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Biomedical subjects

F Lamy

Publications and source records attributed to F Lamy.

At least 55 records · Page 3Linked to original sources
PubMed1987

Photolysis and ozonolysis of (iso)desmosine-containing crosslinked peptides from porcine aorta elastin.

This report describes the use of photolysis and ozonolysis as a means of achieving complete cleavage of the pyridinium ring of (iso)desmosine in crosslinked elastin peptides. Although photolysis leads to the opening of the ring with concomitant formation of lysine, the peptide chains remain attached. Subsequent ozonolysis is able to completely achieve the cleavage of the rest of the ring skeleton, thus leading to the separation of the peptide chains. Formation of new amino acids, i.e. alpha-aminoadipic and glutamic acids, is emphasized. Localization of these amino acids within the released peptides should be of help in structural investigations on the crosslinking zones involving either isodesmosine or desmosine. However, other amino acids such as tyrosine and phenylalanine are sensitive to this procedure and side reactions occur which are responsible for peptide bond cleavage with the formation of breakdown products.

Amino Acids↗
PubMed1986

Determination of proteins and sulfobetaine with the Folin-phenol reagent.

This paper describes a method for the quantitative analysis of solutions containing a mixture of proteins and sulfobetaine. In a preliminary step the proteins, which interfere with the detergent assay, are separated by precipitation with trichloroacetic acid (8%). The insoluble fraction, dissolved in NaOH (1.0 N), and the soluble fraction, containing the detergent, are treated with the Folin-Ciocalteu phenol reagent, essentially following the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275). The absorbance of the protein fraction is read, as usual at 750 nm, while that of the detergent solution is read at 342 nm. At this wavelength, sulfobetaine, treated with the Folin reagent, absorbs strongly, the absorbances being proportional to its concentration up to 1.5 mg/ml.

Betaine↗
PubMed1986

Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum.

Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. The analysis of the proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr approximately equal to 81 000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6') while TSH specifically decreased the labeling of four polypeptides (proteins 1'-4') and increased the labeling of one polypeptide (protein 5') whose synthesis was decreased by EGF and serum. The specific effect of TSH on one protein labeling (protein 7; Mr approximately equal to 39 000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture.

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum-free and thyrotropin-free conditions. Thyrotropin was then added for varying periods of time (6-96 h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one- and two-dimensional gel electrophoresis, followed by autoradiography. The results were as follows. After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively. Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin. A modification in the ratio between alpha and beta tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30 000-40 000) and the isoelectric points of tropomyosins. Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP. In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.

Actins↗
PubMed1985

Analysis of proteins synthesized by fibroblasts from patients with cystic fibrosis by two-dimensional gel electrophoresis and double label autoradiography.

Mucoviscidosis, the most frequently lethal genetic syndrome of Caucasian population, is a recessive disease with multiple tissue involvement. Although the major pathological changes are observed in lungs and pancreas, abnormalities have also been detected in several other exocrine glands. For many reasons, such as the ready availability of tissue material, the absence of secondary changes and the potential for prenatal diagnosis, cultured skin fibroblasts could be the tissue of choice to search for the primary defect. Several abnormalities have been reported in CF fibroblasts, suggesting that the genetic abnormality is expressed in these cells. To search for potentially mutant protein(s) we have compared the protein composition of normal and CF fibroblasts by two dimensional gel electrophoresis and double-labeling autoradiography using 35S and 75Se methionine as tracer. The results demonstrate the power of the method; however, we have not found one protein spot consistently missing in CF cells. Possible reasons for the absence of a single common identifiable defect are discussed.

Adolescent↗
PubMed1985

Patterns of protein synthesis in endometrial tissues from ovariectomized rats treated with oestradiol and progesterone.

Groups of ovariectomized rats were taken as controls or given hormonal treatment mimicking the successive steps in the sequence of ovarian secretions leading to implantation. Total endometrium or separated epithelium and stroma were incubated in vitro with [35S]methionine. Dissolved proteins were submitted to two-dimensional polyacrylamide gel electrophoresis (pH range 5-7), followed by autoradiography. Priming with oestradiol (2 days) and subsequent treatment with progesterone (3 days) enhanced the synthesis of 12 and 14 polypeptides, respectively, which are specific for each of these treatments. Progesterone also suppressed the production of 10 oestrogen-dependent proteins both in the epithelium and the stroma. When an oestrogen-progesterone-oestrogen treatment was given, synthesis of all but 4 of the progesterone-induced polypeptides in the epithelium was inhibited while 5 of the proteins abolished by progesterone in this tissue compartment reappeared. These results are compatible with a mechanism of implantation acting at the epithelial level by lifting of intrauterine inhibition and stimulation by embryotrophic substances.

