Biomedical subjects

J Gautron

Publications and source records attributed to J Gautron.

At least 37 records · Page 2Linked to original sources

PubMed1998

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro.

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3-15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.

Animals↗

PubMed1998

In vitro secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline9]GnRH from the rat hypothalamus exhibits a differential sensitivity to castration and second messengers.

The decapeptide [hydroxyproline9]GnRH (HypGnRH) has been characterized as an endogenous posttranslational product of the gonadotropin-releasing hormone (GnRH) precursor in a wide range of mammalian brains. Despite consistent biological effects, its secretion by the hypothalamus remains hypothetical. We report here in vitro secretion of HypGnRH and GnRH by the hypothalamus from intact and castrated male rats and provide evidence that they are differentially regulated. Both peptides were identified by two anti-GnRH antibodies of different specificities after separation under two high-performance liquid chromatography conditions. Calcium dependency of HypGnRH release was demonstrated under stimulation with KCl in the absence or presence of Ca2+, as well as with Bay K 8644, veratridine, methoxyverapamil, or tetrodotoxin. Activation of signaling pathways involving adenylate cyclase and protein kinases A and C (PKC) induced HypGnRH release. Expression of data as percentage of release over tissue stores revealed a two- to threefold higher release of HypGnRH than of GnRH under the different modes of stimulation used, except under PKC activation which triggered a comparable recruitment of both peptides. Castration selectively affected PKC-coupled GnRH secretion which showed a twofold lesser release than in intact rats, while the HypGnRH release was unaffected. We conclude that HypGnRH and GnRH are not secreted from the hypothalamus according to the same mechanisms.

Adenylyl Cyclases↗

PubMed1997

Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens.

Organic constituents of the uterine fluid, the acellular milieu in which the eggshell is mineralized, were biochemically characterized at initial, mid and final stages of shell calcification in hens. The electrophoretic protein profiles changed at the different stages of shell mineralization. Two major bands (80-kDa and 43-kDa glycoproteins) with calcium affinity were specific to the initial stage. Four protein bands of 180, 150, 116 and 32 kDa, present at the phase of rapid shell formation, coprecipitated with calcium carbonate in vitro. At this stage were also present a calcium-binding glycoprotein of 36-kDa and a 20-kDa protein. Uterine fluid of the final stage was characterized by a darker intensity of the 66-kDa band, which showed calcium-binding ability and by the presence of three additional proteins (72, 13 and 6 kDa). At least seven bands of the uterine fluid showed similar migration patterns to those of eggshell extracts. Western blotting with ovocleidin and ovalbumin antisera demonstrated the presence of these matrix proteins in uterine fluid collected at initial and mid phase, respectively. Total uterine fluid collected at the end of calcification and dialyzed uterine fluid from the various stages delayed the rate of calcium precipitation in vitro. These observations demonstrate the presence of precursors of eggshell matrix in the uterine fluid and support the hypothesis of their involvement in the process of eggshell mineralization.

Animals↗

PubMed1996

Growth factors in skeletal muscle regeneration.

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

Animals↗

PubMed1996

Soluble matrix of hen's eggshell extracts changes in vitro the rate of calcium carbonate precipitation and crystal morphology.

1. Extra and intramineral eggshell matrix proteins were solubilised before and after demineralisation by sequential extractions using guanidine hydrochloride and EDTA. 2. The intramineral electrophoretic profile of SDS-PAGE showed the presence of 80, 66, 43, 36 and 15 kDa bands with a predominance of a 17 kDa band. In the extramineral part, the major protein was the 15 kDa band. 3. The introduction of intramineral extract to a metastable solution of calcium carbonate delayed the rate of crystal growth. The delay in the rate of precipitation was elicited by a single fraction (MW 50-80 kDa), isolated by gel filtration chromatography, of eggshell extracts. Extramineral extracts had no effect. 4. Addition in vitro of intramineral eggshell extracts modified the morphology of calcite; the crystals aggregated and showed irregular surfaces. 5. These observations suggest that constituents of the eggshell matrix are involved in the control of calcite growth and crytallographic structure of the hen's eggshell.

