DEMONSTRATION OF THYROGLOBULIN IN LYMPH VESSELS OF TSH STIMULATED GUINEA PIGS BY THE INDIRECT IMMUNOFLUORESCENCE TECHNIQUE

1973 ◽  
Vol 74 (4) ◽  
pp. 666-674 ◽  
Author(s):  
W. Böcker ◽  
H. Lietz
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Double Diffusion ◽  
Diffusion Method ◽  
Second Step ◽  
Fluorescence Staining ◽  
Lymph Vessels ◽  
Indirect Immunofluorescence Technique

ABSTRACT The presence of thyroglobulin in intra- and extrathyroidal lymph vessels of TSH-stimulated guinea pigs was demonstrated immunohistochemically. Guinea pig thyroglobulin was purified by gel filtration on Sephadex G-200 and tested by the double diffusion method and by polyacrylamide gel electrophoresis. Antibodies to guinea pig thyroglobulin were obtained in rabbits by immunisation of the animals with this antigen. Indirect fluorescence staining was performed on formol fixed paraffin embedded tissue with rabbit anti-guinea pig thyroglobulin serum in the first step and fluorescein-labelled antirabbit IgG from the goat in the second step of the incubation process.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

Purification and antibacterial activity of recombinant human lactoferrin in milk of transgenic mice

2009 ◽  
Vol 6 (3) ◽  
pp. 253-256
Author(s):  
Miao Ming-Xing ◽  
Yuan Yu-Guo ◽  
An Li-You ◽  
Zhao Jun-Hui ◽  
Bai Ya-Jun ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Transgenic Mice ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Lactoferrin ◽  
Diffusion Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Human Lactoferrin ◽  
Agar Disc

AbstractTo verify its antibacterial activity, recombinant human lactoferrin (rhLF) was extracted from the milk of transgenic mice (Mus musculus) (PCL25and AP) by gel filtration chromatography and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA). In addition, its bacteriostatic properties were tested using the agar disc diffusion method. ELISA analysis showed that the concentration of rhLF in the milk of transgenic mice ranged from 7 to 8 mg/ml, and the recombinant protein expressed in the milk had the same molecular weight as the native protein (~78 kDa), indicating that the rhLFs had a strong antibacterial activity onEscherichia coliandSalmonella.

Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells

1996 ◽  
Vol 270 (6) ◽  
pp. L1002-L1007
Author(s):  
M. Mansour ◽  
M. Karmilowicz ◽  
S. J. Hawrylik ◽  
B. Nalcerio ◽  
J. Angilly ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Pcr Amplification ◽  
Airway Function

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

scholarly journals Purification and properties of guinea-pig submandibular-gland kallikrein

1983 ◽  
Vol 209 (1) ◽  
pp. 125-134 ◽  
Author(s):  
F Fiedler ◽  
M J C Lemon ◽  
C Hirschauer ◽  
G Leysath ◽  
F Lottspeich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Ethyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Purification And Properties ◽  
Order Of Magnitude

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

scholarly journals Purification and immunochemical characterization of malate synthase from Euglena gracilis

1978 ◽  
Vol 173 (1) ◽  
pp. 95-101 ◽  
Author(s):  
E Woodcock ◽  
M J Merrett
Keyword(s):  
Euglena Gracilis ◽  
High Speed ◽  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Double Diffusion ◽  
Ultrasonic Waves ◽  
Malate Synthase ◽  
Ph Optimum

Malate synthase (EC 4.1.3.2) from dark-grown Euglena gracilis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. The enzyme was released from acetate-grown cells by treatment with ultrasonic waves and purified from broken-cell suspensions by high-speed centrifugation and (NH4)2SO4 fractionation, followed by gel-filtration on Sepharose 6B. The final enzyme preparation was purified 190-fold compared with the crude extract. The mol.wt. of the enzyme was about 350000 as determined by gel filtration on Sepharose 6B. Treatment with sodium dodecyl sulphate and urea dissociated the enzyme into subunits of mol.wt. 175000. The pH optimum for the enzyme was 8.0 and the Km values for glyoxylate and acetyl-CoA were 50 and 80 micron respectively. Antibodies raised to the purified enzyme were shown to be monospecific by radiochemical immunoassay. Euglena anti-(malate synthase) tested on Ouchterlony double-diffusion gels gave a sharp precipitation band against acetate-grown Escherichia coli, but no immunological correspondence was observed with acetate-grown Chlorella fusca, Zea mays (maize) scutella or purified malate synthase from Ricinus communis.

