Search PubMed⌕ Search

Biomedical subjects

P Brachet

Publications and source records attributed to P Brachet.

At least 91 records · Page 5Linked to original sources
PubMed1988

Serum contains a macromolecular effector promoting the synthesis of nerve growth factor (NGF) in L cells.

Addition of serum to the culture medium of murine L cells increased both the cellular level of NGF mRNA and the secretion of mature factor. Stimulation of NGF production by the serum was dose-dependent and appeared mediated by some specific factor(s). After gel filtration chromatography of serum, most of the biological activity formed a major peak with an apparent MW of about 160 kDa. This promoting factor was sensitive to heat at neutral pH, but resisted after heating at pH4. An activity inducing NGF synthesis, and displaying a comparable thermal sensitivity was also detected in Cohn fraction IV of human or bovine plasma.

Animals↗
PubMed1988

Messenger RNAs of beta-amyloid precursor protein and prion protein are regulated by nerve growth factor in PC12 cells.

The effect of the neurotrophic factor NGF on the expression of two genes involved in the accumulation of amyloid deposits in neurodegenerative disorders was studied in a clonal cell line, PC12. Use of hybridization methods showed that NGF increased the cellular pool of the mRNA of the prion protein, a macromolecule known to generate fibrillary aggregates in the brain of scrapie-infected animals. Maximal levels of prion mRNA were obtained after 7 days of treatment, but a significant increase was already detectable after 48 hr of exposure to NGF. In contrast, the factor did not increase the cellular content of the transcripts coding for the precursor of the beta-amyloid peptide (APP), which participates in the formation of neuritic plaques in human brains affected by Alzheimer's disease. However, NGF caused a drop in the molecular weight of that mRNA. This change, which is likely to result from a loss of 100-200 bp, was already detected after 24 hr of treatment. These results indicate that NGF induces in target neuronal cells a quantitative and a qualitative modification of the transcription products encoding two different amyloid precursor proteins.

Amyloid↗
PubMed1987

Retinoic acid increases the expression of NGF gene in mouse L cells.

Retinoic acid (RA), the acid form of vitamin A, is shown to enhance the synthesis of nerve growth factor (NGF) in cultures of mouse L cells. Maximal stimulation was observed in cells growing in a serum-free medium supplemented with 10(-6)M RA during 48 h. The drug increased both the level of NGF mRNA and the amount of mature NGF protein secreted by the cells. RA was previously reported to increase the number of NGF receptors on some neuroblastoma cells (Haskell et al., 1987 Cell and Tiss. Res., 247, 67-73). It seems, therefore, that RA may influence nerve cell differentiation by promoting both the synthesis of the neurotrophic factor and the responsiveness of target cells.

Animals↗
PubMed1987

Transport of methionine hydroxy analog across the brush border membrane of rat jejunum.

Brush border membrane vesicles and everted rings of rat jejunum were used to study the absorption of 2-hydroxy-4-methylthiobutyric acid, the alpha-hydroxy analog of methionine (DL-MHA). Uptake by membrane vesicles was found to be unaltered by an inwardly directed sodium gradient in contrast to that of L-methionine, D-methionine and L-lactate, which is sodium stimulated and exhibits a characteristic "overshoot" phenomenon. Initial uptake rates of both L- and D-methionine (0.77 mM) were respectively 6.8 and 1.5 times higher than that of DL-MHA (0.80 mM) under Na+-gradient conditions. However, the hydroxy analog significantly inhibited L-lactate accumulation into membrane vesicles. Uptake of DL-MHA into rat jejunal rings was the sum of a saturable Michaelian component [KappM = 4.4 mM and Vappmax = 587 nmol/(g X min)] and a diffusive component [Kd = 47 microL/(g X min)]. Furthermore, L-lactate completely inhibited the carrier-mediated uptake of DL-MHA. All this demonstrates that jejunal uptake of DL-MHA in the rat is mediated by an Na+-independent carrier system associated with L-lactate transport and thus is quite distinct from the L-methionine pathway. This difference could help explain numerous findings with respect to a lower biological utilization of the hydroxy analog in some species when fed as the sole source of methionine in purified crystalline amino acid diets.

Animals↗
PubMed1987

Kinetic evidence for separate systems in transport of D- and L-methionine by rat small intestine.

