Biomedical subjects

G Desoye

Publications and source records attributed to G Desoye.

88 records · Page 5Linked to original sources

PubMed1986

Fluorescence topography in biology. III: Characteristic deviations of tryptophan fluorescence in sera of patients with gynecological tumors.

The near-ultraviolet region of the total fluorescence (excitation-emission matrix) of human serum reflects essentially the fluorescence of protein-bound tryptophan. We examined topographically the tryptophan fluorescence of human serum. In comparison with fluorescence topograms from sera of healthy donors, sera of patients with gynecological malignancies showed significantly different patterns of tryptophan fluorescence, the major deviations being at 325 and 365 nm. In healthy donors, the tryptophan fluorescence intensity at 365 nm, expressed as percent of the maximum fluorescence intensity (i.e., at 337 nm) varied little, but was markedly lower for sera from patients with malignancies. We found no clear correlation between the extent of the fluorescence deviations and the relative concentration of the protein fractions as determined by electrophoresis. Furthermore, we could rule out inflammation in tumor patients as an explanation for this effect.

Blood Proteins↗

PubMed1981

Quantitative cytospectrophotometrical determination of the total protein thiols with "mercurochrom": optimization and calibration of the histochemical reaction.

Mercurochrom (2,7-dibromo-4-(hydroxymercuri)-fluoresceine-disodium salt) reacts histochemically not only with protein-SH-groups, but is also bound unspecifically to cellular proteins. The amount of the unspecifically staining approximately equals the specific SH-staining. Two methods are described to remove the unspecifically bound Mercurochrom, without influencing the specific reaction with the protein thiols. The first applies 0,1 m thioglycolate pH 4.0 (MT4-method), the other a special tris-cyanide-buffer pH 7.4 (MCN)-method). Aliquots from preparations of rat hepatocytes, Yoshida ascites tumor cells, Ehrlich ascites tumor cells, isolated nuclei of Ehrlich-cells and chicken thymocytes were investigated as well for the protein thiol content of the cells macroscopically with DTNB as microspectrophotometrically for the extinctions of the cells after staining with MT4- or MCN-method. A strong correlation was found between the macroscopically determined total-protein-SH-contents and the microphotometrically determined mean-total-extinctions of the cells. Additionally the molar absorptivities determined macroscopically by Schauenstein and Scheuringer (1980) coincide excellently with the values found microspectrometrically on MT4- and MCN-stained cells.

Animals↗

PubMed1981

The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method.

The protein content of identical samples from 5 different cell types (lymphocytes of the thymus and of the bursa of FABRICIUS of the chicken, EHRLICH ascites tumor cells, YOSHIDA ascites tumor cells, and rat liver cells) have been determined both macroscopically, by the method of LOWRY (1951), and microspectrometrically, by the tetrazonium coupling method of NOHAMMER (1978). In all the different cell types, a strong correlation is shown between the protein values determined by the 2 methods. By comparing the values thus measured, a conversion factor has been obtained such that an extinction of 0.4235, determined microspectrometrically, corresponds to a protein mass of 1 pgm. To test the accuracy of the microspectrometric method of protein determination at the lowest extreme point, the protein content of rat liver mitochondria has been measured and a mean value of 0.239 pgm protein per mitochondrion obtained. This corresponds well with the value of 0.233 pgm per mitochondrion as determined macroscopically by GEAR (1972).

Animals↗

PubMed1981

[Model experiments for quantitative cytophotometry using protein films (author's transl)].

The usefulness of protein films denaturated and fixed with ethanol/ether as cytochemical model systems was investigated. Kinetics of staining with amido black 10B were analyzed as well as the spectroscopic properties of the protein-dye complex. The absorption coefficient of the complex was estimated. Data obtained so far are compared with both stained cells and the protein-dye complex in solution. Discussion focuses on the influence of fixation and the possibilities to use the films as systems for calibrating cytochemical stainings.

Animals↗

PubMed1981

[Thrombopoiesis and blood coagulation in pediatric patients with septicemia (author's transl)].

45 infants and children with thrombocytopenia and septicemia were studied. Many parameters of blood coagulation, the platelet diameters and the megakaryocytes of the bone marrow (Feulgen stained cytophotometry and maturity of the megakaryocytes) were examined. 15 patients had a consumption coagulopathy and 30 were classified as having an isolated septic thrombocytopenia. In both groups the number of the megakaryocytes of the bone marrow smears were normal. 81% of the megakaryocytes were mature. The patient group with isolated septic thrombocytopenia had significantly greater ploidy values of the megakaryocytes than a control group. In both groups the diameters of the platelets were also significantly greater than in an age matched control group of children with a normal platelet count. These results allow the conclusion that the thrombocytopenia in pediatric patients with septicemia is not caused by a diminished production of platelets.

