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Biomedical subjects

J Gautron

Publications and source records attributed to J Gautron.

At least 19 recordsLinked to original sources
PubMed2005

Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens.

The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.

Animals↗
PubMed2004

Sex ratios in mule duck embryos at various stages of incubation.

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.

Animals↗
PubMed2003

Cloning and characterization of HEP21, a new member of the uPAR/Ly6 protein superfamily predominantly expressed in hen egg white.

Using two-dimensional (2D)-PAGE, partial protein internal sequencing, and PCR with degenerate primers, we cloned a novel cDNA named HEP21 from hen egg white. The 0.5-kb cDNA encodes a 106 amino acid protein with a cysteine spacing pattern suggesting that HEP21 is a new member of the uPAR/CD59/Ly-6/ snake neurotoxin superfamily. The closest homology of HEP21 is to mouse Ly-6C. Unlike most members of this protein family, HEP21 is not glycosylphosphatidylinositol (GPI)-anchored but is a secreted protein, as indicated by its localization and the presence of a signal peptide in its sequence. Moreover, HEP21 appears as an original member of this protein superfamily because it is predominantly expressed in a tissue, i.e., the oviduct, and especially the magnum where the egg white components are secreted.

Amino Acid Sequence↗
PubMed2003

Purification of ovocalyxin-32, a novel chicken eggshell matrix protein.

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate and an organic matrix from the acellular uterine fluid. Characterization of the individual matrix components is necessary to determine their influence upon calcite crystal shape, size, and orientation during eggshell calcification. We have purified and sequenced a novel 32-kDa protein, ovocalyxin-32 (OCX-32), which is present at high levels in the uterine fluid during the terminal phase of eggshell formation, and is localized predominantly in the outer eggshell. Database searches identified expressed sequence tags (ESTs) whose alignment yielded the complete cDNA. OCX-32 protein possesses limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in rat cerebral cortex and mast cells, and to a skin protein that is encoded by a retinoic acid receptor-responsive gene, TIG1. The timing of OCX-32 secretion into the uterine fluid suggests that it may play a role in the termination of mineral deposition.

Animals↗
PubMed2002

Differential changes in protein kinase C associated with regeneration of rat extensor digitorum longus and soleus muscles.

We used a model of crush-induced regeneration in rat in order to characterize biochemically and histologically the implication of protein kinase C (PKC) in muscle repair after damage. In this model, slow soleus and fast extensor digitorum longus (EDL) muscle regeneration proceed differently. PKC activity has been assayed in regenerating muscles and their intact contralateral during the first 14 days following crushing. Degeneration (myolysis) occurring shortly after crush was associated with a marked down-regulation of the enzyme in both wound muscles and notable increase in the corresponding contralateral muscles. Muscle fiber reconstruction in EDL was associated with a rise in PKC activity which peaked at day 7 in regenerating muscle where it was twice higher than in intact muscle. At variance, muscle PKC activity in soleus increased slower than that of EDL and reached later intact level. Western blot analysis and immunohistochemical studies of representative members of the three PKC subfamilies were performed. All the isoform tested were much less expressed in regenerating than in control intact muscles suggesting that the overall PKC activity in regenerating muscles was more activable than in controls. We have shown that PKC isoforms were sequentially expressed during regeneration in both muscle types. PKC theta; being present the earliest, then delta, epsilon and alpha and finally zeta, beta and eta. Some isoforms were differentially expressed according muscle type. PKC delta being more expressed in soleus whereas beta and eta appeared earlier in EDL. Histochemical studies have revealed that the isoforms were differently localized in muscle tissue and that fiber regeneration was associated with PKC alpha translocation from sarcoplasma to sarcolemma. Together these data have shown that multiple PKC isoforms are implicated in the regenerative process acting at different in times and location and suggesting that individual isoform may fulfill distinct functions.

Animals↗
PubMed2001

Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization.

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.

Amino Acid Sequence↗
PubMed2001

Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration.

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.

Animals↗
PubMed2001

Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer.

The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80 kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

Amino Acid Sequence↗
PubMed2000

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.

Animals↗
PubMed2000

Extraction and quantification by ELISA of eggshell organic matrix proteins (ovocleidin-17, ovalbumin, ovotransferrin) in shell from young and old hens.

