Antibody production in milk serum after antigen instillation of the goat mammary gland. IX. Sham infection studies with Neisseria meningitidis L.C.D.C. 608B

1975 ◽  
Vol 21 (5) ◽  
pp. 662-667 ◽  
Author(s):  
Arthur E. Pasieka ◽  
G. Calver ◽  
C. P. Kenny ◽  
C. Perusse ◽  
L. F. Guerin ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Neisseria Meningitidis ◽  
Large Scale ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Goat Milk ◽  
Scale Production ◽  
Strain Isolation ◽  
Lactating Mammary Gland ◽  
Immune Globulins

Specific immune globulins have been prepared in goat milk in response to the intramammary gland instillation of Neisseria meningitidis 608, serogroup B strain. Isolation, purification, and characterization of the goat whey by gel filtration, electrophoresis, and analytical ultracentrifugation demonstrated that the active immune component resided in the IgA class of globulins, specifically 9.2-S IgA. The potential of the lactating mammary gland as a "biological factory" for the large-scale production of diagnostic antiserum to killed bacterial whole cell antigen is described.

Antibody production in milk serum of goats experimentally infected with Neisseria gonorrhoeae

1976 ◽  
Vol 22 (8) ◽  
pp. 1113-1119 ◽  
Author(s):  
A. E. Pasieka ◽  
F. E. Ashton ◽  
R. Wallace ◽  
F. Ota ◽  
A. Ryan ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Antibody Response ◽  
Neisseria Gonorrhoeae ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Goat Milk ◽  
Purification And Characterization ◽  
Colony Type ◽  
Milk Serum

Instillation of the goat's mammary gland with Neisseria gonorrhoeae colony type Tl has elicited an antibody response in the goat milk serum (GMS). Purification, and characterization of the GMS by gel filtration, electrophoresis, immunodiffusion, analytical ultracentrifugation. And serological analyses demonstrated that the active immune component was mainly in the IgA and IgG fractions (F2 and F3) of GMS.

scholarly journals Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose

1978 ◽  
Vol 171 (1) ◽  
pp. 137-141 ◽  
Author(s):  
F Auricchio ◽  
A Rotondi ◽  
P Sampaolo ◽  
E Schiavone
Keyword(s):  
Mammary Gland ◽  
Oestrogen Receptor ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Large Aggregate ◽  
Low Salt ◽  
Lactating Mammary Gland ◽  
Stokes Radius

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

scholarly journals Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose

1979 ◽  
Vol 178 (3) ◽  
pp. 581-587 ◽  
Author(s):  
A Rotondi ◽  
F Auricchio
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Large Scale ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Receptor Aggregation ◽  
Low Salt ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In ‘low-salt’ buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

Antibody production in goat milk serum after virus instillation of goat mammary gland. VII. Biochemical studies on sham infection with rabies and other non-propagating agents

1975 ◽  
Vol 21 (5) ◽  
pp. 655-661 ◽  
Author(s):  
Arthur E. Pasieka ◽  
L. F. Guerin ◽  
Chas. A. Mitchell
Keyword(s):  
Mammary Gland ◽  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Goat Milk ◽  
Neutralizing Antibody ◽  
Antibody Activity ◽  
Gel Chromatography ◽  
Milk Serum

This report describes the development of a method for the production and isolation of neutralizing antibodies against rabies virus (ERA strain) and other agents in the goat mammary gland during active lactation. The rabies virus did not propagate in the gland, but neutralizing antibodies were produced by serial instillations of the antigen. This process was termed 'sham infection.' Antibodies first appeared in the milk about the 20th day and the titer increased until the 28th day. The antibodies were found to be associated with the lactogammaglobulin. The milk globulins were fractionated by gel chromatography and the fraction containing the antibody was isolated. This fraction was further purified and characterized by immunodiffusion, immunoelectrophoresis, and an analytical ultracentrifugation technique. The neutralizing antibody activity after the 29th day was associated with the milk serum IgG fraction. This active fraction was found to have a sedimentation coefficient of 6.8 Svedberg units.

