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Biomedical subjects

F Belleville

Publications and source records attributed to F Belleville.

At least 19 recordsLinked to original sources
PubMed2002

Action of boron at the molecular level: effects on transcription and translation in an acellular system.

It has been shown that boric acid has well-defined biological effects such as stimulation of wound healing in vivo, release of growth factors and cytokines, and increase of the extracellular matrice turnover. We examined its action at the molecular level, using cell-free systems of transcription (isolated placenta nuclei) and translation (wheat germ extract). We found that 10 mM boric acid greatly increased RNA synthesis, measured by absorbance at 260 nm (x 6.4) or by [3H]-UTP uptake (x 11). Full-length functional mRNA was produced because proteins of 14-80 kDa were translated. Among these proteins, factors involved in angiogenesis and, subsequently, in wound healing (VEGF and TGFbeta) were identified by slot blot, whereas growth factors such as FGF1 and TNFalpha were not detected. These results demonstrate that boron may contribute to biological cell activities at both the transcription and translation levels. However, the mechanism of action is still not known.

Autoradiography↗
PubMed2000

Screening of new antioxidant molecules using flow cytometry.

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.

Animals↗
PubMed2000

Assay of a seric human hexapeptide (HWESAS) using a monoclonal antibody and ELISA.

Human serum contains low-molecular-weight growth factors potentiating some in vitro biological effects of IGF-I and IGF-II and recently two peptides were mainly identified: HWESAS and WGHE. In order to determine seric HWESAS concentration, a specific monoclonal antibody against HWESAS was prepared. Its specificity was studied by inhibition tests: this antibody cross-reacts with Y-HWESAS, Cys-HWESAS. It does not react with HWESAS when its COOH is blocked, or with HWE, WGHE and tryptophan or with C3f (SSKITHRIHWESASLLR) which is a fragment of human complement containing HWESAS motif. Its affinity was measured by non competitive enzyme immunoassay (3.89+/-2.44.10(8) M(-1)). Then, this antibody was used in enzyme-linked immunosorbent assay (ELISA) and the preliminary assays were performed to detect HWESAS in serum. In contrast to healthy subjects, patients with chronic renal failure exhibited undetectable concentration of hexapeptide while after successful renal transplantation values increased to reach levels found in healthy subjects and varying according to post-operative evolution. These data are a strong hint that the kidney plays an important role in the production of this hexapeptide and underly the clinical interest of HWESAS detection in renal pathology.

Amino Acid Sequence↗
PubMed2000

Effects of boron derivatives on extracellular matrix formation.

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.

Animals↗
PubMed1998

Purification from human plasma of a hexapeptide that potentiates the sulfation and mitogenic activities of insulin-like growth factors.

The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement.

Animals↗
PubMed1998

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts.

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.

Boric Acids↗
PubMed1998

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture.

The effect of low concentrations of hydrogen peroxide (H2O2) (5 x 10(-7)-9.5 x 10(-7) M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H2O2 (8 x 10(-7) M) but H2O2 concentration of 7 x 10(-6) M and above increased cell death. H2O2 stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 x 10(-7) or 1 x 10(-5) M H2O2 was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 x 10(-7) M H2O2 was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H2O2 or peroxides (detected with DCFH-DA) by cells incubated with 1 x 10(-5) M H2O2 which increased with increasing H2O2 until cell death.

Animals↗
PubMed1998

In vivo and in vitro effects of boron and boronated compounds.

Boron is ubiquitously present in soils and water. Associated with pectin it is essential for vascular plants as a component of cell walls, and it stabilizes cell membranes. It is required for the growth of pollen tubes and is involved in membrane transport, stimulating H(+)-pumping ATPase activity and K+ uptake. However, a high boron concentration in the soils is toxic to plants and some boronated derivatives are used as herbicides. An absolute requirement for boron has not been definitively demonstrated in animals and humans. However, experiments with boron supplementation or deprivation show that boron is involved in calcium and bone metabolism, and its effects are more marked when other nutrients (cholecalciferol, magnesium) are deficient. Boron supplementation increases the serum concentration of 17 beta-estradiol and testosterone but boron excess has toxic effects on reproductive function. Boron may be involved in cerebral function via its effects on the transport across membranes. It affects the synthesis of the extracellular matrix and is beneficial in wound healing. Usual dietary boron consumption in humans is 1-2 mg/day for adults. As boron has been shown to have biological activity, research into the chemistry of boronated compounds has increased. Boronated compounds have been shown to be potent anti-osteoporotic, anti-inflammatory, hypolipemic, anti-coagulant and anti-neoplastic agents both in vitro and in vivo in animals.

