Immunization with human thyrotrophin receptor peptide induces an increase in thyroid hormone in rabbits

1992 ◽  
Vol 135 (3) ◽  
pp. 479-484 ◽  
Author(s):  
M. Ohmori ◽  
T. Endo ◽  
M. Ikeda ◽  
T. Onaya
Keyword(s):  
Amino Acid ◽  
Thyroid Hormone ◽  
Western Blot ◽  
Synthetic Peptide ◽  
Matched Control ◽  
Tsh Receptor ◽  
Amino Acid Residues ◽  
Normal Range ◽  
Tsh Receptor Antibody ◽  
Peptide Antibodies

ABSTRACT Eight rabbits were immunized with a synthetic peptide corresponding to the unique N-terminal region (termed N peptide; amino acid residues 29–57) in the extracellular domain of the human thyrotrophin (TSH) receptor. After 10 weeks, all of the eight rabbits produced anti-N peptide antibodies. Western blot analysis revealed that the antibodies recognized rabbit TSH receptor as an approximately 100 kDa protein. We compared the level of thyroid hormone in serum taken before immunization (preimmune sera) with that of serum taken after immunization (postimmune sera) in these immunized rabbits. Postimmune sera from the eight rabbits had higher mean (± s.d.) levels of tri-iodothyronine (T3) and thyroxine (T4) than did preimmune sera (T3, preimmune 0·82 ± 0·26 μg/l vs postimmune 1·33 ± 0·35, P < 0·01; T4, preimmune 33·7 ± 10·0 μg/l vs postimmune 41·0 ± 6·0, P < 0·05). T3 levels in four rabbits and T4 levels in four rabbits after immunization were over the normal range obtained from six age-matched control rabbits. Seven rabbits exhibited thyroid-stimulating antibody (TSAb) activity with various degrees (241–545%). The concentration of T3 and T4 did not increase over 10 weeks in either non-immunized rabbits (T3, preimmune 0·89 ± 0·34 μg/l vs postimmune 0·82 ± 0·22; T4, preimmune 31·1 ± 7·3 μg/l vs postimmune 30·3 ± 5·1) or other peptide-immunized rabbits (T3, preimmune 0·68 μg/l (n = 2) vs postimmune 0·69; T4, preimmune 33·1 μg/l vs postimmune 26·4). These results indicate that experimentally produced anti-TSH receptor antibody with TSAb activity induces an increase in thyroid hormone in rabbits. Journal of Endocrinology (1992) 135, 479–484

Broad spectrum pan-cadherin antibodies, reactive with the C-terminal 24 amino acid residues of N-cadherin

1990 ◽  
Vol 97 (4) ◽  
pp. 607-614
Author(s):  
B. Geiger ◽  
T. Volberg ◽  
D. Ginsberg ◽  
S. Bitzur ◽  
I. Sabanay ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Amino Acid ◽  
Western Blot ◽  
Cardiac Muscle ◽  
Broad Spectrum ◽  
Synthetic Peptide ◽  
Adherens Junctions ◽  
Cross Reactivity ◽  
Amino Acid Residues ◽  
C Terminus

We describe here the preparation and application of antibodies directed against a synthetic, 24 amino acid long, peptide corresponding to the conserved cytoplasmic C terminus of N-cadherin. We demonstrate here that the antibodies to the synthetic peptide react extensively with all known members of the cadherin family and, in addition, recognize novel cadherins in a variety of cells and tissues, suggesting that these antibodies indeed exhibit pan-cadherin reactivity. By Western blot screening of chicken tissues at least 4 different immunoreactive bands were resolved, commonly disclosing 2–3 distinct bands within the same tissue. The pan-cadherin antibodies also displayed a broad interspecies cross reactivity, recognizing cadherins in man, bovine, canine, avian, amphibian and teleost cells. This property renders these antibodies excellent reagents for the cloning and identification of novel cadherins. Immunocytochemical labelling with the pan-cadherin antibodies, at the light- and electron-microscope levels, revealed an extensive reactivity with intercellular adherens junctions in cardiac muscle and in various epithelia. We thus propose that the pan-cadherin antibodies may be used as ubiquitous cadherin probes and serve as markers for adherens junctions.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Plasmin-platelet interaction involves cleavage of functional thrombin receptor