Purification and Characterization of Activation Fragments F1 and F2 from Human Prothrombin

1975 ◽  
Author(s):  
A. P. Ball ◽  
J. Fenton ◽  
D. L. Aronson ◽  
R. B. Franza ◽  
A. M. Young
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Scale Production ◽  
Purification And Characterization ◽  
Large Scale Production ◽  
Human Thrombin ◽  
A Chain

Considerable quantities of the non-thrombin portions of human prothrombin (II) have become available as a byproduct of the large-scale production of human thrombin (Biochim. Biophys. Acta 229, 26). Components not adsorbed on CG-50 are further purified by DEAE-cellulose chromatography and gel filtration, yielding the NH2-terminal fragment (F1) and the inner fragment (F2) which are homogeneous by SDS gel electrophoresis. SDS gel electrophoresis of reduced F1 indicates variable amounts of a two-chain derivative, F1’, with one chain migrating just ahead of F1 and one just ahead of the thrombin A-chain. The F1 → F1’ conversion is catalyzed by thrombin with the creation of a new MH2-terminal threonine. Ultracentrifugal patterns of human F1 and F2 closely resemble those of the bovine fragments. NH2-terminal residues were found to be alanine (± threonine) for F1 and serine for F2. Minor deviations from the reported amino acid compositions of bovine F1 and F2 were observed, primarily in the acidic residues. Other properties include:Immunization of rabbits with F1 gave a precipitating antibody to F1 which cross-reacts with II, but native F2 does not appear to be immunogenic. 3H-F1 is rapidly cleared from the blood of rabbits (T 1/2 20 min), with a major portion detectable in the urine.

Large-scale preparation and characterization of recombinant ovine placental lactogen

1997 ◽  
Vol 152 (2) ◽  
pp. 317-327 ◽  
Author(s):  
E Sakal ◽  
C Bignon ◽  
J Grosclaude ◽  
A Kantor ◽  
R Shapira ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Large Scale ◽  
Stop Codon ◽  
Gel Filtration ◽  
Resonance Method ◽  
Biologically Active ◽  
Placental Lactogen ◽  
Expression Plasmid ◽  
Kinetic Measurements ◽  
High Level

Abstract To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5′ and 3′ primers containing, respectively, Ncol and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(−1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into Ncol/PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-β-d-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30–40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate β-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors. Journal of Endocrinology (1997) 152, 317–327

311 RISK ANALYSIS OF ALPHA-LACTALBUMIN TRANSGENE TRANSFER TO NON-TRANSGENIC CONTROL ANIMALS DURING REARING AND BREEDING

2009 ◽  
Vol 21 (1) ◽  
pp. 252
Author(s):  
M. B. Wheeler ◽  
W. L. Hurley ◽  
S. Lane ◽  
J. Mosley ◽  
G. E. Bressner ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Mammary Gland ◽  
Large Scale ◽  
Initial Step ◽  
Scientific Data ◽  
Bodily Fluids ◽  
Transgenic Livestock ◽  
Lactating Mammary Gland ◽  
Transgenic Swine

Assessment of general risk posed from transgenic (T) animals is important to their future contributions to society. Identification of potentially harmful properties of transgenic livestock is the initial step in a risk assessment. Direct and indirect impacts of potential harmful properties of T livestock need to be evaluated at 3 levels, namely (1) characterization of how the transgene, its product, and the T livestock behave in their immediate environment, that is, in their barn or pen; (2) determination of possible impacts of large-scale release of T livestock, that is, if they were to be integrated into the larger population of food animal livestock; and (3) determination of the more complex environmental and safety consequences of their release into livestock populations. We previously developed and characterized transgenic swine containing a mammary-specific transgene (bovine α-lactalbumin, bALAC) that results in increased milk production in sows. We currently are determining whether bALAC is expressed in tissues of T swine other than the lactating mammary gland and whether the transgene DNA (Tg) crosses into non-transgenic control (C) swine under various physiological and physical conditions. The specific aims addressed in the present study were to determine: (1) whether the Tg can be transferred directly from T animals to C animals by physical association or contact and (2) whether the Tg can be transferred directly from an adult T animal to an adult C animal via mating. The T animals utilized in these studies are in at least generation 10 and have stable incorporation of the Tg. Comparable age- and weight-matched animals, T and C, were housed together allowing for general contact that is normal within swine production, for either 180, 220, or 250 d of age after weaning. Swine due to their behavior ingest saliva, regurgitated food, and stool or urinary products, as well as other bodily fluids and cells during normal housing. In a second study, vaginal, cervical, uterine, oviductal, and ovarian tissues from C females on 2, 7, or 90 d after mating to T males and penis, bulbourethral gland, urethra, testis, and epididymis tissues from C males on 2 or 7 days after mating to Tg females were collected. The presence of Tg in tissues from all C animals was tested via PCR. We have analyzed for the presence of the Tg in various tissues [including mammary gland, salivary gland, skin (sebaceous gland), muscle, lung, liver, kidney, brain, ovary, oviduct, uterus, cervix, vagina, penis, bulbourethral gland, urethra, testis, epididymis, and intestine]. Results indicate no presence of the Tg in tissues of C animals (n = 28) after co-habitation for 180, 220, or 250 d (n = 305 samples analyzed) or at 2 (n = 5), 7(n = 14), or 90 (n = 2) d post-mating (n = 60, 174, or 24 samples analyzed, respectively). The present results suggest that there is no horizontal Tg transmission between T and C pigs due to rearing or mating. This work provides a critical step toward providing rigorous scientific data for risk assessment of transgenic livestock. This project supported by the USDA BRAG Project #2005-03799.