Animals↗
PubMed1985

Photolysis and ozonolysis of desmosine and elastolytic peptides.

This paper describes the combined effects of photolysis and ozonolysis upon the main interchain crosslinks of elastin, desmosine and isodesmosine. Photolysis of purified (iso)desmosines in solution leads to the cleavage of the pyridinium rings to give lysine and an analogue of glutaconic aldehyde which is deformylated to a stable compound absorbing at 242 nm. This photoproduct is subsequently fragmented by ozone into glutamic, alpha-aminoadipic and possibly alpha-amino-delta-oxocaproic acids. However the yields of these different compounds are very low because we observed that numerous competing side reactions (polymerisation, recyclisation) accompany the photoozonolytic decomposition of the pyridinium rings of free (iso)desmosines. The results are clearer when reticulated elastolytic peptides are photoozonolysed. The (iso)desmosines, covalently linked in these peptides, are cleaved into lysine, glutamic and alpha-aminoadipic acids (in a ratio 2:1) with yields corresponding to 80-90% of those expected from the decomposition of the (iso)desmosines originally present in the peptide fraction. We have also observed that ozonolysis alone degraded another crosslink present in these peptides, the aldol condensation product, resulting in the production again of glutamic and alpha-aminoadipic acids in amounts consistent with the known concentrations of this particular crosslink in elastin. Finally we noted that the complete photoozonolytic degradation of the (iso)desmosines present in a semi purified reticulated elastolytic fraction resulted in a shift of the size distribution of these peptides toward lower values. It is not certain however that this shift, indicative of a freeing of the polypeptide chains from their original three dimensional network, is due uniquely to the cleavage of the (iso)desmosines. Indeed we have observed that tyrosine and phenylalanine were also degraded during photoozonolysis. Not knowing the mechanism of this degradation it is impossible to rule out the possibility that concomitant cleavages of peptide bonds did occur.

Amino Acids↗
PubMed1984

The diagnosis of cutaneous T-cell lymphoma by morphometric evaluation of the cellular infiltrate, using semithin sections.

In order to improve the cytological criteria for the diagnosis of cutaneous T-cell lymphomas (CTCL), a number of morphometric assessments in semithin sections have been performed on the dermal infiltrates of twenty-one cases of overt CTCL (group I) and twenty-two cases of well-defined benign dermatoses (group II). In each biopsy, an average of 250 cells were measured for perimeter (P), surface area (S), 'nuclear shape index' (NSI), mean surface and mean NSI. We also determined the percentage of typical 'highly cerebriform lymphocytes' (HCL). Cells of group I patients were found to have mean S and mean NSI that differed significantly from those of group II. HCL were found to have a mean NSI value of less than 0.40. The diagnostic value of these measurements was confirmed in the group I patients. Nineteen of them had a mean NSI value of less than 0.61, together with a mean S greater than 14 sq. micron. None of the twenty-two patients of group II had such values. The discriminating power of these criteria was then tested retrospectively on another group of initially controversial patients who presented with suspected CTCL (group III patients). Nine have since evolved into overt CTCL (group III M) while the other ten have remained benign (group III B). All group III B patients and five patients of group III M were correctly assigned using the above morphometric criteria. This method could improve the diagnosis of the early stage of cutaneous T-cell lymphoma.

Cell Nucleus↗
PubMed1983

Isolation and partial characterization of a complex of several (Pro-) endopeptidases from whole porcine pancreatic extract and from purified zymogen granules.

Pancreatic zymogen granules contain exportable proteins at a very high concentration. The mechanism leading to this condensation is unknown. On the other hand, it is known that aqueous extract of pancreatic acetone powder precipitates at low ionic strength and acidic pH. The precipitable fraction is called 'euglobulin' in the literature. We thought that euglobulin could serve as a simplified model to study the condensation of some of the pancreatic exportable proteins. We have compared quantitatively and qualitatively the composition of euglobulins prepared from porcine pancreatic acetone powder and from lysates of purified pancreatic zymogen granules. They were found to be nearly identical, consisting of glycoprotein(s) and/or proteoglycan(s) associated to a proesterase activity, chymotrypsinogens C and D and proelastase. We conclude therefore, that the interactions between the constituents of euglobulin must be specific, since they can occur in the complex protein mixture of the whole organ to a similar extent as in the zymogen granules themselves. We have tried to identify the nature of these specific interactions. We were able to demonstrate that neither the granule membranes, nor the high-molecular-weight proteoglycan present in the granules (Reggio, H.A. and Palade, C.E. (1978) J. Cell. Biol. 77,288-314) were responsible for the observed aggregation. Electrostatic interactions between acidic and basic proteins (Thomson, A. and Denniss, I.S. (1976) Biochim. Biophys. Acta 429, 581-590) were demonstrated between proelastase and chymotrypsinogens C and D. However, the possible roles of the glycoprotein(s) and/or proteoglycan(s) in the condensation process remain unknown.

