Effects of 17 beta-estradiol and R5020 on glucose-6-phosphate dehydrogenase activity in MCF-7 human breast cancer cells: a cytochemical assay.

Although increasing levels of glucose-6-phosphate dehydrogenase (G6PD) have been widely reported in human breast tumor tissue, the effects of 17 beta-estradiol and progesterone on this key enzyme of cellular growth processes have not been well documented. Cellular heterogeneity of breast tumor tissue, coupled with the low sensitivity of classical biochemical assays of G6PD, are the main sources of difficulties in these studies. In the present report, the effects of estradiol and a progesterone analogue (R5020) on G6PD activity were studied using the MCF-7 cell line and a cytochemical assay of G6PD activity. Our results show that: (a) this assay can be used to perform quantitative measurements of G6PD on individual cells using small number of cells (20-30); and (b) both estradiol (10(-8)-10(-5) M) and R5020 (10(-9)-10(-5) M) stimulate the G6PD activity in dividing MCF-7 cells. Maximal stimulation (20-30%) was obtained after 24 h of treatment. This combination of MCF-7 cells and cytochemical assay is suitable for further studies on the effects of estradiol and R5020 stimulation of G6PD activity in breast tumors.

[Intracoronary administration of antithrombotic agents via a perfusion balloon catheter in patients with ST-segment elevation myocardial infarction presenting with massive intraluminal thrombus and failed aspiration]. / Apport de l'administration intracoronaire d'antithrombotiques par cathéter à ballon de perfusion en phase aiguë d'infarctus du myocarde avec thrombus massif et échec de la thromboaspiration.

BACKGROUND: Massive intracoronary thrombus is associated with adverse procedural results including failed aspiration and unfavourable reperfusion. We aim to evaluate the effect of the intracoronary administration of antithrombotic agents via a perfusion catheter in patients with ST-segment elevation myocardial infarction (STEMI) presenting with a large thrombus burden and failed aspiration. METHODS: We retrospectively analyzed the thrombus burden, the TIMI grade flow, and the myocardial Blush in 25 consecutive STEMI patients with a large thrombus burden and failed manual aspiration, who received intracoronary infusion of glycoprotein IIb/IIIa inhibitors (N=17) or bivalirudine (N=8) via a 6F-infusion catheter (ClearWay™ RX) RESULTS: Mean age was 67±14 years, 16 patients (64 %) presented with anterior STEMI, and 7 (28 %) with cardiogenic shock. Immediately after intracoronary infusion, the TIMI flow grade improved of 2 grades in 7 patients (28 %), and 1 grade in 14 (56 %), a complete resolution of the thrombus was observed in 9 patients, and a >50 % resolution in 12. Blush was improved of 3 grades in 15 patients (60 %), of 2 grades in 7 (28 %), and Blush grade 0 remained in 3. At the end of procedure, we observed normal TIMI 3flow in most patients (92 %), a complete resolution of thrombus in 80 %, and a Blush grade 3 in 68 %. CONCLUSIONS: In STEMI patients presenting with a large thrombus burden and failed aspiration, intracoronary administration of glycoprotein IIb/IIIa inhibitors or bivalirudin via the perfusion catheter ClearWay™ RX significantly reduced the thrombus burden and improved the TIMI flow and the Blush grade, without bleeding.

The Dispatch catheter as a delivery tool for arterial gene transfer.

OBJECTIVE: Most currently available percutaneous delivery methods for arterial gene therapy are limited by the need for a long incubation period, which may lead to unacceptable tissue ischemia, especially in the coronary vasculature. Conversely, shorter incubation times may result in inefficient gene transfer, especially in atheromatous arteries. A new local delivery autoperfusion multichamber catheter is now available which permits local delivery in the coronary arterial system without inducing myocardial ischemia. The present study aimed at evaluating the performance of this catheter for achieving arterial gene transfer using replication-defective adenoviral vectors in normal and atheromatous arteries. METHODS: A replication-defective adenoviral vector carrying a nuclear-targeted beta-galactosidase reporter gene (Ad-RSV beta gal, 5.10(9) plaque-forming units [pfu]) was delivered to the iliac arteries of normal (n = 7) and atheromatous (1% cholesterol diet + arterial abrasion) (n = 6) rabbits, via a multichamber autoperfusion balloon catheter (Dispatch, SciMed). Duration of gene delivery was 60 min. RESULTS: Three days later, marked expression of the reporter gene was detected by histochemistry in the endothelium at the delivery site (percentage of transfected cells: 16 +/- 8% / artery (range 11-25%). There was a low transduction rate in medial smooth muscle cells 0.7 +/- 0.4%/artery (range 0.3-1.1%). In atheromatous arteries, transduction was consistently achieved in the superficial layers of the neointima but was lower (1.1 +/- 0.5%/artery, range 0.3-1.7%). Transgene expression was detected by histochemistry in the liver of 3/13 animals, suggesting that there is a substantial risk of systemic dissemination of the viral vectors. CONCLUSION: Efficient arterial gene delivery to endothelial and superficial smooth muscle cells is feasible using local delivery of adenoviral vectors via the Dispatch autoperfusion catheter, in both normal and atheromatous arteries. This perfusion catheter may be a useful tool for coronary artery gene transfer.

