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A Fleury

Publications and source records attributed to A Fleury.

At least 55 records · Page 3Linked to original sources
PubMed1995

A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila.

Using two antibodies raised against Paramecium axonemal tubulin, a monoclonal antibody, AXO 49 (Callen et al., Biol. Cell 81, 95-119 (1994)), and a polyclonal antibody, PAT (Cohen et al., Biol. Cell 44, 35-44 (1982)), which have been shown elsewhere to detect a new posttranslational modification of tubulin presumably corresponding to polyglycylation, we have analyzed the occurrence of this modification during spermatogenesis in Drosophila. Results obtained by immunofluorescence on cysts isolated by laceration of testes showed that the antibodies reacted on axonemal microtubules of several species within the genus. Observation of different stages of differentiation of D. obscura sperm cells indicated, first, that the epitopes reactive with both antibodies appeared at late stages, and secondly, that they were detected simultaneously along all axonemes within a cyst. Immunofluorescence on semithin sections and electron microscopic immunocytochemistry on ultrathin sections confirmed that the appearance of the epitope recognized by the monoclonal antibody occurred at the time of the individualization process of spermatids in D. melanogaster. These results indicate that the posttranslational modification occurs as a very late event, after complete assembly of axonemal microtubules, and that the axonemal tubulin becomes modified when axonemal microtubules become coupled with the membrane, suggesting that the modification may in some way be induced by the microtubule-membrane interaction.

Animals↗
PubMed1994

Another way of being anisogamous in Drosophila subgenus species: giant sperm, one-to-one gamete ratio, and high zygote provisioning.

It is generally assumed that sexes in animals have arisen from a productivity versus provisioning conflict; males are those individuals producing gametes necessarily small, in excess, and individually bereft of all paternity assurance. A 1- to 2-cm sperm, 5-10 times as long as the male body, might therefore appear an evolutionary paradox. As a matter of fact, species of Drosophila of the Drosophila subgenus differ from those of other subgenera by producing exclusively sperm of that sort. We report counts of such giant costly sperm in Drosophila littoralis and Drosophila hydei females, indicating that they are offered in exceedingly small amounts, tending to a one-to-one gamete ratio after a single mating. As a result, most of them are successfully involved in a fertilization. Hence, the concept of "paternity assurance of individual sperm" arises. Evidence is further provided here that almost the entire sperm is incorporated into the egg during fertilization. Labeling with specific antibodies in fertilized eggs reveals intact axonemes up to late gastrulation. The question, then, is why selection has favored such an unusual strategy. Explanations related to some prefertilization functions are ruled out. It is therefore tentatively proposed that virtually every giant sperm constitutes a "direct paternal legacy to the embryo," which, in contrast to any male-derived nuptial gift, cannot be minimized by female remating. We suggest that dramatic shortage of giant sperm with a high prospect of fusion and increased zygote provisioning is merely another way of being anisogamous.

Animals↗
PubMed1994

Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335.

Microtubular networks are extensively developed in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82-85; Eddé et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules. Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies.

Animals↗
PubMed1994

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium.

Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins. The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of guinea pig cytochrome P450c17 cDNA (steroid 17 alpha-hydroxylase/17,20 lyase): tissue distribution, regulation, and substrate specificity of the expressed enzyme.

In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.

Adrenal Glands↗
PubMed1994

Power and limits of laser scanning confocal microscopy.

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-of-focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and non-destructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a ray-tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photo-bleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for a 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens.

Animals↗
PubMed1994

Significance of highly positive c22-3 "indeterminate" second-generation hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) and resolution by third-generation HCV RIBA.

Second-generation recombinant immunoblot assay (RIBA) is widely used for the validation of anti-hepatitis C virus (HCV) antibody detection. The aims of this work were (i) to determine, in terms of liver disease and HCV replication, the significance of a peculiar "indeterminate" second-generation RIBA pattern characterized by the presence of high titers of antibodies directed to c22-3, a protein bearing core epitopes and (ii) to determine whether a more advanced version of the same strip assay, namely a third-generation RIBA, may solve the problem of such indeterminate patterns. Sixty patients for which c22-3 indeterminate second-generation RIBAs were highly positive were studied. Forty-two of them (70%) were immunocompromised. Serum transaminases were increased in 46 cases (77%), and HCV RNA was detected by PCR in 50 cases (83%). Third-generation RIBA remained highly positive c22 indeterminate for 9 patients (15%) but was positive for 51 (85%), mostly because of increased sensitivity for the detection of both anti-c100 and anti-c33c antibodies. These results suggest that third-generation RIBA may achieve resolution of most of these cases but that highly positive c22 indeterminate third-generation RIBA may persist when used with some patients with very low titers of anti-HCV nonstructural protein antibodies.

Adolescent↗
PubMed1993

A dynamical model for post-translational modifications of microtubules.