Blood Coagulation↗

PubMed1980

[Quantitative determination of proteins in single cells with amidoblack (author's transl)].

In aqueous solution Amido Black B (ASB) forms stable and well-defined complexes with bovine serum albumin (RSA) at pH 5.5. The complexes can be separated by column chromatography. The formation of the complexes consist in a fast reaction during which, after 3 to 5 h approximately, 3 molecules of ASB have been bound per molecule RSA, and of a much slower reaction which, even after a laps of 24 h, is still far from approaching its final stage. With solid films of RSA, after denaturation with ethanol, fast reaction is found to approach its final stage after 10 min reaction time. With these model protein preparations, the molar extinction coefficient of the ASB-protein complexes can be determined: the soluble ASB-RSA complexes can be brought to complete dissociation at pH 12.3. After the additivity of the specific absorptions of both RSA and ASB had been proven, it was possible to determine the content of the solution of ASB and RSA, and therefrom the molar extinction coefficient of the ASB-RSA-complex at Ph 5.5: epsilon 620 = 110,000. ASB-stained ethanol-fixed RSA films show an epsilon 620 of approximately 96,000, if their thickness and specific weight are known. After incubation in watery or ethanolic/TCA solutions of ASB, also animal cells fixed with ether-ethanol show the ASB absorption band to be in the region of 600 nm after removal of the surplus of ASB by thorough washings. As already observed with the RSA films, the kinetics of the staining of the cells show the fast reaction reaching its final stage already after 15 to 20 min. When alcoholic solution of ASB is used, the extinctions are found to be twice or three times higher than those achieved by an aqueous one. After standardization of the staining procedures with both solvents the total extinctions of EATZ, YATZ, rat hepatocytes, chicken thymus and bursa cells were measured and plotted against the macroscopically determined protein content of the respective cells. Highly significant positive linear correlations resulted with staining both in watery and alcoholic solutions, respectively. From the slope of the straight lines, specific extinction coefficient of ASB stained cellular proteins could be calculated up to epsilon' 620 = 1.76 with watery ASB solution and epsilon' 620 = 3.83 with the alcoholic solvent. The soluble ASB-RSA complexes have an epsilon' 620 = 1.67 the ASB stained ethanol denaturated films of RSA an epsilon' 620 of within a range of 1.21 to 1.80.

Amido Black↗

PubMedDate not supplied

Location of insulin receptors in the placenta and its progenitor tissues.

The insulin receptor gene is constitutively expressed, so the presence of insulin receptor proteins might be expected on all mammalian tissues, with the plasma membrane as the predominant site of receptor location. Results reviewed here indicate that insulin receptors are also present in all placental tissues and the placenta's progenitor tissues and cells, i.e., oocytes, spermatozoa, and preimplantation embryos, in most of the species studied. Receptor densities, however, vary among individual cells and cell types and at various developmental stages. Three aspects deserve emphasis. 1) In human placenta, the insulin receptor distribution pattern is characterized by a spatiotemporal change between first trimester and term. At the beginning of pregnancy, insulin receptors are found predominantly on the maternal side (apical membrane of syncytiotrophoblast, low density on cytotrophoblast); at term, however, they are on the fetal side (lining the fetal vessels). This suggests that, in the first trimester, maternal insulin regulates insulin-dependent processes, whereas, at term, it must be fetal insulin mainly controlling these processes. 2) The majority of insulin receptors is expressed on structures that are currently assumed to drive placental growth, i.e., syncytial sprouts and mesenchymal villi in first-trimester placentas and fetal endothelium at term. Therefore, we hypothesize a growth-promoting function, among others, of insulin on the placenta. 3) At present, no histologic evidence is available to demonstrate insulin receptors in structures commonly associated with receptor-mediated endocytosis. Whether placental insulin receptors are internalized, therefore, awaits clarification.

Animals↗

PubMedDate not supplied

Sustained hypoglycemia affects glucose transporter expression of human blood leukocytes.