The eggshell matrix is mainly composed of proteins that are thought to influence shell formation and calcification and, thus, modify the resulting properties of the shell. We investigated the potential of some of these proteins as biomarkers of eggshell quality by developing a competitive indirect ELISA for quantifying ovotransferrin, ovalbumin, and ovocleidin-17 in eggshell extract. Eggshell fragments were demineralized in acetic acid (20%) and freeze-dried. The micro-extraction yield was markedly increased (>50%) when Tween 20 was added to the subsequent extraction and dialysis milieus. Microplates were coated with ovotransferrin and ovalbumin in a 0.1M carbonate-bicarbonate buffer, but ovocleidin-17 was fixed with acetone (-20 C, 20 min). Optimal dilutions of the monoclonal (ovotransferrin) and polyclonal (ovalbumin and ovocleidin-17) antibodies were 1/3,000, 1/25,000 and 1/4,000, respectively. The inhibition curves were optimized by preincubating the antibodies and proteins overnight. The intraassay coefficient (<5%), parallelism of the standards and samples curves, and recovery (101%) were satisfactory for ovotransferrin. Measurements of ovalbumin were less precise because of higher interassay variation and differences between the slopes of standard and sample inhibition curves. Ovocleidin-17 assays showed similar slopes for standard and eggshell extracts. Although the total protein in soluble matrix extracts was not affected by age, the concentrations of these proteins were higher in eggshell extracts from older hens compared with those from young hens: 1.98x for ovotransferrin, 1.86x for ovalbumin, and 1.58x for ovocleidin-17. The quantification of specific eggshell matrix proteins in shell of differing quality is, therefore, a promising tool for analyzing the origin of eggshell faults and may provide useful information for breeding programs.

Aging↗
PubMed2000

The effect of avian uterine fluid on the growth behavior of calcite crystals.

Eggshell formation takes place on the eggshell membrane in an acellular medium, the uterine fluid that contains the inorganic minerals and precursors of the organic matrix. The high degree of eggshell structure could be due to an interaction between calcium carbonate and the organic matrix. The aim of this study was to demonstrate such an interaction by measuring the effect in vitro of uterine fluid collected at various phases of shell formation on precipitation kinetics, size, and morphology of calcite crystals. The SDS-PAGE profiles of the organic constituents differed between the different phases of eggshell formation. The predominant constituents were ovalbumin and ovotransferrin at the initial phase and lysozyme, ovocleidin-17, ovocalyxin-32, 36- and 21-kDa bands, and ovocleidin-116 at the growth phase. These proteins were numerous in the terminal phase and showed an increased staining of the 32- and 66-kDa bands and appearance of very low molecular weight bands. The precipitation lag time was shortened in proportion to the protein concentration at the initial stage. The effect was observed with a lower magnitude in the presence of constituents of growth and terminal phases. Crystal size was smaller in the presence of constituents from the three stages compared with the control. Components from the initial phase induced the formation of twinned crystals and of rounded corners in the rhombohedric crystals. The presence of components from the growth and terminal phases strongly modified the morphology of the calcite crystals. The majority of the corners became rough and developed curved faces. These observations confirm the interaction of the uterine fluid with calcite and its contribution to eggshell structure.

Animals↗
PubMed2000

Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.

Animals↗
PubMed1999

Molecular cloning and ultrastructural localization of the core protein of an eggshell matrix proteoglycan, ovocleidin-116.

The role of avian eggshell matrix proteins in shell formation is poorly understood. This calcitic biomaterial forms in a uterine fluid where the protein composition varies during the initial, calcification, and terminal phases of eggshell deposition. A specific antibody was raised to a 116-kDa protein, which is most abundant in uterine fluid during active eggshell calcification. This antiserum was used to expression screen a bacteriophage cDNA library prepared using mRNA extracted from pooled uterine tissue harvested at the midpoint of eggshell calcification. Plasmids containing inserts of differing 5'-lengths were isolated with a maximum cDNA sequence of 2.4 kilobases. Northern blotting and reverse transcriptase-polymerase chain reaction demonstrated that the 2. 35-kilobase message was expressed in a uterine-specific manner. The hypothetical translational product from the open reading frame corresponded to a novel 80-kDa protein, which we have named ovocleidin-116. After removal of the predicted signal peptide, its N-terminal sequence corresponded almost exactly with that determined from direct microsequencing of the 116-kDa uterine protein (this work) and with that previously determined for the core protein of a 120-kDa eggshell dermatan sulfate proteoglycan (Corrino, D. A., Rodriguez, J. P., and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175-193). Ultrastructural colloidal gold immunocytochemistry of ovocleidin-116 demonstrated its presence in the organic matrix, in small vesicles found throughout the mineralized palisade layer, and the calcium reserve assembly of the mammillary layer. Ovocleidin-116 thus is a candidate molecule for the regulation of calcite growth during eggshell calcification.

