Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.

Female-enriched and thermosensitive expression of steroidogenic factor-1 during gonadal differentiation in Pleurodeles waltl.

In the urodele amphibian Pleurodeles waltl, sex differentiation is genetically controlled, that is, ZZ male vs ZW female, but may be influenced by temperature, which induces a female-to-male sex reversal. We investigated whether steroidogenic factor 1 (SF-1) could be involved in Pleurodeles sex differentiation or in temperature-dependent sex reversal by cloning a Pleurodeles SF-1 cDNA and examining its developmental expression. The 468-amino-acid deduced protein is highly conserved in comparison with other species. In ZZ and ZW control larvae, SF-1 mRNA is detected at the first stage of the thermosensitive period (TSP) in the gonad-mesonephros-interrenal complex (GMI). By the end of TSP at stage 55, SF-1 is expressed in the gonad (Gd) and in the mesonephros-interrenal (MI) both in ZZ and ZW larvae. During this stage, a transient, ZW-specific increase of SF-1 transcription occurs not only in Gd but also in MI, this increase starting earlier in Gd than in MI. Therefore, in P. waltl, an SF-1 upregulation occurs after the onset of the ovarian-specific increase of aromatase mRNA expression. At the end of metamorphosis, the SF-1 transcription level in Gd and MI is nearly the same in both ZZ and ZW larvae. Besides, after long-term heat treatment leading to sex reversal, SF-1 mRNA upregulation is not observed in ZW larvae, in either Gd or MI. However, SF-1 expression is not decreased after a 48-h heat shock applied at the end of the TSP, suggesting that temperature has no inhibitory effect by itself in long-term heat treatment. Estradiol benzoate treatments show that, at the end of the TSP, SF-1 gene transcription could be controlled by the estrogen level. This is in accordance with the female-enriched SF-1 expression and the decreased SF-1 expression following long-term, sex-reversing heat treatment, which is known to decrease aromatase expression and activity. Thus, it is unlikely that SF-1 is directly involved in Pleurodeles temperature-dependent sex reversal.

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines.

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.

Xenopus oocyte maturation: cytoplasm alkalization is involved in germinal vesicle migration.

In Xenopus laevis oocytes a transient increase in intracellular pH has been reported to occur during progesterone-induced maturation. Using a cytological approach, we have systematically analyzed germinal vesicle breakdown and meiotic spindle formation in various experimental conditions either preventing or promoting pHi changes. Injection of a neutral buffer (MOPS pH 6.9) induced a cytosolic acidification of 0.3 pH unit and inhibited by 30% the formation of the maturation white spot after progesterone exposure; in oocytes displaying a white spot, only half showed a spindle, often located far from the plasma membrane. Similar results were observed with a Na-free medium which prevents oocyte alkalization. Injection of an alkaline buffer (Tris pH 9) was able to induce migration of the germinal vesicle in 25% of the oocytes in the absence of progesterone, but failed to induce GVBD. Taken together, these results suggest that the increase in pHi observed during maturation may be involved in the migration of the germinal vesicle towards the plasma membrane. We also incubated oocytes in the presence of procaine, a weak base often used to artificially alkalize the oocyte cytoplasm. The changes induced by exposure to procaine were different from those resulting from alkaline buffer injection. Indeed procaine promoted GVBD, as well as spindle formation and chromosome condensation. However these events appeared without migration of the germinal vesicle, suggesting that the expected alkalization did not occur.

Abnormal tau species are produced during Alzheimer's disease neurodegenerating process.

Tau proteins were detected in human brain using two polyclonal antibodies: anti-paired helical filaments and anti-human native tau proteins. Both antisera detected identically the normal set of tau proteins in control brains. Moreover they detected two abnormal tau variants of 64 and 69 kDa exclusively in brain areas showing neurofibrillary tangles and senile plaques. Tau 64 and 69 were abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment. Therefore, tau 64 and 69 are specific markers of the neurofibrillary degeneration of the Alzheimer type and might be useful tools for studying the first pathological events that lead to neuronal death.

Modelling Alzheimer-specific abnormal Tau phosphorylation independently of GSK3beta and PKA kinase activities.

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti-phospho-Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3beta and PKA contribution during the progesterone or insulin-induced maturation process. Our results demonstrate that a non-modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.

Inhibition of protein tyrosine phosphatases blocks calcium-induced activation of metaphase II-arrested oocytes of Xenopus laevis.

We have studied the effect of a protein tyrosine phosphatases (PTP) inhibitor on calcium-induced activation of Xenopus laevis oocytes arrested at metaphase II. Ammonium molybdate microinjection blocked pronucleus formation following A23187 treatment while cortical granules still underwent exocytosis. Pronuclei still occurred in ammonium molybdate-injected oocytes following 6-DMAP addition. Changes that usually occurred following A23187 exposure were inhibited in the presence of ammonium molybdate in the oocyte: MAPK dephosphorylation, p34(cdc2) rephosphorylation and cyclin B2 and p39(mos) proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin-dependent degradation machinery.

Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl.

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl.

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.

Phosphorylation of Tau proteins: a major event during the process of neurofibrillary degeneration. A comparative study between Alzheimer's disease and Down's syndrome.