Animals↗

PubMed1995

Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration.

We recently described dextran derivatives (RGTA) which stimulate tissue repair in several in vivo models. One of them, RGTA11, has been shown to accelerate crush-induced regeneration and reinnervation of rat EDL and Soleus muscles. In this study we wanted to know if RGTA11 alters the pattern of myosin heavy chain expression during regeneration. In both EDL and Soleus muscles, RGTA11, injected at the moment of the crush, was found to accelerate the shift from neonatal to adult myosin heavy chain isoforms within 2 weeks. The proportion of slow fibers increased considerably, especially in the Soleus where RGTA11 induced a precocious and permanent expression of slow myosin isoform, thus confirming that a more efficient innervation had occurred in the presence of RGTA11. These results illustrate the interesting potential pharmacological use of such dextran derivatives in neuromuscular disease.

Animals↗

PubMed1995

Fast and slow rat muscles degenerate and regenerate differently after whole crush injury.

The whole-crush injured rat skeletal muscle was used as a model to explore the regenerating potentialities of fast and slow muscles. Laminin was chosen to follow changes in basal lamina and desmin to visualize new muscular elements; they were revealed by immunofluorescence on cryostat sections of either fast (extensor digitorum longus) or slow (soleus) regenerating muscle. Soleus myolysis was rapid, extensive and heterogeneous. Basal laminae were nearly destroyed. In contrast, extensor digitorum longus maintained its basal lamina framework during myolysis. Soleus reconstruction began early, following the pattern of remaining basal laminae as closely as possible, but regeneration stagnated from day 16 and the regenerated muscle was fibrotic. In extensor digitorum longus, reconstruction progressed slower than in soleus, but regularly from the periphery toward the centre of the muscle. The regenerated extensor digitorum longus showed a quasi-normal structure from day 16. At the end of the process, the elimination of old basal lamina was completed in extensor digitorum longus, but was not achieved in soleus. We propose that the old basal lamina should help the initiation of reconstruction. This new model also underlines the importance of the turnover of basal laminae in muscular regeneration, and will be useful to understand the background of the different regenerative response of both muscles.

Animals↗

PubMed1995

Calcium solubilization and retention in the gastrointestinal tract in chicks (Gallus domesticus) as a function of gastric acid secretion inhibition and of calcium carbonate particle size.

In chicks, immature pullets and laying hens, the inhibition of gastric acid secretion by omeprazole, an H+,K(+)-transporting ATPase (EC 3.6.1.36) inhibitor, greatly increased proventricular and gizzard pH values. Consequently, gizzard soluble Ca concentration deceased and the insoluble Ca fraction increased. Inhibition of acid secretion increased duodenal pH values in immature pullets and laying hens but not in chicks. Duodenal soluble and ionic Ca concentrations were lowered by gastric acid inhibition in chicks and to a larger extent in immature pullets and laying hens. The use of Ca of coarse particle size increased the gizzard insoluble Ca fraction in chicks and pullets. However, it did not influence its soluble Ca fraction in chicks but tended to reinforce the negative effect of omeprazole on soluble Ca in the gizzard and duodenum of chicks and laying hens. Coarse particles of Ca led to an increase in gizzard and duodenal soluble Ca at the end of eggshell calcification in laying hens. An enhancement in the level of Ca in the diet from 10 to 36 g/kg increased gizzard soluble Ca and duodenal soluble and ionic Ca concentrations in immature and adult hens. Intestinal Ca retention and bone mineralization was unaffected by gastric acid inhibition in chicks but were largely diminished by the use of coarse particles of Ca. Gastric acid inhibition was associated in laying hens with decreased Ca retention to a small extent and with reduced eggshell quality. These observations confirm that gastric acid secretion is of importance for CaCO3 solubilization but question its role as a prerequisite for intestinal Ca retention in chicks and even in hens fed on a high Ca diet.