Liquid scintillation counting for measurement of tritiated uptake in lymphocyte transformation in vitro: a new direct-suspension procedure.

Liquid scintillation counting of trichloracetic acid extracts of [3H] TdR-labelled lymphocytes has been studied by a direct suspension method without previous solubilization. In this new method, mixing of the labelled powder in water suspension with Scintillator Emulsifying Mixture (SEM) results in a firm gel whose mechanical properties provide an heterogeneous sample ready for counting. Counting efficiency and stability of such samples are related to the amount of labelled cells. Comparative studies with standard hyamine procedure have shown that this simple and rapid method gives reliable results.

Systematic search for 1 alpha,25-dihydroxy-vitamin D3 receptors in human breast carcinomas.

We looked systematically for the presence of receptor like binding sites for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in the cytosol from 22 breast cancers. Cytosols were centrifuged on 5-20% sucrose gradients after labeling with tritiated 1,25 (OH)2D3 (3H-1,25(OH)2D3 or 25-hydroxyvitamin D3 (25(OH)D3 alone or in the presence of a large excess of these unlabeled sterols. Binding sites for 1,25 (OH)2D3 migrating in the 3.5-3.7 S region were found in 7 out of 22 cancers, while 5.5-6.5 binding sites for 25 (OH)D3 were found in all cytosols. In a patient in whom cytosol containing a 3.5-3.7 S binding site was in sufficient amount, quantification of 1,25 (OH)2D3 binding indicated a KD of 0.28 nM and a maximal binding capacity of 0.15 pmol/mg prot. No relation was found between the presence or the absence of 1,25 (OH)2D3 binding sites and the histological type, the extension of the cancer, the presence of radiological or histological calcifications, the amount of estrogen or progesterone receptors, the plasma calcium or phosphate concentration and the emergence of metastases after 1 year. The significance of the presence of receptor-like binding sites for 1,25-(OH)2D3 in one third of breast cancers remains therefore unknown at the present time.

[Clinical, experimental and physiopathological aspects of restenosis after coronary angioplasty]. / Aspects cliniques, expérimentaux et physiopathologiques de la resténose après angioplastie coronaire.

Restenosis is still the main limitation of coronary angioplasty. A comparison of clinical data and experimental observations from animal models showed the early, local and transient features of this phenomenon. Several hypotheses have been advanced to explain the mechanism. Intimal hyperplasia remains an important feature of restenosis in the human but probably does not play the exclusive role as used to be believed. Vascular remodelling which has several mechanisms seems to be a major contribution to reduction of the vessel lumen. The role of thrombosis remains controversial in the clinical setting but is an essential factor in the pig which is a very similar model to the human. Each of these different mechanisms participates in the development of restenosis but in proportion which remain to be defined.

Purine biosynthesis de novo by lymphocytes in gout.

1. A method of measurement in vitro of purine biosynthesis de novo in human circulating blood lymphocytes is proposed. The rate of early reactions of purine biosynthesis de novo was determined by the incorporation of [14C]formate into N-formyl glycinamide ribonucleotide when the subsequent reactions of the metabolic pathway were completely inhibited by the antibiotic azaserine. 2. Synthesis of 14C-labelled N-formyl glycinamide ribonucleotide by lymphocytes was measured in healthy control subjects and patients with primary gout or hyperuricaemia secondary to renal failure, with or without allopurinol therapy. 3. The average synthesis was higher in gouty patients without therapy than in control subjects, but the values obtained overlap the normal range. In secondary hyperuricaemia the synthesis was at same value as in control subjects. 4. These results are in agreement with the inconstant acceleration of purine biosynthesis de novo in gouty patients as seen by others with measurement of [14C]glycine incorporation into urinary uric acid.

Binding of cholecalciferol metabolites to rat duodenal mucosa cytosol.