The kinetics of D- and L-methionine uptake by rings of everted intestine in vitro are consistent with a saturable Michaelis-Menten component (Km = 11.7 and 1.7 mM; Vmax = 0.53 and 0.74 mumol X g-1 X min-1 for D- and L-methionine, respectively) plus a linear, diffusional one. All the data could be fit with a diffusion constant (Kd = 3.2 microliters X g-1 X min-1), which was essentially the same, independent of whether it was estimated by iteration or by using the extracellular marker, inulin. Similar results were obtained from in vivo perfusion experiments, except that the diffusional term was negligible. D-Methionine was found to inhibit L-methionine uptake by intestinal rings according to fully noncompetitive kinetics (Ki = 45 mM). Another set of experiments with jejunal brush-border membrane vesicles showed that D-methionine uptake is dependent on a Na+ gradient and is significantly inhibited by L-methionine and L-proline, but not by beta-alanine and alpha-methylaminoisobutyric acid. Our results indicate that, in rat jejunum, D-methionine is taken up through a Na+-dependent pathway distinct from the neutral amino acid (L-methionine) carrier and from the imino acid (L-proline, alpha-methylaminoisobutyric acid, beta-alanine) carrier.

Animals↗
PubMed1986

Molecular cloning of the avian beta-nerve growth factor gene: transcription in brain.

A chicken gene cross-hybridizing with a murine beta-nerve growth factor (beta NGF) cDNA probe was identified by Southern blot analysis and isolated from a genomic DNA library. The DNA sequence coding for the putative mature beta NGF protein was determined, providing direct evidence for the existence in birds of a neurotrophic factor sharing a high degree of sequence homology with mammalian beta NGF. In addition this gene is shown to be transcriptionally active in adult avian brain as demonstrated by Northern blot analysis.

Animals↗
PubMed1986

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines.

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.

Animals↗
PubMed1986

Dexamethasone rapidly reduces the expression of the beta-NGF gene in mouse L-929 cells.

Mouse L-929 cells were treated with dexamethasone, and the cellular levels of beta-NGF mRNA were estimated by hybridization of the RNAs with a beta-NGF cDNA probe. The results revealed that the glucocorticoid decreased specifically, in a dose-dependent manner, the pool of beta-NGF transcripts. After 4 h, L-929 cells cultured with 10(-7) M dexamethasone contained one-fifth as much beta-NGF mRNA as untreated control cells, and as little as one-tenth as much when the glucocorticoid concentration was 10(-6) M. The effect of the hormone became maximal after 8 h of treatment. Amounts of beta-NGF secreted by the cells during 24 h were measured with a two-site enzyme immunoassay. They also appeared reduced in cultures exposed to the glucocorticoid. These data indicate that dexamethasone controls negatively the expression of the beta-NGF gene in L-929 cells at some pre-translational level.

Animals↗
PubMed1986

Pheochromocytoma PC12 cells influence the differentiation of co-cultured skeletal myotubes.

Rat skeletal myotubes were cultured in the presence or absence of pheochromocytoma PC12 cells under experimental conditions permitting the maintenance of the muscle cells for up to 5 weeks. The number of acetylcholine receptors (AChRs), estimated using radioiodinated alpha-bungarotoxin (alpha-BTX), was comparable in both types of culture for the first 2 weeks. After prolonged incubation (4 weeks), however, alpha-BTX binding sites were two times more abundant in the PC12-myotube cultures than in the myotube cultures. This is likely to reflect a higher number of AChRs at the surface of the myotubes, for control experiments showed that the contribution of PC12 cells was negligible. Presence of PC12 cells also increased the number of AChR aggregates. Use of fluorescent alpha-BTX demonstrated that mixed cultures contained two times more receptor patches than did myotubes alone during the first 2 weeks of culture, and up to five times more after 4 weeks. Essentially all the tight contacts established between PC12 cells and myotubes were characterized by a receptor cluster and, in some cases, by an aggregate of an acetylcholine esterase (AChE), as visualized by immunohistochemistry. Furthermore an antiserum specific for the adult (fast) isoform of the myosin heavy chain stained some myotubes in long-term, mixed cultures. The antigen was never observed in sister cultures of myotubes alone. These data show that PC12 cells, which are thought to derive from the medullary part of the adrenal gland, can influence the differentiation of skeletal muscle cells in several ways.

Animals↗
PubMed1986

Synthesis of nerve growth factor mRNA and precursor protein in the thyroid and parathyroid glands of the rat.