Animals↗

PubMed1995

[Acceleration of the regeneration of skeletal muscles in adult rats by dextran derivatives].

Dextran derivatives were obtained by controlled successive substitutions of carboxymethyl, carbomethyl-benzylamide and carboxymethyl-benzylamide sulfonate groups on glucose residues. Among these derivatives, RGT11 was selected since it mimicked some properties of heparin or heparan sulfate to stabilise and protect heparin binding growth factors such as FGFs and TGF-beta. Furthermore RGT11 inhibited plasmine and leukocyte elastase. In previous works, we have explained the healing effects of RGT obtained in skin or bone repair models by these protecting and inhibiting properties. We now present the results obtained after a single injection of RGT11 in a regenerating crushed extensor digitorum longus (EDL) muscle. After 8 days, RGT11 injected muscles contained 10 times the number of fibers than controlled injected muscles. Fibers were organized in bundles of normal size. Histological analysis indicated that regeneration was comparable to that observed after 3 weeks without RGT. Hence in vivo, RGT11 could act by protecting the heparin binding growth factors involved in the natural process of muscle regeneration and therefore favour their actions. This family of polymers should offer a new pharmaceutical potential for treating muscle atrophy and destruction and is worth studying further.

Animals↗

PubMed1995

[Effects of substituted dextran on reinnervation of a skeletal muscle in adult rats during regeneration].

RGTA11 is a chemically substituted dextran that mimics some of the properties of heparin or heparan sulphates towards heparin binding growth factors as well as inhibits some heparin binding proteases. In vivo RGTA11 has been shown to enhance muscle regeneration after crush. We now present evidence that RGTA11 can alos favour reinnervation of fast (EDL) as well as slow (soleus) crushed muscles. Both types of muscles were injected with RGTA11 after crushing and nerve cutting. In EDL muscles, after 16 days, motor end plates were more rapidly reformed and choline acetyl-transferase activity was 2 fold higher than in controls. In soleus muscles, after 16 days, motor end plates were reformed at normal size while controls were on average 30% smaller, the 16S form of acetyl-cholinesterase and choline acetyl-transferase activity were twice those of non injected regenerating controls. In conclusion, RGTA11 favours axonal growth and synaptic differentiation, allowing a more rapid reinnervation and maturation of the regenerated fibers. RGTA may present a new family of drugs against neuromuscular degenerescence.

Acetylcholinesterase↗

PubMed1993

Gastric acid secretion in the chicken: effect of histamine H2 antagonists and H+/K(+)-ATPase inhibitors on gastro-intestinal pH and of sexual maturity calcium carbonate level and particle size on proventricular H+/K+ ATPase activity.

1. Cimetidine was more potent 4 hr after a single injection of 25 or 100 mg/kg body wt in increasing gastric pH than other H2 receptor antagonists, ranitidine and famotidine but was less efficient than H+/K(+)-ATPase inhibitors. Omeprazole rose proventricular and gizzard pH at a lower dose than SCH 28080 and Ro 18-5364 (30, 50 and 200 mg/kg body wt, respectively). 2. Proventricular and gizzard pH values were maximal 1 and 4 hr after a single injection of 7.5 mumol/kg body wt omeprazole. Inhibition of acid secretion was maintained for 24 hr after an injection of 100 mumol/kg. 3. H+/K(+)-ATPase activity in vitro was 10 mumol Pi/hr/mg protein in the microsomal fractions of the proventriculus. It was doubled by nigericine and inhibited by SCH 28080. However, western blots by high specific H+/K(+)-ATPase monoclonal antibody 95-A3 and 95-111 recognized a 42 kDa band but hardly exhibited the specific 95 kDa band recognition. 4. Chickens and immature pullets showed a higher H+/K(+)-ATPase activity than laying hens. Calcium level of the diet did not affect the enzyme activity but coarse particles of calcium fed to pullets or laying hens enhanced the H+/K(+)-ATPase activity when compared with ground particles.

