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C Lacombe

Publications and source records attributed to C Lacombe.

At least 109 records · Page 6Linked to original sources
PubMed1990

Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex.

In order to study the erythropoietin receptor in its native state, we solubilized erythropoietin-receptor complexes from spleen cell membranes of mice infected with the anemia strain of Friend virus using mild detergents. Among 11 tested detergents, Triton X-100 and Lubrol PX were the most effective. Triton X-100 was therefore selected for this study. The solubilized complexes appeared to be well representative of the total membrane receptor population as indicated by cross-linking experiments and affinity measurements. The hydrodynamic characteristics of the complexes were determined by gel filtration chromatography and ultracentrifugation through sucrose gradients prepared with H2O or D2O. Although erythropoietin-receptor-detergent complexes exhibited some heterogeneity, we determined the following minimal hydrodynamic values: sedimentation coefficient (s20,w): 11.7 +/- 0.8 S, Stokes radius: 7.7 +/- 0.2 nm, partial specific volume: 0.774 +/- 0.017 ml/g, giving a molecular mass of 458 +/- 66 kDa. The contribution of the detergent was estimated to be 28% from the measured partial specific volume, giving an estimated molecular mass of 330 +/- 48 kDa for the erythropoietin-receptor complex. The minimal molecular mass value was significantly greater than those obtained by polyacrylamide gel electrophoresis under denaturing conditions, strongly suggesting that the erythropoietin receptors were present as multimeric complexes. The nature of these complexes is discussed. Beside this major component our results revealed the presence of higher-molecular-mass erythropoietin binding components. We also demonstrated that erythropoietin-receptor complexes could be precipitated with anti-erythropoietin antibodies. This property should greatly improve the purification of erythropoietin receptors.

Animals↗
PubMed1990

Glycosylation of the murine erythropoietin receptor.

Murine erythropoietin-responsive Rauscher Red 5-1.5 cells were used to determine the contribution of glycosylation to the size and function of the erythropoietin receptor. The half life of the receptors was determined to be 4 h. The number of receptors was not significantly decreased in cells treated for 48 h with inhibitors of glycosylation (tunicamycin, glucosamine or swainsonine) and their affinity was slightly enhanced in tunicamycin- or glucosamine-treated cells. Erythropoietin was cross-linked with two proteins of 104 and 86 kDa. Their molecular masses were not significantly reduced in cells treated with the glycosylation inhibitors. When immunoprecipitated cross-linked receptors were digested with endoglycosidases, the molecular masses of both proteins were only slightly modified giving values of 100 and 82 kDa. Thus we can conclude that the proteins cross-linked to erythropoietin are very weakly glycosylated.

Alkaloids↗
PubMed1990

Metabolism of low-density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells.

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Adenocarcinoma↗
PubMed1990

Tumor cells are the site of erythropoietin synthesis in human renal cancers associated with polycythemia.

One to five percent of human renal cell carcinomas are associated with polycythemia. It is generally assumed that polycythemia results from the secretion of erythropoietin (Epo) by the malignant cells. However, there is no direct proof supporting this hypothesis. Three patients with typical renal adenocarcinoma and polycythemia were studied. All three exhibited high Epo serum levels as measured by radioimmunoassay (RIA). A strong Epo signal was observed on Northern blot analysis of total RNA extracted from the renal tumors. The Epo message seemed to be of normal size and no Epo gene rearrangement was observed with the restriction enzymes tested. Using the in situ hybridization technique, a significant labeling was constantly observed on the tumor cells. Immunohistochemical studies showed that these tumor cells, known to be of tubular origin, were labeled by an anti-cytokeratin antibody and therefore were of epithelial nature. Thus, this study demonstrated that malignant cells of tubular origin were able to produce Epo constitutively, whereas in the mouse hypoxic kidney, peritubular cells (probably capillary endothelial cells) were the major site of Epo synthesis.