Nerve growth factor (NGF) mRNAs were detected in the thyroid and parathyroid glands by hybridization to a preproNGF and a NGF-beta cDNA probe. The thyroid NGF transcript was identical in size to that found in the mouse submaxillary gland. Mature NGF-beta was not detected in the thyroid tissue either by immunoprecipitation or by immunohistochemical methods. In contrast, an antiserum directed against the predicted precursor protein sequence immunoprecipitated a protein of Mr 30,000-31,000 in thyroid extracts. A protein of identical molecular weight was also detected by this serum in the mouse submaxillary gland extracts. In vitro translation of hybrid-selected NGF RNAs detected two peptides of Mrs 35,000 and 28,000. These peptides closely correspond to the proposed translation products of the preproNGF mRNA sequence, suggesting that two different initiation codons can be utilized, at least in vitro. The proNGF antiserum also cross-reacted with the in vitro synthesized Mr 35,000 peptide. The antigenic determinant recognized by the precursor-specific antibody was localized to the convoluted granular tubules of the mouse submaxillary gland, known to be the site of synthesis of the NGF-beta. We present evidence for the localization of unprocessed proNGF in the parathyroid gland and in some parafollicular cells of the thyroid.

Animals↗
PubMed1985

Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells.

Mouse L cells synthesize and secrete a neurotrophic factor related to the beta subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the beta-NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the beta-NGF gene locus of these cells. The abundance of the beta-NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of beta-NGF transcript by an additional factor of 4. The presence of 10(-7) M testosterone in the serum-free medium did not modify the level of beta-NGF mRNA, while addition of 10(-7) M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the beta-NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.

Animals↗
PubMed1984

Precursors of the nerve growth factor gamma subunit and renin bind to microtubules.

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.

Animals↗
PubMed1984

Synthesis and partial maturation of the alpha- and gamma-subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes.

Sera raised against the alpha-, beta- and gamma-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti-beta NGF sera did not cross-react with any material. In contrast, the precursors of the alpha- and gamma-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the alpha- and gamma-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of alpha NGF, gamma NGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.

Animals↗
PubMed1984

L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC12 pheochromocytoma cells.

Two mouse cell lines, 3T3 and L, reported to secrete an NGF-like activity in the culture medium were co-cultured with the pheochromocytoma cell line PC12 which responds to NGF in vitro. In these co-cultures mitomycin-treated L or 3T3 cells were employed at low cell density (1 000 cells/cm2). L cells, but not 3T3, promoted efficiently neurite outgrowth of PC12. The response of the PC12 cells was blocked by an antiserum to male mouse submaxillary gland beta NGF. The NGF secreted by the L cells and immunoprecipitated by this antiserum co-migrated with the submaxillary gland beta NGF monomer in SDS-polyacrylamide gels. Surprisingly the neurite-promoting activity of media conditioned by L or by L-PC12 co-cultures was at most one-tenth of that expected on the basis of the response of PC12 cells in the co-cultures. This was not due to proteolytic degradation of the NGF-like factor or to losses by manipulation of the media. It seems therefore that co-cultures provide conditions which enhance the effect of the factor. Possible mechanisms responsible for this effect are discussed.

Animals↗
PubMed1983

[The neurotrophic factor nerve growth factor (NGF) synthetized by murine L-929 cells is not associated with alpha and gamma subunits].

L-929 cells secrete a neurotrophic factor which is immunologically related to the beta NGF present in the male Mouse submaxillary gland. In order to determine whether L-929 cells also synthesize the alpha and gamma subunits which are associated with the beta NGF in the gland, immunoprecipitations were performed with appropriate antisera. Our results indicate the absence of alpha and gamma subunits in the extracellular medium or in the cell extracts and suggest that the synthesis and secretion of the beta NGF by the L cells does not require these two proteins.

Animals↗
PubMed1982

Cellular responsiveness to cyclic AMP in a phosphodiesterase-defective mutant of Dictyostelium discoideum.

Responsiveness of Dictyostelium discoideum amoebae to cAMP, a chemotactic mediator, was investigated in a strain defective in cAMP-phosphodiesterase production. Cells were subjected to a high cAMP signal (10(-6) M) in the presence or absence of exogenous phosphodiesterase, and the changes of intracellular cAMP and cGMP concentrations and of adenylate cyclase activity were measured. In the presence of cAMP hydrolysis, both adenylate and guanylate cyclases are transiently activated. In the absence of hydrolysis, the high and constant extracellular cAMP concentration is sufficient to elicit a re-activation of adenylate cyclase a few minutes after the first transient response. In contrast, levels of cGMP remain basal for at least 20 min after termination of the initial response to the cAMP addition.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae.

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.

3',5'-Cyclic-GMP Phosphodiesterases↗