Six different brain areas from 6 patients with Down's syndrome (DS) of different ages were studied in respect of their Tau protein content using the western-blot technique. They were also studied histologically using a Palmgren (silver staining) method in order to reveal the presence of NFT and SP. The results of these studies show that Tau 64 and 69, two pathological Tau variants recently described in the brains of patients with Alzheimer's disease (AD), are also present in the brains of patients with DS. Alkaline phosphatase treatment demonstrates that their heavy molecular weight is due, as in AD, to an abnormal phosphorylation of Tau proteins. The results of this study show that the detection of Tau 64 and 69 in the brain of these patients is correlated with the presence of neurofibrillary tangles (NFT) and senile plaques (SP). These findings confirm that DS can act as a model for the study of the pathological events that occur in AD. Moreover, they suggest that the abnormal phosphorylation of Tau proteins, enhancing a shift of their electrophoretic mobility, might be an important step among the sequence of events that characterize neurofibrillary degeneration.

Characterization of two pathological tau protein, variants in Alzheimer brain cortices.

Tau proteins were detected in brain tissue homogenates from 10 patients with Alzheimer's disease versus 10 age-matched controls using the immunoblot technique and 2 polyclonal antibodies: anti-paired helical filaments (PHF) and anti-human native Tau proteins. In control brains, both antisera detected identically the normal set of Tau proteins, with molecular weight (MW) ranging from 45 to 62 kDa. Moreover, in association areas of neocortex from Alzheimer brains, the antisera detected 2 additional Tau variants of 64 and 69 kDa. Tau 64 and 69 were not found in regions of Alzheimer brains where the Alzheimer pathology was absent (caudate nucleus or cerebellum for example). The heavy MW of Tau 64 and 69 is due to their phosphorylation state as shown by the decrease of their MW after alkaline phosphatase treatment. Therefore, Tau 64 and Tau 69 are specific markers of the Alzheimer's disease neuronal degenerating process and their characterization demonstrates that an abnormal phosphorylation of Tau really occurs during the disease. Tau 64 and 69 were isolated with normal Tau proteins while the PHF were insoluble. Therefore, Tau proteins are likely to be abnormally phosphorylated prior to their incorporation in the PHF structure. Consequently, they might appear before the lesions and might be instrumental for the search of biochemical deregulations that precede the neurofibrillary degeneration.

Effect of cadmium on gonadogenesis and metamorphosis in Pleurodeles waltl (urodele amphibian).

In the amphibian Pleurodeles waltl, steroid hormones play a key role in sex differentiation. Since cadmium has been reported to block receptors of sex steroid hormones, we analyzed the effects of this heavy metal on Pleurodeles larvae gonadogenesis. At stage 42, larvae die in the presence of 10.9 microM Cd in the rearing tap water, with TL(50) of 46.3 h, but the concentration of 5.5 microM is tolerated for more than 60 days. When used at 5.5 microM cadmium accumulation measured by atomic absorption spectrophotometry (AAS) in total homogenates of larvae at stage 54 (after 77 days of exposure to the heavy metal) reached 58.1 microg/g of dry weight. At stage 54, we did not detect inhibitory effects on gonadogenesis in larvae reared in the presence of 5.5 microM Cd since stage 42. When the exposure to 5.5 microM Cd was lengthened after stage 54, metamorphosis was delayed and could not be completed. When larvae were exposed to 10.9 microM Cd from stage 54, metamorphosis did not occur and gonad development was stopped. Our study demonstrates a lack of a direct effect of cadmium on sex determination-differentiation but a strong inhibitory effect on metamorphosis, which impairs further gonadal development.

[Towards the development of an in vivo study model of Alzheimer-type neurofibrillary degeneration]. / Vers la mise au point d'un modèle d'étude in vitro de la dégénérescence neurofibrillaire de type Alzheimer.

Progress in the search for the cause of Alzheimer's disease is considerably hampered by the lack of animal or in vitro model. We have shown that in Alzheimer's disease two pathological variants of Tau proteins, called Tau 64 and Tau 69, are regularly present in neural tissue undergoing neurofibrillary degeneration. Beside their diagnostic value, Tau 64 and Tau 69 might enable such a model to be devised at long last. It now seems possible to investigate for biochemical disorders capable of inducing the emergence of these two Tau proteins in neuron cultures or among transgenic animals. The innumerable pathogenetic tracks of Alzheimer's disease (aluminium, zinc, superoxide dismutase and free radicals, proteases and antiproteases, beta protein A4 precursor, etc.) should then be opened to exploration.

Busulfan-mediated germ cell depletion does not alter gonad differentiation in the urodele amphibian Pleurodeles waltl.

It is not known in urodele amphibians whether germ cells (GCs) are indispensable for gonadal differentiation. In order to address this question in the newt Pleurodeles waltl, we first cloned the ortholog of VASA which is known as a GC marker in many species. Male (ZZ) and female (ZW) larvae were then exposed to the alkylating agent busulfan (25 µg/ml for 3 days) just after hatching (stage 36). In the main body of busulfan-treated larvae, PwVASA mRNA expression decreased before gonad differentiation in both sexes (at stage 50). This suggested GC depletion which was confirmed by histology, with a complete absence of GCs observed slightly earlier in females (stage 54) than in males (stage 54 + 60 days). In busulfan-treated ZW larvae, the presence of the typical central cavity and expression of a high level of aromatase mRNA confirmed the ovarian phenotype. In busulfan-treated ZZ larvae, the presence of a medulla surrounded by a thin cortex and a low level of aromatase mRNA confirmed the testis phenotype. At the juvenile stage, efferent ducts and lobules were present in the first testis lobe. Taken together, these data suggest that GC depletion does not alter gonad differentiation in P. waltl.

Sexual development of the urodele amphibian Pleurodeles waltl.

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Mullerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes.

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes.

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.