Glycogen in guinea pig neutrophils: Ultrastructural study of neutrophils at the intradermal site of BCG injection

1983 ◽  
Vol 41 ◽  
pp. 726-727
Author(s):  
Corazon D. Bucana
Keyword(s):  
Bone Marrow ◽  
Guinea Pig ◽  
Electron Density ◽  
Peritoneal Cavity ◽  
Ultrastructural Study ◽  
Guinea Pigs ◽  
Random Distribution ◽  
Glycogen Content ◽  
Polymorphonuclear Neutrophils ◽  
Ultrastructural Studies

In the circulating blood of man and guinea pigs, glycogen occurs primarily in polymorphonuclear neutrophils and platelets. The amount of glycogen in neutrophils increases with time after the cells leave the bone marrow, and the distribution of glycogen in neutrophils changes from an apparently random distribution to large clumps when these cells move out of the circulation to the site of inflammation in the peritoneal cavity. The objective of this study was to further investigate changes in glycogen content and distribution in neutrophils. I chose an intradermal site because it allows study of neutrophils at various stages of extravasation.Initially, osmium ferrocyanide and osmium ferricyanide were used to fix glycogen in the neutrophils for ultrastructural studies. My findings confirmed previous reports that showed that glycogen is well preserved by both these fixatives and that osmium ferricyanide protects glycogen from solubilization by uranyl acetate.I found that osmium ferrocyanide similarly protected glycogen. My studies showed, however, that the electron density of mitochondria and other cytoplasmic organelles was lower in samples fixed with osmium ferrocyanide than in samples fixed with osmium ferricyanide.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

Inhibition of Human and Animal Platelet Adhesiveness to Glass Bead Columns by Adenosine, Dipyridamole, Chlorpromazine and Acetylsalicylic Acid

1976 ◽  
Vol 36 (02) ◽  
pp. 401-410 ◽  
Author(s):  
Buichi Fujttani ◽  
Toshimichi Tsuboi ◽  
Kazuko Takeno ◽  
Kouichi Yoshida ◽  
Masanao Shimizu
Keyword(s):  
Guinea Pig ◽  
Acetylsalicylic Acid ◽  
Guinea Pigs ◽  
Glass Bead ◽  
Animal Species ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Platelet Adhesiveness

SummaryThe differences among human, rabbit and guinea-pig platelet adhesiveness as for inhibitions by adenosine, dipyridamole, chlorpromazine and acetylsalicylic acid are described, and the influence of measurement conditions on platelet adhesiveness is also reported. Platelet adhesiveness of human and animal species decreased with an increase of heparin concentrations and an increase of flow rate of blood passing through a glass bead column. Human and rabbit platelet adhesiveness was inhibited in vitro by adenosine, dipyridamole and chlorpromazine, but not by acetylsalicylic acid. On the other hand, guinea-pig platelet adhesiveness was inhibited by the four drugs including acetylsalicylic acid. In in vivo study, adenosine, dipyridamole and chlorpromazine inhibited platelet adhesiveness in rabbits and guinea-pigs. Acetylsalicylic acid showed the inhibitory effect in guinea-pigs, but not in rabbits.

Comparative Study of Proactivators of the Fibrinolysin System in Three Mammalian Species

1969 ◽  
Vol 21 (03) ◽  
pp. 594-603 ◽  
Author(s):  
Y Takada ◽  
A Takada ◽  
J. L Ambrus
Keyword(s):  
Guinea Pig ◽  
Comparative Study ◽  
Human Plasma ◽  
Mammalian Species ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
The Other ◽  
Rabbit Plasma ◽  
Plate Test ◽  
Fibrin Plate

SummarySephadex gel filtration of human plasma gave results suggesting the presence of two proactivators of plasminogen, termed proactivators A and B.Activity resembling that of proactivator A was found in rabbit plasma, but not in guinea pig plasma.Plasminogen activators produced by the interaction of proactivator A of human plasma with streptokinase had no caseinolytic or TAMe esterolytic effect.Proactivator A can be separated in a form apparently free from plasminogen, as shown by the heated fibrin plate test and by immunological analysis. On the other hand, proactivator B concentrates prepared so far are contamined with plasminogen.Human proactivators appear to be far more susceptible to streptokinase than are rabbit proactivators.Inhibitors of the fibrinolysin system were observed in the plasmas of all 3 species. These inhibitors are not present in the euglobulin fraction of plasma. Sephadex fractionation of euglobulin fractions results in proactivator preparations that do not contain inhibitors.

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