In vitro binding of 25-hydroxycholecalciferol (25-(OH)D3) and 1alpha,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the duodenal mucosa cytosol of rachitic rats: both 25-(OH)D3 and 1,25-(OH)2D3 bind to a macromolecule (sedimentation coefficient 5.5 to 6 S in low or high ionic strength) with a high affinity (KD at 4 degrees C = 1.2 X 10-9M and 2 x 10-9M, respectively). In addition, it is concluded from competition and chase experiments that 25-(OH)D3 binding sites differ from that for 1,25-(OH)2D3.

[De novo purine biosynthesis. In vitro measurement in hyperuricemia (author's transl)]. / Mesure de la biosynthèse de novo des bases puriques dans les hyperuricémies.

De novo purine biosynthesis has been investigated in circulating blood lymphocytes in vitro. N-formyl-glycinamide ribonucleotide (FGAR) has been mesured using 14C-formate incorporation in the presence of azaserine, a metabolic inhibitor blocking the metabolical pathway at the level of FGAR synthesis. Such a synthesis was measured in 20 healthy controls, 24 patients with primary gout (11 on allopurinol therapy) and 26 patients with chronic renal failure and secondary hyperuricemia (8 on allopurinol therapy). Among gouty patients without allopurinol therapy, FGAR synthesis was normal in 5 and increased in the others. FGAR synthesis was decreased in patients with renal failure whatever the therapy. However, FGAR synthesis remained increased in patients with a primary gout complicated with renal insufficiency. The test we propose for de novo purine biosynthesis measurement is simple and of value to analyse the patho-physiology of hyperuricemia and its therapy. The test allows an acurate discrimination between primary and secondary hyperuricemia in the presence of renal insufficiency.

Comparative effects of 17 beta-estradiol, progestin R5020, tamoxifen and RU38486 on lactate dehydrogenase activity in MCF-7 human breast cancer cells.

The effects of 17 beta-estradiol (estradiol), synthetic progestin R5020 and their antagonists, tamoxifen (Tam) and synthetic RU38486 on lactate dehydrogenase (LDH) activity in MCF-7 human breast cancer cells during the growth period were studied. A specially developed quantitative cytochemical assay was used; LDH activity is expressed per cell, and is thus independent of the positive and negative growth effects of the hormones and antagonists. Estradiol and R5020 stimulated LDH activity after similar exposures (6-48 h) and the stimuli were concentration dependent over the range 10(-7) M to 10(-10) M. As for the antagonists, RU38486 stimulated LDH activity in much the same way as estradiol and R5020; Tam alone, on the other hand, does not stimulate LDH, but when added to estradiol, Tam inhibits estradiol mediated LDH activation. When present at half-stimulant concentration, estradiol + R5020 and estradiol + RU38486 exhibit additive effects on LDH activity. Thus LDH appears to be an interesting tool for the study of hormone and antagonist effects in MCF-7 breast cancer cells.

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry.

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.

Developmental changes of Ca2+, PO4, and calcitriol metabolism in spontaneously hypertensive rats.

We have measured Ca and P balance, serum calcitriol, and vitamin D-dependent intestinal calcium-binding protein (CaBP9k) in the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY) at 4-5 and again at 13-14 wk of age. In rats on a 1% Ca diet, P balance was significantly more positive in the 5-wk-old SHR than WKY (P less than 0.01); Ca balance tended to be greater in the 5-wk-old SHR. In contrast, in the 14-wk-old SHR, P and Ca balance were less positive than in the WKY (P was 5.8 +/- 1.3 vs. 13 +/- 1.7 mg/day, P less than 0.01, and Ca was 53 +/- 4.6 vs. 67 +/- 2.7 mg/day, P less than 0.05). On the 1% Ca diet, plasma calcitriol levels of the 5-wk-old SHR were higher than those of the WKY (58 +/- 3.2 vs. 40 +/- 2.1 pg/ml, P less than 0.002), whereas at 12 wk there was no difference. On Ca-deficient (0.1%) diets, plasma calcitriol was increased in 5-wk-old and 12-wk-old SHR and WKY, compared with the 1% Ca diet (P less than 0.001). Calcitriol metabolic clearance rate was the same in the 13-wk-old SHR and WKY on either Ca diet. Intestinal CaBP9k content of the 5-wk-old (38 +/- 2.6 vs. 40 +/- 3.9 micrograms/mg) and the 12-wk-old (11 +/- 1.6 vs. 14 +/- 1.4 micrograms/mg) SHR and WKY were similar on the 1% Ca diet.(ABSTRACT TRUNCATED AT 250 WORDS)