Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN-gamma receptor gene.

To investigate the role of interferon-gamma (IFN-gamma) in experimental autoimmune thyroidits (EAT), H-2k mice with a disrupted IFN-gamma receptor (IFN-gamma R) gene were immunized with porcine thyroglobulin (pTg). We observed that EAT occurred on day 19 and remitted on day 35 in IFN-gamma R-deficient (IFN-gamma R(0/0)) mice, whereas in wild-type mice, EAT occurred on day 21 and remitted on day 42-49. Moreover, EAT in the mutant mice was attenuated and accompanied by diminished Tg-specific cytotoxic and proliferative responses and decreased titers of anti-Tg antibodies, notably of the IgG2a and IgG2b isotypes. In contrast, Tg-specific IgG1 was increased in the IFN-gamma R(0/0) mice. In supernatants from T cells further stimulated in vitro by Tg, IFN-gamma levels were higher in IFN-gamma R(0/0) than in wild-type mice throughout the course of the disease, whereas interleukin-10 was transiently increased prior to EAT onset in both groups of mice. Finally, using IFN-gamma R(0/0) mice, we demonstrate that induction of EAT does not require an intact IFN-gamma system, while progression to full-blown disease depends on the action of IFN-gamma.

Electroblotting proteins onto carboxymethylcellulose membranes for sequencing.

Proteins and peptides separated by polyacrylamide gel electrophoresis either in acetic acid-urea or in sodium dodecyl sulfate (SDS)-containing gels were electroblotted onto carboxymethylcellulose membrane and eluted from the membrane by dilute acid. Polypeptides thus recovered with a high yield can be used to get structural or biological data.

Amino acid sequence of the human intermediate basic protein 2 (HPI2) from sperm nuclei. Structural relationship with protamine P2.

Human intermediate basic protein 2 (HPI2) is a low-molecular-mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N-terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys-C and Glu-C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N-terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues. The amino acid sequence of HPI2 (81 residues) is identical to the sequence of the C-terminal region of another minor sperm nuclear protein, human intermediate basic protein 1 (HPI1, 101 residues), which was sequenced previously [Martinage, A., Arkhis, A., Alimi, E., Sautière, P. & Chevaillier, P. (1990) Eur. J. Biochem. 191, 449-451]. Due to this structural similarity, HPI2 must be considered as an intermediate in the maturation of proprotamine HPI1 limited proteolysis.

[Purification and characterization of precursors of mouse protamine mP2]. / Purification et caractérisation des précurseurs de la protamine mP2 de Souris.

Mouse protamine 2 (62 residues) is synthesized as a precursor pmP2 (106 residues) which is subsequently processed during the end of spermiogenesis. Three proteins with molecular and electrophoretic properties similar to those of pmP2 were isolated from testis purified nuclei. The comparison of their amino acid composition and of their N-terminal sequence with those of pmP2 shows that the three isolated proteins are products of pmP2 precursor processing. The three intermediate proteins pmP2/5, pmP2/11 and pmP2/20 which contain 102, 96 and 87 residues respectively, are generated from pmP2 precursors after N-terminal excision of 4, 10 and 19 residues, respectively.

Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3.

The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.