Adenocarcinoma↗
PubMed1990

Hemoglobin Dhonburi alpha 2 beta 2 126 (H4) Val----Gly: a new unstable beta variant producing a beta-thalassemia intermedia phenotype in association with beta zero-thalassemia.

While investigating the mechanism of a beta-thalassemia intermedia phenotype in a 34 year old Thai male, a new Hb variant beta 126 Val----Gly named Hb Dhonburi was discovered. Genetic and structural studies revealed the existence of a beta zero-thalassemia genotype in association with the beta variant. The new variant is unstable but exhibits normal oxygen binding properties. Hb Dhonburi was also discovered in the mother of the propositus in association with Hb E.

1-Propanol↗
PubMed1990

Determination of the red blood cell apparent membrane elastic modulus from viscometric measurements.

Rhelogical measurements on a dilute suspension of red blood cells (RBCs) are interpreted by means of a microheological model that relates the shear evolution of the apparent viscosity to the intrinsic properties of the suspended particles. It is then possible to quantify the average deformability of a RBC population in terms of a mean value of the membrane shear elastic modulus, Es. Dilute suspensions of erthrocytes exhibit shear-thinning behavior with a constant high shear viscosity. This behavior is identical to the one predicted for a suspension of spherical capsules where the same phenomena of deformation and orientation prevail. A comparison between theoretical and experimental curves yields a mean value of Es, assuming all other cell properties--internal viscosity, geometry--to be otherwise equal. In Dextran, the values of Es for normal RBCs are found to be of order 3.10(-6) N/m. For erythrocytes hardened by heat exposure for 15 minutes at 48 degrees C, the increase in Es reaches 45 percent. This procedure of shear elastic modulus determination is easy to perform and seems to give a good discrimination between normal and altered erythrocytes.

Biomechanical Phenomena↗
PubMed1990

New results of hemoglobin variant structure determinations by fast atom bombardment mass spectrometry.

Fast atom bombardment mass spectrometry has already been used for the identification of mutations in abnormal human hemoglobin chains. This paper presents new results obtained with this technique. The methodology used here is compared with more conventional biochemical techniques and automated microsequencing. In every case, a well-chosen combination of peptide-high performance liquid chromatography, mass spectrometry, amino acid analysis, and sequence analysis led rapidly to the identification of the mutant. The high sensitivity of these techniques holds great promise for the analysis of molecular abnormalities in various genetic disorders presently detectable only by the application of a molecular biological approach.

Adult↗
PubMed1990

Erythroid cultures and erythropoietin assay. Clinical and diagnostic value.

In vitro cultures of erythroid progenitors and radioimmunoassay of erythropoietin (Epo) are 2 recently available techniques. It is possible to assess their relevance in various hematological disorders. Erythroid cultures can be performed in the investigation of polycythemias, pure red cell aplasias (PRCA) and refractory anemias. In primary polycythemias "spontaneous" colonies appear in vitro whereas this phenomenon is never observed in secondary polycythemias. These so called "spontaneous" colonies have been demonstrated with a lower incidence in all myeloproliferative disorders. Therefore, if the absence of spontaneous colonies does not permit us to eliminate the presence of a myeloproliferative syndrome aside from polycythemia vera, their presence does seem pathognomonic of a myeloproliferative disorder. In acquired chronic pure red cell aplasia in adults, a strong correlation is found between the in vitro growth of erythroid colonies and the results of immuno-suppressive treatment. In refractory anemias erythroid cultures do not have either diagnostic, or prognostic interest. Serum epo level does not have a high discriminatory value in distinguishing between primary and secondary erythrocytosis. Indeed in PV, the Epo level is generally low or normal, in secondary polycythemias Epo level is high or normal. There is an important overlap between the two groups. Epo level determination can have a therapeutic incidence. Administration of recombinant Epo seems justified only in patients both sufficiently anemic to warrant transfusions and in whom Epo level is low in comparison with the degree of anemia.

