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M Thiry

Publications and source records attributed to M Thiry.

At least 55 records · Page 3Linked to original sources
PubMed1994

Recombinant protein fragments from haemorrhagic septicaemia rhabdovirus stimulate trout leukocyte anamnestic responses in vitro.

This work shows that viral protein fragments are capable of stimulating fish anamnestic immunological responses in leukocytes from the rainbow trout (Oncorhynchus mykiss, W.). Recombinant protein fragments of glycoprotein and nucleoprotein from the rhabdovirus causing viral haemorrhagic septicaemia of trout (VHSV), were cloned and expressed in Escherichia coli, Yersinia ruckeri (a trout pathogen) and Saccharomyces cerevisiae. The recombinant protein fragments stimulated anamnestic responses in leukocyte cultures derived from the anterior kidney of survivors of VHSV infection but not from uninfected trout. Two types of stimulatory anamnestic responses were detected, (i) a stimulation of lymphoproliferation as measured by thymidine incorporation assays and (ii) an increase in number, spreading and size of cells as determined by fibrin-clot and/or flow cytometry techniques. The evidence presented suggests that both adherent and non-adherent trout cell populations are needed for the immunological response to VHSV in this primitive vertebrate. The possible use of in vitro lymphoproliferation assays as a preliminary screening method for candidate fish vaccines prior to their testing in vivo is discussed.

Animals↗
PubMed1994

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout.

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M(r) of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculovirus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.

Animals↗
PubMed1993

Ultrastructural distribution of DNA within the ring-shaped nucleolus of human resting T lymphocytes.

The precise distribution of DNA within the resting human T lymphocyte nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecular immunocytochemical techniques. The nucleolus is partially enveloped by a layer of condensed chromatin which, at several places, penetrates into the nucleolar body until in close contact with the fibrillar centers. Morphometric analysis reveals that 32% of the fibrillar center surface is essentially occupied by condensed chromatin. Using the in situ terminal deoxynucleotidyl transferase-immunogold procedure for detecting DNA, we further show that evident label is exclusively found over the condensed chromatin and over the fibrillar centers, whereas no significant label is detected over the dense fibrillar component of the nucleolus.

Cell Nucleolus↗
PubMed1993

Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase.

We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.

Animals↗
PubMed1993

Structure, function and assembly of the nucleolus.

Most events of ribosome biogenesis--such as transcription of the ribosomal RNA (rRNA) genes, processing of their primary transcripts into mature rRNAs and assembly with ribosomal and nonribosomal proteins to form the preribosomes--are confined to a special nuclear compartment, the nucleolus. Immunogold labelling and in situ hybridization at the ultrastructural level are providing novel insights into structure-function relationships of the nucleolus, and in vitro systems are beginning to shed light on the molecular mechanisms involved in the reforming of nucleoli after mitosis.

Journal Article↗
PubMed1993

Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

Animals↗
PubMed1993

Ultrastructural distribution of DNA and RNA within the nucleolus of human Sertoli cells as seen by molecular immunocytochemistry.

The precise distribution of DNA and RNA within the human Sertoli cell nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecular immunocytochemical techniques. In Sertoli cells, the nucleolar components show a typical spatial distribution. The fibrillar centres are not surrounded by a layer of dense fibrillar component, but come in contact only with strands of dense fibrillar component. These fibrillar parts of strands are the extensions of granular strands connected to a large granular mass. These strands delimit numerous nucleolar interstices in which chromatin fibres are clearly obvious. Using the in situ terminal deoxynucleotidyl transferase/immunogold procedure for detecting DNA, we find evident label exclusively over the chromatin fibres enclosed in the nucleolar interstices and over the fibrillar centres, and no significant label over the dense fibrillar component and granular component of the nucleolus. Furthermore, using the polyadenylate nucleotidyl transferase/immunogold procedure for detecting RNA, we show that label is deposited not only over the granular component and dense fibrillar component, as expected, but also quite obviously over the fibrillar centres. No label is seen over the interstices containing chromatin.

Cell Nucleolus↗
PubMed1993

Differential location of nucleic acids within interchromatin granule clusters.