1996 ◽  
Vol 271 (1) ◽  
pp. C54-C60 ◽  
Author(s):  
M. Kimura ◽  
T. T. Andersen ◽  
J. W. Fenton ◽  
W. F. Bahou ◽  
A. Aviv
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Platelet Activation ◽  
Synthetic Peptide ◽  
Time Dependent ◽  
Thrombin Receptor ◽  
Amino Acid Residues ◽  
Enzymatic Action ◽  
High Concentrations ◽  
Inhibition Of Thrombin

We tested the hypothesis that the inhibition of thrombin-induced platelet activation by plasmin is mediated via the enzymatic action of plasmin on the functional thrombin receptor. We monitored the binding of the anti-thrombin receptor antibody [anti-TR-(34-46)] to platelets; this binding is sensitive to the cleavage of the thrombin receptor at amino acid residues Arg-41 to Ser-42. Plasmin inhibited anti-TR-(34-46) binding in dose- and time-dependent manners. The inactive synthetic peptide with the amino acid sequence 40-55 of the thrombin receptor (D-FPRSFLLRNPNDKYEPF) was similarly cleaved by thrombin and plasmin to an active peptide (SFLLRNPNDKYEPF) that produced robust cytosolic Ca2+ responses. At high concentrations, plasmin itself can activate platelets. We explored this effect with the use of anti-TR-(1-160). This antibody abolished the cytosolic Ca2+ responses to thrombin and to the thrombin receptor-activating peptide SFLLRN but did not attenuate the plasmin-induced cytosolic Ca2+ response. Thus plasmin inhibits thrombin-evoked platelet activation by cleaving the thrombin receptor, but the plasmin-induced cytosolic Ca2+ response is not due to the generation of the tethered peptide of the thrombin receptor.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

1999 ◽  
Vol 276 (2) ◽  
pp. R627-R631 ◽  
Author(s):  
Carles Garriga ◽  
Nativitat Rovira ◽  
Miquel Moretó ◽  
Joana M. Planas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Brush Border ◽  
Synthetic Peptide ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

Immunolocalization ofTaenia soliumgap junction innexins

Parasitology ◽  
2008 ◽  
Vol 135 (9) ◽  
pp. 1125-1131 ◽  
Author(s):  
R. ZURABIAN ◽  
A. LANDA ◽  
L. ROBERT ◽  
K. WILLMS
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Small Molecules ◽  
Synthetic Peptide ◽  
Polyclonal Antibodies ◽  
Taenia Solium ◽  
Peptide Sequence ◽  
Rabbit Igg ◽  
Membrane Peptide ◽  
Cytoplasmic Glycogen

SUMMARYIn previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues ofTaenia soliumtapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adultT. soliumcontain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.

The human anion transport protein, band 3, contains a CD36-like binding domain forPlasmodium falciparum-infected erythrocytes

Parasitology ◽  
1996 ◽  
Vol 112 (3) ◽  
pp. 261-267 ◽  
Author(s):  
I. Crandall ◽  
I. W. Sherman
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Blood Cells ◽  
Synthetic Peptide ◽  
Epitope Mapping ◽  
Protein Band ◽  
Band 3 ◽  
Anion Transport ◽  
Transport Protein ◽  
Amino Acid Residues

SUMMARYEpitope mapping of a murine monoclonal antibody (mAb), 5H12, prepared against livePlasmodium falciparum-intected red blood cells indicated that the epitope consisted of amino acid residues 474–487 of the human anion transport protein, band 3. mAb 5H12 enhanced cytoadherence, but inhibited the CD36-like mediated resetting. A synthetic peptide based on the sequence of the epitope (FSFCETNGLE) blocked both resetting and cytoadherence, suggesting that this amino acid sequence may form the CD36-like receptor. The CD36-like region of band 3 was antigenically distinct from platelet or endothelial CD36.

scholarly journals Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins.

1990 ◽  
Vol 110 (1) ◽  
pp. 27-34 ◽  
Author(s):  
S J Gould ◽  
S Krisans ◽  
G A Keller ◽  
S Subramani
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Rat Liver ◽  
Synthetic Peptide ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal ◽  
Peptide Antibodies

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.

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