Adult↗
PubMed1997

Effect of boric acid solution on cartilage metabolism.

Pelvic cartilage of chick embryo was used to demonstrate that presence of boron in culture medium decreases synthesis of proteoglycans, collagen and total proteins but on the other hand increases the release of these macromolecules. However, when glucose concentration in culture medium is brought to 22mM, the synthesis decrease is no longer observed, whereas release increase persists. Proteins released into the culture medium included heat shock proteins (70 hsp) and tumor necrosis factor alpha (TNF alpha). The amount of phosphorylated proteins was enhanced in presence of boron while endoprotease activity in cartilage and in culture medium was significantly augmented. The in vitro effects of boric acid may explain its in vivo effect on wound healing.

Animals↗
PubMed1997

Alcoholic cirrhosis and cobalamin metabolism.

The cobalamin status of 27 patients suffering from alcoholic cirrhosis and 20 control subjects was analyzed. Plasma cobalamin (p < 0.005), total corrinoids (p < 0.005) and their analogs (p < 0.05) were all significantly elevated in the cirrhosis patients. These differences were due to increased haptocorrin (HC)-bound corrinoid (p < 0.02), which could be explained by a deficient hepatic clearance of cobalamin bound to HC. The increase in the concentration of true cobalamin was greater than that of its analogs. There were positive correlations between cholestasis (serum alkaline phosphatase) and plasma analog concentrations (p < 0.05), HC-bound cobalamin (p < 0.005) and total corrinoids bound to HC (p < 0.005). The plasma concentrations of the indicators of cobalamin deficiency, homocysteine (p < 0.05) and methylmalonic acid (p < 0.001), were increased, which could indicate poor cellular penetration of vitamin B12 or a defect in the activation of the two vitamin-B12-dependent enzymes.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Crohn's disease and vitamin B12 metabolism.

The concentrations of vitamin B12, its analogs, and the haptocorrin and transcobalamin carriers in 21 patients suffering from Crohn's disease and a group of controls (20 adults) were measured. There were no significant differences in the mean values for vitamin B12, total corrinoids (vitamin B12 + analogs), or vitamin B12 or total corrinoids bound to haptocorrin or transcobalamin of the Crohn's and control patients. There was a significant increase in the binding capacity of transcobalamin in the Crohn's patients compared to the controls (P < 0.001), but there was no difference in the binding capacities of haptocorrin. The serum concentrations of the markers of vitamin B12 status, homocysteine and methylmalonic acid, showed an increase (P < 0.01) in homocysteine in the Crohn's disease patients, but no change in methylmalonic acid. As the hyperhomocysteinemia was associated with normal folate concentrations, there may have been a defect in the activation of the enzyme due to altered intracellular vitamin B12 status.

Adult↗
PubMed1995

Purification from human plasma of a tetrapeptide that potentiates insulin-like growth factor-I activity in chick embryo cartilage.

Human plasma has been shown to contain a low molecular weight factor that potentiates human IGF-I stimulation of glycosaminoglycan synthesis in chick embryo cartilage. The peptide was purified and characterized by Edman degradation and electrospray mass spectrometry. The primary structure determined was: Trp-Gly-His-Glu. A homologous synthetic peptide similarly promoted matrix biosynthesis in cartilage exposed to IGF-I.

Amino Acid Sequence↗
PubMed1995

Methods for reducing the ammonia in hybridoma cell cultures.

