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J Bourguignon

Publications and source records attributed to J Bourguignon.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transverse formamide gradients as a simple and easy way to optimise DNA single-strand conformation polymorphism analysis.

Although widely used, the detection of DNA mutations by the single-strand conformation polymorphism (SSCP) method is often hampered by the need to examine a large set of electrophoretic conditions in order to select the one suited to the DNA sequence under study. We show here that the use of transverse chemical gradient gels allows for a quick and easy optimisation of SSCP analysis, as exemplified on two mutations in exon 2 of the alpha-1-antitrypsin gene.

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

The gene encoding T protein of the glycine decarboxylase complex involved in the mitochondrial step of the photorespiratory pathway in plants exhibits features of light-induced genes.

We have isolated and characterized a genomic clone encoding the 41 kDa monomer T-protein. This gene called gdcT spans approximately 3 kbp and is composed of four exons interrupted by three introns (321, 691 and 114 bp). The splice sites for donor and acceptor are in agreement with the canonical GT/AG rule. Primer extension strongly suggests the presence of two major transcription start sites. The first transcription start site around 43 bases downstream of a putative TATA box was assigned the +1 position. The second (+31) is not correlated with a putative TATA box, but revealed a pyrimidine-rich region which is very similar to the initiator element. Sequence analysis of the 5'-upstream region of the gene reveals three consensus regions found in the nuclear genes encoding the chloroplastic proteins of ribulose-1,5-bisphosphate carboxylase (rbcS) and the chlorophyll a/b-binding protein (cab) such as an AT-rich sequence localized at -539 to -530, a box II core sequence GGTTAA (-123 to -118) and between -364 and -354 a tandem GATA motif. These elements are known to be involved respectively in the regulation of light-responsiveness and cell-type specificity expression of plant genes. Gel shift assays indicate that the box II core sequence could bind protein nuclear factors similar to the trans-acting factor which interact with corresponding promoter region of rbcS gene.

Amino Acid Oxidoreductases↗
PubMed1997

The origin of hyaluronectin in human tumors.

The origin of tumor stroma hyaluronectin (HN), a glycoprotein that binds to hyaluronan (HA), has long remained unknown. Histological observations of human tumors suggest that tumor HN could originate from stroma fibroblasts, and in some cases from inflammatory cells. The fibroblast origin was confirmed by the discovery of HN-like antigen along with hyaluronan in culture medium of tumor-derived fibroblasts. An HA-binding protein was characterized in the culture medium of peripheral blood mononuclear cells (PBMC) in both normal subjects and tumor-bearing patients and was found to be human HN. Cultivated monocytes did not produce HA. HN was not related to the HA-binding site CD44. Sequencing of brain HN-derived peptides demonstrated that each determined peptide sequence was similar to a sequence of the proteoglycan PG-M/versican, suggesting that HN is the HA-binding moiety of the proteoglycan. One probe was synthesized from human PBMC by polymerase chain reaction with primers derived from HN sequences also found in versican. Northern blots were positive only with HN-producing cells. The main RNAs were in the 6-8 kb range, and there was a limited proportion of smaller RNA, which was compatible with the size expected from the HN molecular mass. Southern blotting of monocytes and tumor cells demonstrated that the gene was limited to a unique band. We conclude that HN, an extracellular component of brain, connective embryonic, inflammatory and tumoral tissues, is a PG-M/versican-derived molecule. Our results suggest that tumor HN, which originates from fibroblasts and monocytes of tumor stroma, is a molecular component of the host-tumor relationship and could play a role in the regulation of HA activity in oncogenesis.

Adenocarcinoma↗
PubMed1997

Structural studies of the glycine decarboxylase complex from pea leaf mitochondria.

The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions since its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. The X-ray crystal structure of different forms of the H-protein has shown a unique conformation of the protein. This leads to the hypothesis of a three-dimensional recognition of the H-protein by the other components of the system and also by the ligase which lipoylates the H-protein. Striking structural similarities are observed between the H-protein and other lipoate domains of 2-oxo acid dehydrogenases and with the biotin carrier protein of acetyl-CoA carboxylase. In the H-protein, the lipoamide arm is free to move in the solvent when oxidized but is pivoted and tightly bound into a cleft at the protein surface when methylamine-loaded. This implies that the H-protein and the T-component form a stable complex during the catalytic transfer of the methylene unit to the tetrahydrofolate cofactor of the T-protein. This complex has been detected by small angle scattering experiments. In conclusion, in the glycine decarboxylase system, the lipoamide arm does not swing freely from one catalytic site to another as was proposed in other systems.

