Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc.

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA-Zn(II) column was observed for P1 protamines with one histidine in the N-terminal sequence (ram and boar protamines). The strong binding was found to be related to the presence of tyrosine, serine and threonine closely spaced to the histidyl side chain. In the case of human protamine P2, the strong retention on the IDA-Zn(II) column seems to result from the additive contribution of all the histidine residues of the molecule. Thus, strong retention of protamines in IMAC seems to depend on an additive contribution of amino-acid side chains: histidine, tyrosine, serine, threonine and perhaps arginine. The high affinity of protamines, more especially P2 protamines, for zinc suggests that this metal ion could play a role for their correct folding and binding to DNA.

Hydrodynamic characterization and DNA-binding properties of the liganded and unliganded forms of the retinoic-acid receptor alpha from human HL-60 cells.

The hydrodynamic parameters of the retinoic-acid receptor from human myeloblastic leukemia HL-60 cells were accurately investigated. The ligand-bound retinoic-acid receptor (RAR) has a Stokes radius of 3.5 nm when analyzed by size-exclusion chromatography. A 53-kDa protein was detected by Western blot analysis using a polyclonal antibody directed against the F domain of hRAR alpha, in the fractions containing the 3.5-nm complex. Fractionation of a crude nuclear extract from HL-60 cells, untreated with retinoic acid, yielded antibody-revealed material with Stokes radii ranging from 3.5 nm to 6 nm. From the hydrodynamic data, a molecular mass of 52 kDa was calculated for the liganded receptor, whereas no precise value could be deduced for the unliganded receptor form, since it dissociates rapidly into the 3.5-nm form. Gel-retardation experiments showed that the 3.5-nm form of hRAR alpha bound specifically to DNA, whereas binding of the unliganded receptor form was sharply reduced. These findings suggest that the unliganded inactive receptor form dissociates upon ligand binding and acquires a ligand-dependent DNA-binding activity.

Auto-antibodies to human sperm basic nuclear proteins in infertile and vasectomized men: characterization of antigens and epitopes recognized by antibodies.

The sera of vasectomized men and of patients with immune infertility were used to study the antigens and epitopes of sperm nuclear proteins that bind antibodies in these sera. No reaction with sperm histones was observed except for one serum. P1, P2 protamines and pro-P2 protamines were recognized by auto-antibodies. Studies with peptides derived from P1 and P2 protamines and with mammalian protamines related to HP1 showed that antibodies are mainly specific for a folded protamine molecule, more especially antibodies from vasectomized men. These results disagree with the random coil model proposed for protamines by several previous works. A cross-reactivity between P1 and P2 protamines was observed only for the whole molecules and not for peptides derived from them. This observation suggests that the two classes of protamines, different in sequence, may have a similar folding and thereby may be functionally equivalent.

The immune response to synthetic peptides of human protamines HP1 and HP2.

Peptides representing the amino-terminal sequence of protamines HP1 (sequence 1-12) and HP2 (sequence 1-11), the two major nuclear proteins of human sperm, have been synthesized. Rabbits were immunized either with peptide conjugated with a carrier or with free peptide. The resulting antisera were examined for their capacities to bind the homologous peptide, other peptides from protamines HP1, HP2, from ram protamine, a protein resembling HP1, and finally with the whole protamine. Only free peptides were immunogenic. Antisera were found to react with the homologous peptide, but also with some other peptides. More especially, antibodies to peptide HP1 1-12 were found to recognize an epitope shared by the homologous peptide, peptide HP1 37-49 and peptide 1-12 of ram protamine. The common antigenic determinant seems to depend on the conformation of the peptides, rather than strictly related to common sequences. Anti-peptide antibodies react poorly and in a non-specific manner with the parent protein. The failure of reactivity with the protamines strongly suggest that these small basic proteins are folded and probably globular molecules in contrast with the totally random model postulated by several previous works.

Pathological proteins Tau 64 and 69 are specifically expressed in the somatodendritic domain of the degenerating cortical neurons during Alzheimer's disease. Demonstration with a panel of antibodies against Tau proteins.

