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J Bourguignon

Publications and source records attributed to J Bourguignon.

At least 73 records · Page 4Linked to original sources

Human inter-alpha-trypsin inhibitor. Synthesis and maturation in hepatoma HepG2 cells.

In hepatoma HepG2 cells, human inter-alpha-trypsin inhibitor (ITI) was synthesized as three heavy chains, H-1 (100 kDa), H-2 (110 kDa) and H-3 (113 kDa), and light hybrid chain (49.5 kDa) composed of alpha 1-microglobulin and HI-30 (ITI derivative, human inhibitor of 30 kDa). The association of at least two heavy chains, H-1 and H-3, with the HI-30 part of the light chain gave rise to a molecule similar to serum ITI. A composite protein (approximately 250 kDa) including heavy and light chains was also secreted, while alpha 1-microglobulin and ITI H-2 protein were released as separate entities. Light chain synthesis could be the limiting factor for ITI maturation.

Alpha-Globulins↗

Human plasma inter-alpha-trypsin inhibitor is encoded by four genes on three chromosomes.

Human inter-alpha-trypsin inhibitor is a plasma protein of Mr 180,000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379-383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et. al. (1985) Biochem. Biophys. Res. Commun. 131, 1146-1153; Salier, J.P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272-8276]. In the present study, we show that human poly(A)-rich RNAs hybrid-selected with various heavy-chain-encoding cDNA clones translate three different heavy chains, designated H1 (Mr 92,000), H2 (Mr 98,000) and H3 (Mr 107,000). We previously characterized two heavy-chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)-rich RNAs with the three heavy-chain cDNAs as probes clearly identified a single major mRNA population of 3.3 +/- 0.1 kb. Chromosomal localization by in situ hybridization shows that inter-alpha-trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211-p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light-chain gene is located in the q32-q33 region of chromosome 9. These results indicate that heavy and light chains of inter-alpha-trypsin inhibitor are encoded by at least four functional genes.

Alpha-Globulins↗

Improvement of the sensitivity of mono- and bi-dimensional immunoelectrophoretic techniques by transfer onto nitrocellulose.

The sensitivity of crossed immunoelectrophoresis (CIE) and rocket immunoelectrophoresis (RIE) does not usually permit the study of diluted protein solutions or the detection of trace components. Antigen-antibody precipitates obtained after immunoelectrophoresis were therefore dissociated and transferred from the agarose gel onto nitrocellulose filter by electrophoresis at pH 11. Immunochemical detection of proteins was then performed with the peroxidase-anti-peroxidase method. Diluted (1/100) solutions gave patterns similar to those obtained with the starting biological fluid. As an example, this technique was applied to human inter-alpha-trypsin inhibitor present at concentrations in the nanogram range. Other proteins, differing in charge and/or molecular weight, were also identified.

Alpha-Globulins↗

Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase.

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

Amino Acid Oxidoreductases↗

Isolation and characterization of renin-producing human chorionic cells in culture.

Chorionic tissue is one of the major extrarenal sites of renin production, and as such, cultured chorionic cells are a potential model for in vitro studies of renin biosynthesis and regulation. Human chorionic cells were isolated from four chorions and maintained in tissue culture for a total of eight subcultures. Total renin production was considerable in the primary cultures, but fell gradually with successive passages. The cells could be frozen and thawed without losing their ability to divide or produce renin. Both the primary cultures and the subcultures contained a single type of elongated cell containing abundant rough endoplasmic reticulum and myofibrils, but no renin granules, suggesting that the cells had smooth muscle-like features. Immunocytochemistry indicated that they contained both renin and prorenin. The renin produced by the chorionic cells was not stored within the cells, but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had biochemical and immunological properties similar to those of pure human renin. The cells contained a renin mRNA that had the same size as that for renal renin (1.6 kilobases), confirming the synthesis of renin by these cells. The cells were also examined for the presence of other components of the renin-angiotensin system. Angiotensinogen and angiotensin I were not detected, but angiotensin-converting enzyme was present in extracts of primary and secondary cultured cells. beta hCG and progesterone were also found in the medium of primary culture. However, the production of beta hCG and progesterone fell after the primary culture, and beta hCG and progesterone were indetectable in secondary and tertiary cultures, respectively. These experiments suggest that these two hormones do not influence renin synthesis or vice versa. Thus, these cultures of human chorionic cells synthesized considerable quantities of prorenin and can provide a permanent source of nonrenal prorenin-producing cells.

Cell Separation↗

Human inter-alpha-trypsin inhibitor. Isolation and characterization of heavy (H) chain cDNA clones coding for a 383 amino-acid sequence of the H chain.

A cDNA library cloned in lambda gt 11 expression vector was screened with specific anti-inter-alpha-trypsin inhibitor (ITI) heavy (H) chain antiserum. Among seven positive clones selected, two overlapping clones were sequenced. The corresponding consensus sequence encompasses a stretch of bases with one single open reading frame, and the deduced amino-acid sequence displays no homology with the full length cDNA-deduced amino-acid sequence of ITI-light (L) chain. Furthermore, analysis of poly A+ mRNAs hybrid-selected by the corresponding cDNA from clone lambda gt11 revealed an immunoprecipitable polypeptide of Mr 91,000. Northern blot analysis showed a single population of mRNAs of 3.3 kb. These data provide new evidence for the presence of distinct H and L chains in the 180 kDa ITI molecule.