We have examined in great detail the distribution of nucleic acids within interchromatin granule clusters in different cell types by means of various immunocytochemical approaches. Using the in situ polyadenylate nucleotidyl transferase-immunogold technique for RNA detection or anti-RNA antibodies, we decisively demonstrate the presence of appreciable amount of RNA in clusters of interchromatin granules of untreated cells. Neither the in situ terminal deoxynucleotidyl transferase-immunogold technique nor anti-DNA antibodies, nor the in situ nick-translation technique for DNA detection have revealed any DNA in the interchromatin granule clusters. However, dispersed chromatin sensitive to DNase I are found at the borders and in the close vicinity of interchromatin granule clusters. The results indicate that interchromatin granule clusters should not be nuclear structures directly involved in RNA transcription but rather in some other steps of RNA metabolism.

Animals↗
PubMed1992

New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.

Animals↗
PubMed1992

Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry.

This paper describes a new technique for locating DNA on semithin or ultrathin sections of aldehyde-fixed and plastic-embedded cells or tissues. Sections were incubated in a medium containing bromodeoxyuridine (BUdR) triphosphate and terminal deoxynucleotidyltransferase. The labeled nucleotides bound at the surface of the sections were subsequently detected with an anti-BUdR antibody and immunoglobulin-gold complex. On semithin sections, labeled nucleotide detection was achieved by an amplification step with silver enhancement. This technique was applied to a wide variety of biological materials allowing a sensitive detection of DNA-containing structures, even where these are present in very low amounts. Examples of high resolution and sensitive detection include the DNA present in mitochondria, chloroplasts, mycoplasmas, and DNA viruses. Special attention focused on the location of DNA inside the nucleolus. In Ehrlich tumor cell nucleoli, DNA was detected in the fibrillar centers and not in the dense fibrillar component. Identical results were found in the nucleoli of other cell types. These results contradict earlier data but conform with other recent immunocytochemical observations concerning the correlation between structure and function in the nucleolus. This method provides a useful tool for investigations requiring highly precise correlations between a molecular function and a given ultrastructural morphology.

Animals↗
PubMed1992

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.

Animals↗
PubMed1992

Location of DNA within the nucleolus of rat oocytes during the early stages of follicular growth.

We have investigated the DNA distribution within the rat oocyte nucleolus during the early stages of follicular growth by means of the in situ terminal deoxynucleotidyl transferase method. In the fibrillogranular nucleolus, label is visualized on small clumps of peri- and intranucleolar chromatin. Such labeled clumps are frequently observed inside the interstices surrounding the fibrillar centers. Label is also consistently found in the fibrillar centers whereas the dense fibrillar component and the granular component are devoid of gold particles. These results contradict earlier data but conform with other recent immunocytochemical observations, obtained in nucleoli of a variety of somatic cell types, concerning the correlation between structure and function in the nucleolus.

Animals↗
PubMed1991

Sequence of a cDNA carrying the glycoprotein gene and part of the matrix protein M2 gene of viral haemorrhagic scepticaemia virus, a fish rhabdovirus.

A cDNA clone encoding for the glycoprotein of the viral haemorrhagic scepticaemia virus, a fish rhabdovirus, has been sequenced. The cDNA was 2035 bp long and contained two open reading frames (ORF). A 1523 bp ORF corresponded to the glycoprotein and was adjacent, on its 5' side, to an incomplete 372 bp ORF. Although the protein encoded by this ORF displayed no similarity with other rhabdovirus proteins, it was supposed that the cDNA had been reverse-transcribed from a readthrough mRNA encoding successively for the M2 and the G proteins.

Amino Acid Sequence↗
PubMed1991

Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144.

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.

Cloning, Molecular↗
PubMed1991

Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144.

A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced. The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772. This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility. The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.

Amino Acid Sequence↗
PubMed1991

Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy.

Immunoelectron microscopy and in situ hybridization have been used to investigate the precise location of transcribed and non-transcribed rDNA spacer sequences. Whereas a 5'-external transcribed spacer sequence is predominantly visualized in the fibrillar centers of nucleoli, a non-transcribed spacer sequence is preferentially detected in the interstices, in close contact with the fibrillar centers and which interrupt the surrounding dense fibrillar component. Occasionally these two spacers are also observed in clumps of dense nucleolus-associated chromatin. These observations provide insights into the organization of ribosomal repeats within the nucleolus.

Animals↗
PubMed1991

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.

Animals↗