANOVA revealed the different behavior of PAG-1 and PAG-2, and significant effects of Neospora seropositivity, cool season and twin pregnancy on plasma PAG-2 concentrations throughout gestation (between-subject effects). In addition, based on the odds ratios, the likelihood of abortion increased in Neospora-seropositive cows (by a factor of 7.0) compared to seronegative animals and decreased in cows with a high plasma PAG-2 concentration (>4.5 ng/ml) on Day 120 of pregnancy (by a factor of 0.24), compared to the remaining cows. In conclusion, there is a relationship between plasma PAG-2 concentrations and the risk of abortion in Neosporainfected dairy cows. Thus, plasma PAG concentrations measured using anti-boPAG-2 antiserum on Day 120 of gestation could serve as an indicator of the abortion risk in N. caninum infected animals; values <4.5 ng/ml indicating a high risk of abortion in chronically infected animals. [less ▲]

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See detailPurification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.
Barbato, Olimpia; Melo de Sousa, Noelita ULg; Barile, Vittoria Lucia et al

in BMC Veterinary Research (2013), 9

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of ... [more ▼]

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n = 33) and non-pregnant (n = 26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards. [less ▲]

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See detailMonitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse
Bohak, Zs; Szabo, F.; Beckers, Jean-François ULg et al

in Domestic Animal Endocrinology (2013), 45(1), 38-42

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was ... [more ▼]

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was performed to definitively establish options for testing adrenocortical function. To the best of our knowledge, this is the first study to evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during a 24-h period. Twenty horses were examined under the same conditions. Blood and saliva samples were taken every 2 h for 24 h to determine the daily changes in cortisol concentrations of saliva and serum at rest and to determine the relationship between salivary and serum cortisol levels. Cosinor analysis of group mean data confirmed a significant circadian component for both serum and salivary cortisol concentrations (P < 0.001 in both cases). The serum cortisol circadian rhythm had an acrophase at 10:50 AM (95% CI, 10:00 AM-11:40 AM), a MESOR of 22.67 ng/mL, and an amplitude of 11.93 ng/mL. The salivary cortisol circadian rhythm had an acrophase at 10:00 AM (95% CI, 9:00 AM-11:00 AM), a MESOR of 0.52 ng/mL, and an amplitude of 0.12 ng/mL. We found a significant but weak association between salivary and serum cortisol concentrations; the Pearson correlation coefficient was 0.32 (P < 0.001). The use of salivary cortisol level as an indicator of hypothalamic-pituitary-adrenal axis activity may be warranted. However, the salivary cortisol levels are more likely to be correlated with free plasma cortisol than with the total plasma cortisol concentration. [less ▲]

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See detailRelationship between plasma progesterone and pregnancy-associated glycoprotein concentrations during early pregnancy in dairy cows
Barbato, O; Merlo, M; Celi, P et al

in Veterinary Journal (2013)

The relationship between the concentration of plasma progesterone (P4) during embryo attachment or at recognition of pregnancy, and that of pregnancy-associated glycoprotein (PAG) was assessed in dairy ... [more ▼]

The relationship between the concentration of plasma progesterone (P4) during embryo attachment or at recognition of pregnancy, and that of pregnancy-associated glycoprotein (PAG) was assessed in dairy cows. The outcome of artificial insemination (AI) was classified as positive (AI+), negative (AIÿ), or late embryonic mortality (EM) by measuring circulating PAG concentrations and by ultrasonography. Based on P4 concentrations at either day 21 or day 15, AI+ and EM cows were classified into ‘low’ (P4 concentrations < mean) and ‘high’ (P4 concentrations > mean) P4 groups. In both experiments, the threshold of P4 concentration between the ‘low’ and ‘high’ groups was approximately 6 ng/mL. PAG concentrations were lower in the ‘low’ group only when P4 concentrations were below the threshold. The study findings suggest that a possible P4 threshold exists below which PAG secretion may be impaired [less ▲]

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See detailPlacental and gastric aspartic proteinases: new insights from bovine species
Bella, Amina ULg; Beckers, Jean-François ULg; Melo de Sousa, Noelita ULg

in Proceedings of the 2nd Scientific Meeting of the Faculty of Veterinary Medicine (2012, October 19)

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See detailAssessment of pregnancy-associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera.
Nguyen, V. H.; Barbato, O.; Bui, X. N. et al

in Animal Science Journal (2012), 83(10), 683-689

Pregnancy-associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to ... [more ▼]

Pregnancy-associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three RIA-PAG systems: RIA-1 (raised against bovine PAG67kDa; AS#497), RIA-2 (raised against caprine PAG55+62kDa; AS#706) or RIA-3 (raised against buffalo PAG; AS#859). Samples were collected at slaughterhouse (n = 67). PAG concentrations determined by RIA-2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/ml and 24.0 ± 7.3 ng/ml, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA-2 (21.8 ± 2.4 ng/ml and 20.2 ± 2.5 ng/ml, respectively) and RIA-3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/ml, respectively) were higher than those obtained by RIA-1 (15.5 ± 1.4 ng/ml and 16.1 ± 1.8 ng/ml, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of PAG-RIA systems to determine PAG concentration in swamp buffalo samples. [less ▲]

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See detailPregnancy-associated glycoprotein, chymosin and pepsinogen immunoreactivity of proteins extracted from fetal gastric tissue in bovine species.
Bella, Amina ULg; Melo de Sousa, Noelita ULg; Dehimi, M. L. et al

in Research in Veterinary Science (2012), 92(3), 378-386

The objective of this work was to investigate the expression of gastric aspartic proteinases in fundic and pyloric mucosa removed from bovine fetuses. For this purpose, fractions issued from classical ... [more ▼]

The objective of this work was to investigate the expression of gastric aspartic proteinases in fundic and pyloric mucosa removed from bovine fetuses. For this purpose, fractions issued from classical biochemical protocols were analyzed by proteolytic method, by PAG-RIA and by Western blot with the use of antisera raised against both pepsinogens and PAG. A strong reaction of proteins extracted from the fundic mucosa collected at the beginning of pregnancy was revealed with both anti-bPAG-I and anti-bPAG-II antisera, suggesting the expression of pepsinogen F in bovine species. Concerning pyloric mucosa, the analysis by Western blot highlighted a very strong immunoreaction with the anti-bovine chymosin serum. Amino-terminal sequencing allowed to identify bovine fetuin and albumin in fundic extracts, chymosin in the pyloric mucosa extracts, as well as some unknown proteins in both mucosa. Despite no N-terminal microsequence corresponding to the hypothetical pepsinogen F could be identified, it cannot be excluded that an existing bovine pepsinogen F-like molecule could be degraded during the purification procedure or that co-purified proteins could be responsible for masking its N-terminal microsequence. [less ▲]

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