Amino Acid Oxidoreductases↗
PubMed1997

Scale development in zebrafish (Danio rerio).

In the course of an extensive comparative, structural and developmental study of the cranial and postcranial dermal skeleton (teeth and scales) in osteichthyan fishes, we have undertaken investigations on scale development in zebrafish (Danio (Brachydanio) rerio) using alizarin red staining, and light and transmission electron microscopy. The main goal was to know whether zebrafish scales can be used as a model for further research on the processes controlling the development of the dermal skeleton in general, especially epithelial-mesenchymal interactions. Growth series of laboratory bred specimens were used to study in detail: (1) the relationship of scale appearance with size and age; (2) the squamation pattern; and (3) the events taking place in the epidermis and in the dermis, before and during scale initiation and formation, with the aim of searching for morphological indications of epithelial-mesenchymal interactions. Scales form late in ontogeny, generally when zebrafish are more than 8.0 mm in standard length. Within a population of zebrafish of the same age scale appearance is related to standard length, but when comparing populations of different age the size of the fish at scale appearance is also related to age. Scales always appear first in the posterior region of the body and the squamation then extends anteriorly. Scales develop in the dermis but closely apposed to the epidermal-dermal boundary. Cellular modifications occurring in the basal layer of the epidermis and in the dermis before scale formation clearly indicate that the basal epidermal cells differentiate first, before any evidence of differentiation of the progenitors of the scale-forming cells in the dermis. This strongly suggests that scale differentiation could be initiated by the epidermal basal layer cells which probably produce a molecular signal towards the dermis below. Subsequently dermal cells accumulate close to the epidermis, and differentiate to form scale papillae. The late formation of the scales during ontogeny is due to a late colonisation of the dermis by the progenitors of the scale-forming cells. Because of their late formation during ontogeny and of their regular pattern of development, scales in zebrafish represent a good model for further investigations on the general mechanisms of epithelial-mesenchymal interactions during dermal skeleton development, and in particular for the study of the gene expression patterns.

Animals↗
PubMed1996

Expression, lipoylation and structure determination of recombinant pea H-protein in Escherichia coli.

A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sativum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form. Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-(1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The three-dimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme.

Amino Acid Oxidoreductases↗
PubMed1996

Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis.

In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.

Amino Acid Oxidoreductases↗
PubMed1996

Substitution of carbonate by acetate buffer for IgG coating in sandwich ELISA.

The first step of sandwich ELISA, namely adsorption of antibodies to plastic microtiter plates, was studied as a function of the pH of the coating buffer. Coating efficiency was assessed in terms of maximum signal (absorbance) observed in ELISA and also estimated by measuring the amount of functional antibodies adsorbed to the plate. While goat antibodies displayed better results after coating with acetate pH 5 buffer, rabbit IgGs generally worked well at pH 7.4. On average, the classical carbonate pH 9.6 buffer was only 50% as efficient.

Acetates↗
PubMed1995

Frequency and prognostic evaluation of 3p21-22 allelic losses in non-small-cell lung cancer.

Previous loss of heterozygosity (LOH) studies of chromosome 3p loci have displayed a 60% deletion frequency in non-small-cell lung cancers (NSCLC), as opposed to small-cell lung cancers, in which the 3p deletion is consistently found. However, the high stromal-cell admixture found in NSCLC and the use of the Southern-blot method lead to under-evaluation of this frequency. In this study, we used a very precise microdissection technique followed by PCR amplification of 6 3p21-22 polymorphic genomic sequences to analyze LOH in 86 NSCLC and in normal adjacent tissue. We found the sensitivity of the microdissection-PCR-based LOH technique higher than the sensitivity of the Southern-blot technique: 87% of the squamous-cell carcinomas and 84% of the large-cell undifferentiated carcinomas showed a clear LOH for a 3p21-22 locus. All doubly informative cases but 4 showed concordant deletion at all 3p21-22 loci. The analysis of 3p microsatellite sequences displayed only 2 cases of genomic instability, one of them also displaying features of tumoral heterogeneity as regards the instability genotype. Four carcinomas in situ adjacent to these NSCLC showed the same allelic profile as the invasive tumors. The only prognostic factors in this study were the disease stage and histology. The 3p21-22 deletion was not related to the stage of the disease and did not appear to be a significant prognostic factor of survival. 3p21 loss appears, so far, to be the most frequent and the earliest genetic alteration described in NSCLC, but does not seem to carry significant prognostic information in invasive tumors.