Animals↗

PubMed1991

Changes in the protein kinase C activity or rat sternomastoid muscle during development and after denervation.

The relationship between the activity of protein kinase C (PKC) and muscle innervation was explored in the rat sternomastoid muscle (SM) from day 18 of gestation (E18) to adult age. Between E18 and birth, PKC activity rose 5-fold, and during the day after birth, diminished to a level characteristic of the mature muscle. The rise chiefly occurred in the neural part of the muscle, in both the membrane and the cytosol fractions. Between E18 and day 5 after birth, the ratios of membrane to cytosol PKC activity rose from 0.5 to 10 and 3 respectively in the neural and aneural parts of the muscle. Denervation of adult SM reduced PKC activity by half in the membrane fraction of the neural part but did not significantly change it in the membrane or cytosol fractions of the aneural parts. These results suggest that innervation plays an important part in determining the level of PKC activity in muscle.

Aging↗

PubMed1991

Whitening of brown-shelled eggs: mineral composition of uterine fluid and rate of protoporphyrin deposition.

Changes in the mineral composition of uterine fluid during shell formation and the rates of color appearance and porphyrin deposition on the shell were measured in two subpopulations of brown egg-laying hens with familial histories of low or high incidences of shell whitening. Increases in shell weight and shell breaking strength were correlated with, and proportional to, time spent by the egg in the uterus and were similar in both subpopulations. Shell reflectance decreased and the amount of porphyrin deposited increased linearly 20 to 24 h after oviposition of the preceding egg. Porphyrin deposition was slightly higher at the 23-h stage in the high whitening population but similar amounts of porphyrin were deposited on the shell during the final stage of shell formation in both groups. The coating on the shell responsible for whitening was deposited during the hour prior to oviposition. Uterine fluid pH, pCO2, bicarbonate, and ionized Ca concentrations changed during shell formation but these changes were not related to the incidence of whitening. A milieu supersaturated with calcite solubility product was observed whatever the stage of shell formation. Inorganic phosphorus was not detectable in the uterine fluid whatever the stage of shell formation. The soluble phosphorus fraction of uterine decreased 22 h after oviposition and phosphorus deposition on the shell increased. At the end of egg formation uterine fluid could not be collected. It was concluded that shell whitening was associated with changes in the kinetics of porphyrin deposition rather than with changes in the amount of porphyrin deposited or modifications of uterine fluid composition.

Animals↗

PubMed1990

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.

Antibodies↗

PubMed1990

Slow and fast rat skeletal muscles differ in their plasminogen activator activities.

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.

Animals↗

PubMed1990

Characterization of myogenesis from adult satellite cells cultured in vitro.

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.

Amphibians↗

PubMed1989

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation.

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.

Animals↗

PubMed1988

Differential expression of acetylcholinesterase molecular forms in neural retina and retinal pigmented epithelium during chick development.

Neural retina (NR) and retinal pigmented epithelium (RPE) were used as a model for studying acetylcholinesterase (AChE) expression in neuronal and non-neuronal tissues during development. In chick embryo retina, increasing AChE activity appeared from day 7 to hatching, first in NR and then in RPE. NR contained 3 main AChE forms, 11.3S, 6.5S and 4.5S, resolved by sucrose density gradient centrifugation. An additional 19S form was exclusively detected in RPE whatever the extraction procedure followed. During differentiation, the proportions of AChE molecular form changed until they reached a steady state characteristic of mature neural retina, whereas in RPE, patterns of AChE molecular forms did not change significantly during development. Thus, 19S AChE appeared to be characteristic of non-neuronal retinal tissue. The expression of the AChE molecular forms in NR and RPE, and particularly the 19S form, was independent of the presence of the lens during retinal differentiation.

Acetylcholinesterase↗