Alpha-Globulins↗

Isolation and characterization of cDNAs encoding the heavy chain of human inter-alpha-trypsin inhibitor (I alpha TI): unambiguous evidence for multipolypeptide chain structure of I alpha TI.

Human inter-alpha-trypsin inhibitor (I alpha TI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that I alpha TI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of I alpha TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum I alpha TI. RNA blot analyses of liver RNAs with H- or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000. These results unambiguously establish that I alpha TI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiol-proteinase inhibitors. These data indicate that I alpha TI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

Alpha-Globulins↗

[High-density lipoproteins, total cholesterol, triglycerides and serum trypsin activity. Study in chronic alcoholic and withdrawn subjects].

Apolipoprotein is controlled by proteolytic processus and serum trypsin-like activity (STA) may be elevated in some chronic alcoholic subjects. STA, apoA lipoprotein, HDL-cholesterol, total cholesterol and triglycerides were tested in 44 men dealt in 4 groups (subjects with normal or elevated STA, alcoholic or withdrawn). Significantly lower apoA lipoprotein (p less than 0,02) and HDL-cholesterol (p less than 0,001) levels as well as significantly higher triglyceride levels (p less than 0,01) were evidenced in the group with elevated STA compared to the group with normal STA. In another way, a negative correlation between HDL-cholesterol and STA (p less than 0,01) and a positive correlation between triglycerides and STA (p less than 0,001) were noted. The different factors known to modify these lipidic parameters cannot account for such disturbances. The role of elevated STA is evoked.

Alcoholism↗

Human inter-alpha-trypsin-inhibitor: characterization and partial nucleotide sequencing of a light chain-encoding cDNA.

A synthetic Inter-alpha-Trypsin-Inhibitor (ITI) -specific oligonucleotide probe was used to isolate a clone from a human liver cDNA library. The amino-acid sequence deduced from partial nucleotide sequencing of the corresponding cDNA insert perfectly matched a known ITI sequence, apart from an as yet unreported C-terminal dipeptide. Hybridization on Northern blots evidenced that this cDNA insert originated from an ITI light chain-encoding mRNA whose size was estimated to be 1 300 bases.

Adult↗

The effects of 17 alpha-ethynyloestradiol and of acute inflammation on the plasma concentration of rat alpha 1-acid glycoprotein and on the induction of its hepatic mRNA.

We measured the serum concentration of alpha 1-acid glycoprotein (alpha 1-AGP) and we evaluated the content of its hepatic mRNA in rats after 17 alpha-ethynyloestradiol treatment or after turpentine-induced acute inflammation, or after both treatments performed simultaneously. We have also studied the affinity of serum alpha 1-AGP for concanavalin A under these conditions. Both types of stimuli induce a marked retention of the glycoprotein on free concanavalin A. The serum concentration of alpha 1-AGP is increased about 14-fold compared with that in control rats when a single pharmacological dose (50 micrograms) or multiple injections of 17 alpha-ethynyloestradiol are administered. This increase is greater in turpentine-oil-injected rats (about 21-fold) and reaches a maximum (about 32-fold) in rats injected with 17 alpha-ethynyloestradiol plus turpentine oil; this increase in alpha 1-AGP corresponds to the addition of the effects of the two inducing agents. Similar changes are also observed either in the alpha 1-AGP mRNA content as estimated by using an alpha 1-AGP-specific cDNA probe, or in the amount of translatable alpha 1-AGP mRNA. The results indicate that: after a high dose of 17 alpha-ethynyloestradiol and after acute inflammation, the increase of the alpha 1-AGP serum concentration is due to an accumulation of the alpha 1-AGP mRNA; different mechanisms and/or pathways are probably involved in regulating the synthesis of alpha 1-AGP under various stimuli; 17 alpha-ethynyloestradiol as well as acute inflammation seem to control the glycosylation process of alpha 1-AGP in an identical manner.

Animals↗

Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.

A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.

Antigens, Viral, Tumor↗

Inter-alpha-trypsin-inhibitor (ITI): two distinct mRNAs in baboon liver argue for a discrete synthesis of ITI and ITI derivatives.

Human serum inter-alpha-trypsin-inhibitor (ITI) has so far been assumed to be comprised of a single polypeptide chain which can undergo fragmentation, whereby inhibitory ITI derivatives are released into the blood stream. In contrast, the analysis of the baboon liver mRNA translation products showed that ITI is made up of heavy and light chain(s). The latter may be excreted independently and very likely corresponds to the so-called ITI derivatives.

Alpha-Globulins↗

Inter-alpha-trypsin inhibitor (ITI): use of new antisera for qualitative studies and discrete quantitation of ITI and its derivatives.

Inter-alpha-trypsin inhibitor (ITI) is a human protease inhibitor characterized by its coexistence with several physiological derivatives displaying immunological cross-reactivity when analyzed in plasma with usual anti-ITI antisera. Taking advantage of the presence in urine of a particular ITI derivative, antisera with restricted specificity for either ITI or its derivatives could be prepared. Some applications of these new reagents are given, including qualitative studies and discrete quantitation--by electroimmunoassay--of ITI and ITI derivatives. The procedures herein described should prove useful for qualitative and quantitative analysis to investigators dealing with mixtures of antigenically related proteins.

Alpha-Globulins↗