Amino Acid Sequence↗
PubMed1999

Features of eggshell formation in guinea fowl: kinetics of shell deposition, uterine protein secretion and uterine histology.

1. Rate of calcium carbonate deposition, duration of eggshell formation, organic composition of the uterine fluid, morphology of the egg shells and histochemistry of the uterus were studied in guinea fowl to analyse the origin of such thick, strong egg shells. 2. The egg shell was linearly deposited from 6.4 h to 21.8 h after the oviposition of the previous egg. The rate of egg shell deposition was similar to that in laying hens. However, the duration of linear shell deposition was increased by 2.1 h relative to that in hens. This explained the increased egg shell weight observed in the guinea fowl. 3. Intervals between oviposition of intra-clutch eggs were 24 h throughout the laying period. Ovulation occurred just after oviposition of the previous egg in the guinea fowl, as previously observed in hens but the duration of egg white protein deposition, of plumping and of initiation of shell mineralisation were all 1.5 h shorter than in domestic hen. 4. Uterine fluid can only be collected during the growth and terminal phase of shell formation. The electrophoretic profiles of the uterine fluid differed between phases and were somewhat different from those previously observed in the hen. Ovalbumin and ovocleidin-17 were both present in the uterine fluid and also in egg shell extract. Ovocleidin-17 was predominant during the growth phase. 5. The histology of the uterus differed slightly in guinea fowl compared to hens. Ovocleidin and ovalbumin are both secreted by the tubular glands. 6. Examination of radial ultrathin sections of eggshell showed, above the mammillary layer, intricate interlacing of adjacent exospherite in guinea fowl in contrast to the continuous columnar microstructure in hens. 7. The kinetics of egg shell deposition largely explains the increased egg shell weight of guinea fowl. The organic matrix proteins may be associated with the contrast between the structural organisation of the guinea fowl egg shell and that of the hen egg shell.

Animals↗
PubMed1999

Organic matrix composition and ultrastructure of eggshell: a comparative study.

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.

Animals↗
PubMed1999

A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle.

Ischemia and denervation of EDL muscle of adult rat induce a large central zone of degeneration surrounded by a thin zone of peripheral surviving muscle fibers. Muscle regeneration is a complex phenomenon in which many agents interact, such as growth factors and heparan sulfate components of the extracellular matrix. We have shown that synthetic polymers, called RGTA (as regenerating agents), which imitate the heparan sulfates, are able to stimulate tissue repair when applied at the site of injury. In crushed muscles, RGTA were found to accelerate both regeneration and reinnervation. In vitro, RGTA act as protectors and potentiators of various heparin binding growth factors (HBGF). It was postulated that in vivo their tissue repair properties were due in part to an increase of bioavailability of endogenously released HBGF. In the present work, we show that ischemic and denervated EDL muscle treated by a unique injection of RGTA differs from the control after 1 wk in several aspects: 1) the epimysial postinflammatory reaction is inhibited and the area of fibrotic tissue among fibers is reduced; 2) the peripheral zone, as measured by the number of intact muscle fibers, was increased by more than twofold; and 3) In the central zone, RGTA enhances the regeneration of the muscle fibers as well as muscle revascularization. These results suggest that RGTA both protects muscle fibers from degeneration and preserves the differentiated state of the surviving fibers. For the first time it is demonstrated that a functionalized polymeric compound can prevent some of the damage resulting from muscle ischemia. RGTA may therefore open a new therapeutic approach for muscle fibrosis and other postischemic muscle pathologies.

Animals↗