The scarce data available on leukocyte glucose transporter expression are contradictory and nothing is known about its regulation by glycemic state. Therefore, cytospin preparations of blood leukocytes were searched immunocytochemically for the high-affinity glucose transporters GLUT1, 3, and 4. Hypoglycemia-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3, and 4. Granulocyte GLUT4 levels were increased by 73% (P < 0.05) under hypoglycemic conditions, which was paralleled by a reduction in GLUT1 and a rise in GLUT3. In monocytes, GLUT3 was elevated by 134% (P < 0.05), whereas GLUT1 and GLUT4 remained unaffected upon hypoglycemia. Apart from a minor subpopulation, lymphocytes were negative for these carriers. In conclusion, GLUT1, 3, and 4 are abundantly expressed in granulocytes and monocytes. The differential response of individual isoforms to hypoglycemia may represent a mechanism to protect the cells from the stress of glucose deprivation.

Adaptation, Biological↗

PubMedDate not supplied

The distribution of intermediate filament proteins, actin and desmoplakins in human placental tissue as revealed by polyclonal and monoclonal antibodies.

The distribution of intermediate filament proteins (cytokeratin, vimentin, desmin), actin, and desmoplakins in various placental compartments was studied by immunofluorescence microscopy using polyclonal and monoclonal antibodies. Trophoblast cells (cytotrophoblast, syncytiotrophoblast, isolated trophoblast cells, trophoblastic giant cells) were strongly stained by all types of cytokeratin antibodies. Antibodies to desmoplakins revealed the presence of desmosomes at all membranes, except the basal membrane of cytotrophoblast cells, and at the basal as well as the lumen-oriented membrane of the syncytiotrophoblast. After disappearance of the cytotrophoblast cell layer the distribution of desmosomes in the syncytiotrophoblast was unaltered. Isolated trophoblast cells contained desmosomes around their entire circumference. Amnion epithelial cells were heterogeneous with respect to cytokeratin composition as revealed, for example, by polyclonal antibodies with a broad range of cytokeratin reactivity and by monoclonal antibodies to cytokeratin No. 18. With the latter, a heterogeneous staining of amnion epithelial cells was achieved. Desmosomes (spots reactive with desmoplakin antibodies) were present at the lateral membranes of the amnion epithelial cells. In addition, vimentin filaments were coexpressed in these cells. Large vessels of the chorionic plate and stem villi showed thick walls consisting of vimentin-, desmin- and actin-positive cells. They were surrounded by mantles rich in vimentin-, desmin- and actin-positive cells, resembling myofibroblasts. This indicates that these cells may play a role in villous contractility and modulation of the intervillous space with effect on both maternal and fetal placental circulation.

Actins↗

PubMedDate not supplied

Glycogen distribution in the capillaries of the placental villus in normal, overt and gestational diabetic pregnancy.

Glycogen distribution has been examined in placental capillaries in normal (N; 11), overtly diabetic (OD; 8) and gestational diabetic (GD; 7) pregnancies using light and electron histochemistry. Light microscopy revealed a wide variation in glycogen deposition in all three groups, but the method was too insensitive to visualize deposits in the villous capillaries. An ultrahistochemical technique, however, rendered glycogen detectable. Endothelial cells (EC) and pericytes (PERI) in 100 capillaries per specimen were scored as to whether their glycogen was dispersed (D) or aggregated (A). Again, a large range in the amounts of both types of glycogen was seen. When EC and PERI comprising a capillary cross-section were considered together as 'functional units', a higher (P < 0.11) relative number of cells containing A was found in OD (51 +/- 8 per cent) than in N (41 +/- 17 per cent) or GD (43 +/- 18 per cent). In OD more EC and PERI, considered separately, tended to contain A than those in N and GD, but large variations in the data precluded statistical significance. The proportion of PERI containing aggregated glycogen was higher (P < 0.0002) than that of EC in all groups. We suggest that, on average, placental EC and PERI contain higher glycogen levels in OD than in N or GD. Moreover, these cells might contribute to the high glycogen levels in placentae from diabetic pregnancies.

Capillaries↗

PubMedDate not supplied

DNA microarrays: a novel approach to investigate genomics in trophoblast invasion--a review.

The events that regulate trophoblast invasion need to be characterized at the transcriptional level. Several types of gene products may be involved in various stages oftrophoblast infiltration, including integrins, matrix metalloproteases (MMPs) and extracellular matrix (ECM) proteins. Autocrine or paracrine regulators of cytotrophoblast proliferation or differentiation in vitro (e.g. growth factors and cytokines, as well as oxygen tension) could be characterized mechanistically at the transcriptional level. Large-scale gene expression profiling of trophoblasts of distinct invasive stages could be carried out on fixed tissue obtained by laser-directed microdissection. This information may shed light on physiological implantation and placentation, as well as on the interpretation of pathological processes such as pre-eclampsia. The applications of DNA microarrays are ideal for studies of genomic structure (e.g. mutation and polymorphism analyses) and monitoring of gene expression. The ultimate goal is to understand the critical events underlying growth, development, homeostasis, 'behaviour and the onset of disease at a genomic level. Microarrays detect gene expression levels in parallel by measuring the hybridization of labelled, single-stranded DNA to many thousands of partial or whole gene sequences immobilized on a glass surface (the 'chip'). Microarrays are available both commercially and can be manufactured in house.