Erythroid Precursor Cells↗
PubMed1990

Erythropoietin: physiology and clinical experience.

Erythropoietin is a glycoprotein hormone of primarily renal origin that promotes the proliferation and differentiation of erythrocyte precursors. Technological advances have resulted in the production of recombinant hormone suitable for therapeutic use and have permitted significant progress in the characterization of the physiologic and pathologic processes involved in endogenous erythropoietin production. In situ hybridization studies have shown that erythropoietin production in the hypoxic kidney occurs primarily in peritubular cells, most likely endothelial cells. In renal carcinoma associated with polycythemia, however, erythropoietin mRNA has been detected in the tumor cells, which are tubular in origin. New information regarding the biochemistry of the erythropoietin receptor has been gleaned subsequent to the cloning of the gene encoding the receptor; however, much remains to be learned about the interaction of the hormone with its target cells. With regard to clinical experience, recombinant erythropoietin has been shown to correct the anemia associated with chronic renal failure in patients requiring dialysis, having a significant beneficial effect on the overall physical and psychological state of the patient; the major adverse effect of such treatment is hypertension. The role of recombinant erythropoietin in predialysis patients, patients with anemias of other origin, and other clinical settings is currently being evaluated.

Anemia↗
PubMed1989

Effect of 3,4-dihydroxybutyl-1-phosphonate on cardiolipin synthesis in B. subtilis.

Endogenous phosphatidylglycerol is rapidly transformed into cardiolipin when B. subtilis 168 cells were incubated in a buffer without an energy source. Upon addition of 3,4-dihydroxybutyl-1-phosphonate (DHBP), a synthetic glycerol 3-phosphate analogue, this synthesis was completely blocked after a short lag; if the cells were grown in the presence of the analogue, there was no lag. When membrane fractions were incubated with exogenous [32P]phosphatidylglycerol, free DHBP and glycerol 3-phosphate had no effect on [32P]cardiolipin synthesis, but phosphatidyl-DHBP and phosphatidylglycerolphosphate were potent inhibitors. These results are consistent with our hypothesis that phosphatidylglycerolphosphate, the phosphatidylglycerol precursor, might also be a physical inhibitor of cardiolipin synthesis.

Bacillus subtilis↗
PubMed1989

Cellular localization of erythropoietin gene transcription.

Erythropoietin producing cells were identified in the murine hypoxic kidney by in situ hybridization. The positive cells were peritubular cells, most likely endothelial cells of the cortex and outer medulla. Glomerular and tubular cells were not labelled. In three patients with renal adenocarcinomas associated with polycythemia, a strong Epo message was observed on Northern blot analysis. Using in situ hybridization, a strong labelling was observed in all cases on the tumor cells which are of tubular origin.

Animals↗
PubMed1989

Serum IgG subclass levels in patients with primary immunodeficiency syndromes or abnormal susceptibility to infections.

Serum IgG subclass levels were measured using an indirect competitive immunoenzymatic assay with monoclonal antibodies in 221 patients affected with definite immunodeficiency (ID) syndromes and 229 patients presenting with infection patterns suggestive of ID, but with normal immunoglobulin class levels and no clear evidence of ID. In common variable ID and IgG-IgA deficiency with normal or high IgM, subclass imbalance (mostly IgG1-IgG3 or IgG2-IgG4 deficiency) was the rule, with a higher incidence of severe infections in IgG2-IgG4 defects. One-fifth of patients with IgA deficiency, especially those with autoimmune cytopenia, had subclass deficiencies with no significant correlation with the occurrence of infections. Subclass (mostly IgG2-IgG4) deficiencies were also observed in severe combined ID, defective expression of HLA class II antigens, chronic mucocutaneous candidiasis, and IgM deficiency. Subclass levels were normal in all but one (who was IgG3 deficient) patient with the Wiskott-Aldrich syndrome and in the Buckley's syndrome, except for an unusual patient who presented with low IgG and IgA levels. Subclass (mainly IgG2) deficiency occurred in 24% of infected patients without known ID.