In many cases, post-translational modifications constitute indirect markers of non-dynamic microtubules. The correlation between stability of microtubular systems and post-translational modifications suggests that they may act as a signal in patterning mechanisms. However, a crucial question remains as to how the repertoire of distinctly modified microtubules is generated. We propose here an ubiquitous mechanism of spatial and temporal differentiation of microtubules. In this model, the diversity of post-translational modifications results from a dynamical pathway separation which is ensured by biochemical switches between self-regulated mechanisms of differentiation. Thus, it does not require any hypothetical subcellular compartmentalization of enzymatic activities responsible for the various post-translational modifications. These results are discussed in relation to experimental evidences for a temporal and spatial regulation of microtubule modifications.

Microtubules↗
PubMed1993

A scaffold for basal body patterning revealed by a monoclonal antibody in the hypotrich ciliate Paraurostyla weissei.

In the hypotrich ciliate Paraurostyla weissei, the infraciliature consists of basal bodies on which cilia are anchored together with associated dense material and microtubular rootlets. This is renewed at each morphogenesis, which occurs both in the fully differentiated cell and in a transient dedifferentiated stage, the zygocyst. In both situations, the cell must reconstitute its typical ciliature by properly patterning its basal bodies. Insights into these morphogenetic processes were obtained through an immunocytochemical study using an antipericentriolar material antibody displaying remarkable properties. In Paraurostyla, this antibody decorates the electron-dense material associated with the basal bodies in the interphase cell (as it does with centrosomes on metazoan cells) but, during morphogenesis, the antibody decorates a transient system of tracks which appear prior to basal body patterning and along which basal bodies will later be distributed. In all cases, tracks are initiated close to parental organelles and then elongate to form a system linking the anlagen together during their migration. During zygocyst morphogenesis, they extend along three main cellular meridians. As this antibody decorates an antigen associated with early steps of morphogenesis, it visualizes a proteinaceous system upstream to basal body patterning and provides a structural continuity between parental and newly assembled basal body systems.

Animals↗
PubMed1993

[Proposition of a new classification of the phylum of the protozoa Ciliophora Doflein, 1901].

It is proposed to use all the presently available molecular, ultrastructural, biological and ecological data on ciliates for the new classification of this group of protists. On the basis of the cortical organization and the mode of cilia anchoring as well as the degree of conservation of the cortical pattern during the process of morphogenesis, eleven classes (Karyorelictea, Heterotrichea, Hypotrichea, Oligotrichea, Plagiopylea, Colpodea, Litostomatea, Vestibuliferea, Phyllopharyngea, Nassophorea and Oligohymenophorea) have been distinguished. These classes, in turn, were, according to the "shell theory" of Fleury and coworkers (1992), grouped into three subphyla: Tubulicorticata (presence of the cortical microtubular lattices), Filicorticata (characteristic ecto-endoplasmic microfibrillar boundary) and Epiplasmata (presence of a sub-membraneous epiplasmic layer).

Animals↗
PubMed1991

Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells.

The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre- and post-embedding EM immunocytochemistry with a variety of anti-tubulin antibodies. The 6-11B-1 monoclonal antibody, specific for the post-translational acetylation of Lys 40 of alpha-tubulin, and a polyclonal antibody raised against Paramecium axonemal tubulin (anti-PA tubulin antibody) both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti-PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti-tubulin antibody and the anti-PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti-PA tubulin one. This provided a strong indication that the anti-PA tubulin antibody is directed against a post-translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol-treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti-PA tubulin antibody however, suggesting that in Metazoa the post-translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post-translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.

Animals↗
PubMed1988

The use of correlated ultrastructural and morphogenetic characters in evolutionary taxonomy of hypotrich ciliates.

A new morphogenetic criterion, the "unit of turn-over" emerges from the dynamic analysis of hypotrich morphogenesis. The unit corresponds to the set of morphogenetically interdependent somatic structures whose turn-over is correlated to nuclear modifications. Hypotrichs are seen to conform to one of two very general modes of turn-over of their ciliary structures during a variety of morphogenetic processes, one in which all somatic cilia and their infraciliature are replaced, and the other in which only the cirral structures (not the dorsal dikinetids) undergo replacement. A strong correlation is observed between the type of turn-over occurring and ultrastructural features of the corresponding species. From an evolutionary point of view, these data lead to a new subdivision of hypotrichs, into the Euhypotrichina and the Pseudohypotrichina.

Animals↗
PubMed1987

Studies on the antiobesity activity of tetrahydrolipstatin, a potent and selective inhibitor of pancreatic lipase.

In summary, THL is a potent inhibitor of pancreatic lipase, which is both selective and irreversible in its action in vitro. It is also likely that THL inhibits pancreatic lipase in vivo, since its acute administration to mice and squirrel monkeys suppresses absorption of dietary triglycerides, without an apparent alteration in the absorption of fatty acids. The marked loss in body weight and carcass fat of DIO rats with subchronic administration of THL strongly suggests that inhibition of pancreatic lipase is a feasible strategy for the treatment of obesity. Whether obese humans will overeat in response to a reduction in body energy stores is not known. However, it is probable that compliance to a therapy which maintains the present level of food intake while inducing excretion of dietary fat, will be greater than a reducing diet alone or conjunction with the currently available antiobesity drugs.

Animals↗