Animals↗

PubMedDate not supplied

Endothelin A and B receptors change their expression levels during development of human placental villi.

Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development.

Abortion, Legal↗

PubMedDate not supplied

Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation.

In the third trimester of normal pregnancy, the mother tolerates daily shedding of several grams of dying placental trophoblast into the maternal circulation. The balance between apoptotic and necrotic shedding is presently unknown. Since pre-eclampsia is characterized by an altered placental oxygenation and increased trophoblast shedding, we investigated the role of oxygen on the balance of apoptotic versus necrotic trophoblast shedding in vitro. We studied human trophoblast turnover in explanted villi from late first and third trimester placentas in low oxygen (2 per cent) and higher oxygen tensions (6 per cent and 18 per cent) for up to 72h. Trophoblast turnover including apoptosis and necrosis were assessed by histology, immunolocalization of Mib-1 (proliferation marker), Bcl-2 (apoptosis inhibitor), activated caspase 3 (apoptosis promoter), cytokeratin 18 neo-epitope formation (M30 antibody), TUNEL test (DNA degradation), and (3)H-cytidine and(3) H-uridine incorporations. Culture in 2 per cent oxygen increased cytotrophoblast proliferation and syncytiotrophoblast shedding by necrosis. The proteins necessary for execution of apoptosis were mostly retained in the cytotrophoblast due to lack of syncytial fusion. Culture in 6 per cent and 18 per cent oxygen reduced cytotrophoblast proliferation. Syncytial fusion occurred and activity of caspase 3 was found in the syncytiotrophoblast; the latter remained intact demonstrating physiologic turnover, including apoptotic shedding. We conclude that severe placental hypoxia favours necrotic rather than apoptotic shedding of syncytial fragments into the maternal circulation. Since uteroplacental ischaemia is a significant risk factor for pre-eclampsia, these findings may explain the link between reduced uteroplacental blood flow and the systemic clinical manifestations of this disease.

Adult↗

PubMedDate not supplied

Selective cholesteryl ester uptake from high density lipoprotein by human first trimester and term villous trophoblast cells.

As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.

Adult↗

PubMedDate not supplied

Ultrastructural localization of insulin receptors in human placenta.

PROBLEM: Investigations to date on localization of placental insulin receptors (IR) at the ultrastructural level focused on the maternal facing side of the placenta but did not allow localization of IRs to intracellular compartments. METHOD: The ultrastructural localization of IR in placentae from early and term gestation was studied using the immunogold technique and a monoclonal antibody against the external domain of the IR (clone MA20). RESULTS: At 10 wk gestation, there were high levels of IR in both cytotrophoblast and syncytiotrophoblast, with sparse microvillous labeling. Fetal vessels contained various amounts of IR while stromal cells were also labeled. By 13 wk, this pattern was unchanged; however, at term, fewer IR were present in the syncytiotrophoblast, with more label on microvilli than before. Fetal vessels were well labeled, while stromal cells appeared unchanged. Intense labeling of fetal erythrocytes provided an internal positive control, while omission of the primary antiserum produced loss of binding. CONCLUSION: The data suggest a spatiotemporal alteration in placental IR distribution during pregnancy, possibly reflecting a shift in control of placental growth and metabolism from mother to fetus.

Chorionic Villi↗

PubMedDate not supplied

[Influence of pH, temperature and polarity of the solvent on the absorption of NADH+ and NADH at 260 nm (author's transl)].

Assuming a reversible equilibrium of an unfolded and a base-stacked conformation of both NADH and NAD+ the influence of pH, temperature and methanol on this equilibrium was studied as indicated by the UV-absorption of the adenine, band at 260 nm. By the addition of methanol as well as by an increase of temperature the equilibrium is shifted to the unfolded conformation. An increase of the H+ concentration seems to favor the unfolded conformation in the case of NAD+ while for NADH a decrease of the absorption coefficient is pointing to a higher percentage of the stacked conformation at lower pH. From the temperature variation of the absorption coefficients the enthalpies for the conformational change were calculated and compared with data obtained by different methods.

Hydrogen-Ion Concentration↗