Alleles↗
PubMed1995

Involvement of the three inter-alpha-trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family.

Partial cDNAs coding for each of the three human inter-alpha-trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3-related ITI form and a low-amount H1-related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.

Alpha-Globulins↗
PubMed1995

The human inter-alpha-trypsin inhibitor genes respond differently to interleukin-6 in HepG2 cells.

The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL-6, suggesting that IL-6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL-6-treated cells confirmed these data. The nuclear run-on assay supports the regulatory effect of IL-6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL-6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre-alpha-trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL-6.

Alpha-Globulins↗
PubMed1995

Gene expression and protein distribution of inter-alpha-trypsin inhibitor in three human hepatoma cell lines.

In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno-precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously.

Alpha-Globulins↗
PubMed1995

Blockade of the discriminative stimulus effects of gamma-hydroxybutyric acid (GHB) by the GHB receptor antagonist NCS-382.

The present study was designed to assess the ability of the newly synthetized, selective gamma-hydroxybutyric acid (GHB) receptor antagonist, NCS-382, in blocking the discriminative stimulus effects of GHB in a T-maze, food-reinforced drug discrimination procedure. Two groups of rats were trained to run the left arm of the maze 30 min after the i.g. administration of either 300 or 700 mg/kg GHB and the right arm after water. Once discrimination was acquired, combination of different doses of NCS-382 (0, 12.5, 25.0 and 50.0 mg/kg, IP) and GHB training doses were tested for blockade of GHB discrimination. NCS-382 dose-dependently blocked GHB-appropriate responding in both the 300 and 700 mg/kg GHB rat groups. The results of the present study indicate that the discriminative stimulus properties of GHB are mediated via stimulation of GHB receptors.

Animals↗
PubMed1994

Tandem orientation of the inter-alpha-trypsin inhibitor heavy chain H1 and H3 genes.

The inter-alpha-trypsin inhibitor H1 (ITIH1) and inter-alpha-trypsin inhibitor H3 (ITIH3) genes have both previously been mapped to chromosomes 3 and 14 in the human and mouse, respectively. We now present evidence that these genes are physically linked. By using cDNA probes, a recombinant DNA phage has been isolated from a bacteriophage DNA library, which contains sequences flanking the 5' end of the ITIH3 gene and the 3' end of the ITIH1 gene. Restriction endonuclease mapping, PCR analysis and DNA sequence determination of the recombinant phage and comparison to genomic DNA revealed that the genes are in tandem, 2721 base pairs apart, with the ITIH1 gene to the 5' side of the ITIH3 gene. Their respective transcriptional units are thus on the same strand of DNA and most probably arose in evolution as the consequence of a duplication of a common ancestral gene.

Alpha-Globulins↗
PubMed1994

Post-translational processing of the inter-alpha-trypsin inhibitor in the human hepatoma HepG2 cell line.

In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.

Alpha-Globulins↗
PubMed1994

Plant mitochondrial NAD+-dependent malic enzyme. cDNA cloning, deduced primary structure of the 59- and 62-kDa subunits, import, gene complexity and expression analysis.

The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.39) were purified from Solanum tuberosum L. (potato). NH2-terminal and internal amino acid sequence information was used to identify cDNAs encoding the two subunits. Comparison of the nucleotide sequences revealed that the subunits have 60% identity at the DNA level and 65% identity at the deduced amino acid level, implying that they are derived from a common ancestral gene. The plant NAD+-dependent malic enzymes belong to a family of related enzymes, including cytosolic and chloroplastic NADP+-dependent malic enzymes (EC 1.1.1.40) and bacterial NAD+-dependent malic enzymes (EC 1.1.1.38). The cDNAs were transcribed and translated in vitro and the resultant polypeptides imported into isolated mitochondria and shown to be processed. Southern blot analysis of potato genomic DNA revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits. Northern blot analysis of RNA from a range of potato tissues has shown that the steady state levels for the two subunits are equivalent, suggesting that they are coordinately expressed.

Amino Acid Sequence↗
PubMed1994

Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads.

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.

Alginates↗