The factors which limit the proliferation of eukaryotic cells in vitro are still not well known. Ammonia is believed to be toxic for mammalian cell proliferation and secretion. We have tried two approaches to reducing the ammonia in the medium. We first limited the ammonia produced by the cells by replacing glutamine by glutamate. Then, we used two chemical engineering methods to eliminate accumulated ammonia. In one the used medium was passed through a natural cation exchanger: the clinoptilolite. In the other, the culture medium was passed through a hydrophobic microporous hollow fiber module. Replacing the glutamine by glutamate reduced the medium ammonia concentration. The physicochemical removal of ammonia induced a better cell growth, but not a better specific antibody secretion.

Ammonia↗
PubMed1995

Measurement of plasma testosterone by gas chromatography-negative-ion mass spectrometry using pentafluoropropionic derivatives.

Plasma testosterone was measured by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-MS). The testosterone was extracted from plasma using home-made Extrelut columns and diethyl ether elution. It was quantified as the pentafluoropropionate (PFP) derivative by selected-ion monitoring at m/z 560 (testosterone) and 563 (d3-testosterone), accounting for about 34% of the total ion. The characteristics of the method were: extraction recovery about 95%; linearity over the range 1.7-71.5 nmol l-1 with linear regression equation y = 1.41x + 0.0217, r = 0.999; detection limit 3.5 fmol injected with a signal-to-noise ratio of 7.4; within-day variation, 3% for GC-MS, and 5.8% for the whole process; day-to-day coefficient of variation, 6.6-11%, depending on the concentrations. There was a good correlation between the results obtained by GC-MS and RIA (r = 0.994), but the GC-MS values were significantly lower (p < 0.05) than those obtained by RIA.

Adult↗
PubMed1995

A seric low molecular weight factor modulates IGFs effects on chick cartilage metabolism.

The effects of recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) alone and in combination with a partially purified serum low molecular weight growth factor (LMW-GF) were studied by measuring proteoglycan (PG) and total protein synthesis by chick embryo cartilage. rhIGF-I alone did not increase the incorporation of L[3H]serine or [35S]Na2SO4 into whole cartilages. LMW-GF + rhIGF-I markedly increased the incorporation of these precursors. rhIGF-I alone stimulated D[3H]glucosamine uptake, and LMW-GF increased the effect of rhIGF-I. These results for whole cartilage were reproduced with glycosaminoglycans (GAG) extracted from cartilage. LMW-GF acted in synergy with rhIGF-I to stimulate PG core protein synthesis, xylosyl transferase activity and sulfation. Total protein synthesis, as measured by [35S]methionine uptake, was not altered by rhIGF-I. LMW-GF plus rhIGF-I increased the incorporation of this precursor into whole cartilage. The effect of rhIGF-II on PG synthesis was different from that of rhIGF-I. rhIGF-II alone stimulated GAG chain lengthening and sulfation. LMW-GF did not modify the effect of rhIGF-II on these steps. In contrast, rhIGF-II did not stimulate the synthesis of core protein. LMW-GF plus rhIGF-II increased the [3H]serine incorporation into the whole cartilage, but this combination did not stimulate the uptake of [3H]serine into extracted GAG. rhIGF-II plus LMW-GF were also without effect on xylosyl transferase activity. The combination of these factors increased the [35S]methionine incorporation into cartilage total proteins. These results suggest that rhIGF-II does not regulate the PG core protein synthesis, but in combination with LMW-GF, stimulates the synthesis of proteins other than proteoglycan core protein.

Animals↗
PubMed1995

Stimulation of translation by reactive oxygen species in a cell-free system.

Wheat germ lysate was used as a model system for in vitro translation. We show that an increase of the exchange surface between the reaction mixture and the atmosphere enhanced the amount of incorporated cysteine, indicating that early arrest of protein synthesis previously observed in such a system was due to oxygen starvation in the reaction mixture. This hypothesis was confirmed since the amount of proteins synthesized and the rate of translation increased when oxygen was added. We show that an addition of hydrogen peroxide to the translation mixture had the same effect as oxygen, allowing us to postulate that stimulation could be due to a common property between both molecules: the oxidizing behaviour. Free radicals in in vitro translation were believed to be involved since the utilization of iron chelating agents inhibited translation. This hypothesis was emphasized by the positive effect of a free radical generating system and the negative effect of free radical scavengers. These experiments suggest that the superoxide radical plays an important role in in vitro translation.

Cell-Free System↗