Bundles of paired helical filaments (PHF) accumulate in the pyramidal neurons that degenerate during Alzheimer's disease. This neurofibrillary degeneration is highly correlated with clinical signs of dementia. During the degenerating process, Tau proteins, which are the major antigenic components of PHF, are abnormally phosphorylated and two pathological isoforms named Tau 64 and 69 are expressed. We have studied their immunoblot distribution in the cortical gray and white matter from different regions of normal and Alzheimer brains, to determine if the degenerating process preferentially affects the somatodendritic or the axonal domain. Two categories of antibodies were used. The first category consisted of anti-human native Tau, anti-Tau proteins from different vertebrates, anti-PHF, monoclonal antibody Alz-50 and an anti-C terminal repeated region of Tau. In control brains, these antibodies strongly detected normal Tau proteins in the gray matter while Tau immunodetection was weak in the white matter. In Alzheimer brain cortices, each antibody detected Tau 64 and 69 in gray matter extracts but not at all in white matter extracts. The second category of anti-Tau consisted of the anti-PHF saturated with normal brain protein extracts. This antiserum only probed the abnormally phosphorylated Tau proteins. It detected Tau 64 and 69 exclusively in the cortical gray matter of Alzheimer brains. Moreover, a 55-kDa Tau protein was also immunolabelled, which might be an intermediary form between normal Tau and Tau 64 and 69. Our results demonstrate that Tau proteins are normal and major components of the somatodendritic domain and that Tau pathology, reflected by the presence of Tau 64 and 69, affects preferentially this domain during Alzheimer's disease.

An antiserum to the N-terminal subsequence of the Alzheimer amyloid beta protein does not react with neurofibrillary tangles.

Polyclonal antibodies were raised against a synthetic peptide corresponding to a subsequence for the first 10 residues of the beta amyloid protein A4 (1-10 beta PA4). In an immunoperoxidase study of Alzheimer brain tissue, these antibodies immunostained senile plaque cores, amyloid vessel walls, and amyloid fibrils surrounding senile plaques and angiopathic vessels. Neurofibrillary tangles stained with thioflavin S or immunostained with anti-Tau immune serum were never immunodetected with the anti 1-10 beta PA4. We confirm that the neurofibrillary tangles do not contain epitopes corresponding to the first 10 residues of the beta PA4.

Alzheimer's disease: glycolytic pretreatment dramatically enhances immunolabeling of senile plaques and cerebrovascular amyloid substance.

In Alzheimer's disease, three types of pathologic lesions are stained by thioflavin: neurofibrillary tangles, senile plaques, and amyloidaceous vessels. We have used anti-beta protein amyloid A4 and anti-tau protein antisera and compared immunolabeling with thioflavin staining. Anti-tau detected only neurofibrillary tangles; anti-beta-PA4 immunostained senile plaques and amyloidaceous vessels. Glycolytic pretreatment (2% periodic acid overnight or glycosidases digestion) dramatically enhanced the anti-beta-PA4 immunolabeling of senile plaques, amyloidaceous vessels, and a previously undetected extracellular substance; neurofibrillary tangles were never immunostained. Therefore, glycolytic pretreatment exposes buried epitopes in the amyloid and is a good method for amplification of immunostaining. The nature of the interaction between saccharides and beta-protein amyloid A4 is unknown.

Antibodies to sperm basic nuclear proteins detected in infertile patients by dot-immunobinding assay and by enzyme-linked immunosorbent assay.

The auto-antibody response in infertile men was investigated by means of immunoenzymatic methods, dot-immunobinding assay (DIBA), and ELISA, using, as antigens, human sperm basic nuclear proteins. Comparison was made, for the same patients, with antibody response to membrane antigens, detected by tray agglutination test (TAT), spermotoxic test (STT), and immunobead binding test (IBT). A very good agreement was observed between the two kinds of antibody responses. Thus, an ELISA or a dot-immunobinding test with sperm nuclear proteins may be considered as a simple and sensitive method for detection of auto-antibodies in infertile men. The reactivity in ELISA of various synthetic peptides corresponding to sequences of human protamines HP1 and HP2 was also studied: all the sera containing anti-nuclear antibodies do not react with synthetic peptides. This observation suggests that antibodies to sperm nuclear proteins recognize conformational epitopes that are not present on small synthetic peptides.

[Alzheimer's disease: immunohistochemical characterization of cerebral amyloid deposits]. / Maladie d'Alzheimer: caractérisation immunohistochimique des dépôts amyloïdes cérébraux.

In Alzheimer cortex tissue sections, thioflavine stained three patterns of amyloid lesions: neurofibrillary tangles (NFT), senile plaques (SP) and vessel walls (amyloid angiopathy AA). An anti serum against Tau proteins detected NFT but neither SP nor AA. In contrast, an anti serum against beta protein amyloid (BP A4) revealed SP and AA but not NFT. A periodic acid pretreatment dramatically enhanced the anti-BP A4 immunolabelling corresponding to microplaques as well as a large amount of diffuse extracellular amyloid substance, but never stained NFT. Pretreatment of tissue sections with a mixture of endo and exoglycosidases gave identical results and corroborates the extraneuronal processing of BP A4 that appears in a glycosylated form in the extracellular compartment.

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle.

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.