Autoimmune Diseases↗
PubMed1989

Effects of clinostatism and orthostatism on blood viscosity.

The present study was planned to study the magnitude of the effects of body position upon blood viscosity. We compared blood rheological properties in nine healthy subjects after 1 h in a horizontal position and after 1 h in a vertical position. After orthostatism, blood viscosity was 38%, 41%, and 22% higher at 0.05, 1, and 20 s-1 shear rates, respectively. This result was the consequence of the increase of hematocrit (41.2% +/- 2.3% vs 44.2% +/- 2.7) and plasma viscosity (1.55 +/- 0.09 mPa.s vs 1.67 +/- 0.08 mPa.s) induced by orthostatism in all the subjects. The rise of the different plasma proteins and fibrinogen was the cause of the plasma viscosity increase. Blood viscosity at standard hematocrit (45%) was not significantly altered by orthostatism. Rheological studies at standard hematocrit showed that body position did not modify red cell deformability and aggregability. As suggested by the increase in hematocrit and plasma proteins and by the absence of alteration of blood rheological properties at standard hematocrit, the large effect of body position upon blood viscosity is the consequence of the hemoconcentration without alteration of erythrocyte deformability and aggregability.

Blood Viscosity↗
PubMed1989

Effect of dietary restriction on receptor-mediated and independent catabolism of beta-very low density lipoproteins in cholesterol-fed rabbits.

This study was designed to define the effect of dietary restriction and cholesterol feeding on disturbances of very low density lipoprotein (VLDL) catabolism. Three groups of 15 New Zealand rabbits were fed standard or cholesterol-rich diets either ad libitum or in restricted amounts and in vitro binding assays on hepatocyte membranes were carried out after 4, 8, 12 and 20 d. Cholesterol feeding reduced the specific binding of labeled beta-VLDL to liver membranes. Dietary restriction, which enhances the hypercholesterolemia induced by cholesterol feeding, had no additional effect on the down-regulation of beta-VLDL specific binding. In another set of experiments the uptake of labeled beta-VLDL by various tissues was measured after 15 d of feeding the diets. Cholesterol feeding decreased uptake of these lipoproteins by the liver and various extrahepatic tissues, such as skin, intestine, muscle, adipose tissue, kidneys, spleen and aorta. Additional changes occurred in liver and intestine with dietary restriction. In liver beta-VLDL uptake in the group fed a restricted amount of the cholesterol-containing diet was 70% of that in the group fed the same diet ad libitum. Taken together, the results suggest that dietary restriction acts mainly on the receptor-independent catabolism of beta-VLDL.

Adipose Tissue↗
PubMed1989

c-kit mRNA expression in human and murine hematopoietic cell lines.

The c-kit proto-oncogene belongs to the tyrosine kinase receptor family. Although its ligand is still unknown, there is increasing evidence to suggest its involvement in hematopoiesis. In order to detect lineage or differentiation related specificity, we have studied c-kit mRNA expression in both human and murine hematopoietic organs and cell lines. We show that c-kit mRNA expression is found at early stages of erythroid and myeloid differentiation. There is however, no evidence of c-kit expression in the lymphoid lineage. Our results suggest a possible role for c-kit as a receptor in the early stages of the erythroid/myeloid differentiation.

Animals↗
PubMed1989

IgG subclass deficiency in acute myeloid leukemia.

Serum IgG subclass levels were measured using a competitive indirect immunoenzymatic assay with monoclonal antibodies in 22 acute myeloid leukemia patients at presentation and prior to specific therapy. Three patients had moderate IgG1 deficiency and 1 moderate IgG3 deficiency. Two of the three IgG1 deficient patients (and only one other) failed to respond to induction chemotherapy and the third died of bone marrow aplasia during induction.

Adolescent↗