Expression, Purification and Characterization of the Recombinant Hirudin Variant iii in the Bacillus Subtilis

2011 ◽  
Vol 343-344 ◽  
pp. 753-763 ◽  
Author(s):  
Hua You Chen ◽  
Xiang Hui Qi ◽  
Xu Geng ◽  
Qing Gang Xu ◽  
Jing Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Anion Exchange ◽  
Recovery Rate ◽  
Large Scale ◽  
Scale Up ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Scale Production ◽  
Recombinant Hirudin

Hirudin is the most potent natural inhibitor of thrombin and a powerful anticoagulant. Large-scale production of recombinant hirudin is desirable for therapy. In this study, the gene encoding hirudin variant III was redesigned and synthesized by usingBacillus subtilispreferred codons, and the recombinant hirudin variant III (rHV3) was overexpressed inB. subtilisDB403 with strong anticoagulation activity for the first time. The hirudin activity from the supernatant of culture with optimized expression conditions could reach 210 ATU/ml. The protein in culture supernatant was precipitated by trichloroacetic acid, then desalted by ultrafiltration and purified by anion exchange chromatography. Strong anion Q F.F. performed better than weak anion DEAE F.F. The proper pH and conductivity was determined at pH 8 and 6 ms/cm, respectively. The maximum applied sample was 240 ATU/ml to medium of strong anion Q F.F. This optimized procedure was employed in strong anion exchange HiPrep 16/10Q with the 90% recovery rate and 70.2% purity. After gel filtration, the purity of rHV3 checked by HPLC could reach 95.1%, and the recovery rate was 93% for this step. The purified recombinant rHV3 showed a single band in SDS-PAGE. The rHV3 was stable at 100 °C and acidity condition, but was unstable under the condition of both heating and alkalinity. In conclusion, theses studies suggests thatB.subtilismight be useful for the production of biologically active medicine peptides in secretion facilitating purification procedures, and that this isolation method was suitable for scale-up purification process at a low cost.

scholarly journals Enhanced production of inulinase from Xanthomonas campestris pv. phaseoli

2010 ◽  
Author(s):  
◽  
Kameshnee Naidoo
Keyword(s):  
Xanthomonas Campestris ◽  
Large Scale ◽  
Specific Activity ◽  
Carbon Sources ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Scale Production ◽  
Inulin Hydrolysis ◽  
Enhanced Production ◽  
Good Potential

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase on various carbon sources. The highest inulinase production of 9.24 ± 0.03 IU ml¯¹by X. campestris pv. phaseoli was attained using an optimized medium comprising of 3% sucrose and 2.5% tryptone. Inulinase production in X. campestris pv. phaseoli was further enhanced through ethylmethanesulfonate mutagenesis. The resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated enhanced inulinase production of 22.09 ± 0.03 IU ml¯¹after 24 h, which was 2.4 – fold higher than that of the wild type. Inulinase production by this mutant was scaled up in a 5 L fermenter yielding a final activity of 21.87 ± 0.03 IU ml¯¹with an inulinase/invertase (I/S) ratio of 2.6 after 18 h. Maximum volumetric (21 865 IU 1¯¹ h¯¹) and specific (119 025 IU g¯¹ h¯¹) productivities of inulinase were attained in a fermenter after 18h growth. Inulin hydrolysis by the crude inulinase and subsequent detection of mono- and oligosaccharides indicated the presence of an endoinulinase. The extracellular endoinulinase from the mutant KM 24 was purified to homogeneity by gel filtration chromatography and had a specific activity of 174.74U/mg. the optimum pH and temperature of the purified enzyme were found to be 6.0 and 50°C, respectively. The enzyme was stable up to 60°C, retaining over 60% activity for 30 min, but activity rapidly declined at temperatures above 60°C. The pure inulinase enzyme was also found to be stable between pH 6-9. The Lineweaver-Burk plots showed that the apparent Km and Vmax values of the inulinase for inulin were 1.15 mg/ml and 15µM/min, respectively. The Kcat value was found to be 0.145 min¯¹ with an enzyme catalytic efficiency of 0.126 mg¯¹.ml.min¯¹.This mutant demonstrated good potential for large scale production of inulinase and fructooligosaccharides.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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