Amylases↗
PubMed1982

Pattern of protein phosphorylation in rat hepatocytes stimulated by glucagon or by the Ca2+-linked hormones.

We have adapted the high-resolution electrophoretic technique of O'Farrell to analyze phosphorylated proteins from rat hepatocytes. Total proteins were extracted from rat hepatocytes which had been incubated in the presence of [32P]phosphate and with two types of stimuli: glucagon on the one hand and the Ca2+-linked hormones on the other hand. About 200 phosphorylated polypeptides have been separated. Glucagon modifies the incorporation of [32P]phosphate in at least 17 polypeptides and dibutyryladenosine 3',5'-monophosphate mimics this hormonal effect, implying a common mechanism of action. Phenylephrine (in the presence of the beta-antagonist propranolol), vasopressin and angiotensin all modify the incorporation of [32P]phosphate in about 13 polypeptides; since the Ca2+ ionophore A23 187 reproduces the effect of these agents it may be concluded that Ca2+ mediates their effect. Not all the substrates affected by the two types of hormones are identical. Both types of stimuli increase the phosphorylation of a same set of seven proteins and decrease the phosphorylation of a same set of three proteins but seven proteins have their phosphorylation uniquely enhanced by glucagon whereas three other specific proteins get more phosphorylated by the Ca2+ -linked hormones. The clear differences between the patterns of protein phosphorylation observed in the presence of glucagon and dibutyryladenosine 3',5'-monophosphate on the one hand and by the Ca2+-linked hormones on the other hand strongly suggest different mechanisms of action for these two types of stimuli.

Angiotensin II↗
PubMed1982

Changes in the pattern of endometrial protein synthesis during decidualization in the rat.

Ovariectomized rats were given hormonal treatment mimicking progestational ovarian secretions. At maximal sensitivity a decidual reaction was induced by scratching the endometrium. After incubation of tissue with [35S]methionine, proteins were extracted from decidualizing and control endometrium and submitted to two-dimensional polyacrylamide gel electrophoresis (pH range 5--7), followed by staining or autoradiography. A total of about 800 different peptides could be distinguished on the gels. By 24 h after the decidual stimulus the most prominent changes were the appearance of 4 new peptides (mol. wt 12 000, 25 000, 42 000 and 56 000) and the disappearance of 5 others (mol. wt 31 000, 35 000, 100 000, 110 000 and 140 000). This new pattern remained grossly unchanged up to 72 h after decidual induction.

Animals↗
PubMed1981

Freeze-fracture and deep-etching studies on zymogen-granule membranes of the rat pancreas.

Whole pancreatic zymogen granules or their membrane fraction were examined by freeze-fracture or deep-etching under different experimental conditions. The granules were fixed for different time periods, or not fixed, and were cryoprotected with glycerol or DMSO; 3% glutaraldehyde followed by 30% glycerol were finally chosen for giving the best resolution and the highest density of intramembrane particles (IMP). IMP are present on the PF and EF leaflets. Their number decreases with the duration of the fixation. Several granules exhibit IMP-free blebs. Incubation of the granules with protamine sulfate causes an aggregation of IMP and of the rough-textured background on the EF leaflet. A second fracture plane can be formed and has been shown by deep-etching to be intercalated between PF and EF. Deep-etching has also shown that particles attached to the perimeter of the granules and of the blebs are, in fact, large nodules on the PS face which partially extend onto the blebs and do not aggregate with the IMP after protamine treatment. Fusion is also indicated between membrane vesicles. Freeze-fracture of the purified membrane fraction seems to indicate the formation of an IMP cap during the lysis of the granule. Moreover, large nodules remain present on the PS face on these membrane fractions but the majority disappear after washing at pH 11.2 with Na2CO3 and EDTA.

Animals↗