Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein.

The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1 alpha) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein.     

Azide function and polyaddition: reaction of 2-azido-2,4,4-trimethylpentane and monoazidopoly(2-methylpropene) with some dienophiles

The 1,3-dipolar addition of 2-azido-2,4,4-trimethylpentane ( 1 ) to acetylene and ethylene derivatives such as phenylacetylene (2), acrylonitrile, diphenyl fumarate ( 4 ) and N,N′-methylenebis(1,4-phenylene)dimaleimide ( 8 ) leads to the corresponding triazole and triazoline derivatives. This study demonstrates that tertiary azides are able to react with dienophiles, and the application of this reaction for the synthesis of block copolymers was investigated with monoazido-telechelic poly(2-methylpropene) (PMP). It was shown that the azido group is stable under the severe conditions of the polycondensation reaction without decomposition (170°C, 24 h), and the monoadduct PMP- 8 is produced with acceptable yield.     

Scintigraphy with J001X, a Klebsiella membrane glycolipid, for the early diagnosis of chronic berylliosis: results from an experimental model.

A glycolipid (J001X) isolated from the membrane proteoglycans of a non-pathogenic strain of Klebsiella pneumoniae was developed to bind selectively to macrophages. A scintigraphic technique could thus be developed and applied to an experimental model of lung berylliosis. Six baboons were injected intratracheally with a beryllium metal suspension. Three to 24 months later, they were submitted to both an anatomical and a functional respiratory evaluation. Two baboons were explored at the early stage of alveolitis and four baboons at a more advanced stage characterised by a granulomatous disorder. Scintigraphy was performed using J001X labelled with 99mtechnetium administered as an aerosol. In the six baboons, conventional imaging techniques (chest x ray film, computed tomography scan, gallium scintigraphy), failed to show either any lung abnormality or mediastinal lymph nodes consistent with beryllium disease. In the two recently contaminated baboons, J001X scintigraphy showed a well defined parenchymal fixation facing the contaminated lobe. In the four baboons who were at a more advanced stage of berylliosis, J001X fixation was always focused paratracheally without any significant involvement of the lung parenchyma. The subcarinal and laterotracheal lymph nodes seen at necropsy corresponded to J001X scintigraphic fixations. In conclusion, when compared with conventional techniques such as chest x ray film, computed tomography scan, magnetic resonance imaging, and gallium scintigraphy, J001X scintigraphy has proved its ability to detect occult lesions in experimental berylliosis in baboons. By comparison with gallium scintigraphy, scintigraphy with J001X appears to have superior sensitivity and can be performed in four hours.     

Identification and characterization of a rat protein (p 105) auto-antigenic in rats bearing a progressive syngeneic colon carcinoma.

Sera from BDIX rats inoculated with 2 tumor clones derived from a single syngeneic colon carcinoma were assayed by Western blotting for the presence of antibodies against the grafted tumor. The PROb clone is progressive and produces metastases. We observed that rats bearing this tumor developed antibodies against an unglycosylated water-soluble protein of 105 kDa. The magnitude of this humoral response, as assessed by the intensity of the signal on immunoblots, was inversely correlated with survival of the rats. Furthermore, rats inoculated with the REGb clone, which is immunologically rejected, never developed detectable antibodies against the tumor. Antisera from rats injected with PROb tumor detected p105 antigen in cellular extracts from the REGb clone and from a series of rat and human cell lines. This protein was also detected in variable amounts in some normal adult and fetal tissues. Treatment of PROb or REGb cells by either interferon-gamma or heat shock did not significantly alter the expression of the p105 auto-antigen.     

Analyses and perspectives in cancer immunotherapy.

Since the last two decades, rapid progress has been made in the field of cancer immunotherapy relevant to manipulation of adaptative cytotoxic T lymphocytes (CTLs) and innate immunity natural killer (NK) cells as well as antibodies. Many possibilities are now offered for therapeutic purposes contributing to better approaches in treatment of cancer.     

Mechanism of strength increase for a hydrothermal porcelain.

OBJECTIVES: The objectives of this study were to verify the formation of a hydrolyzed surface layer on Duceram LFC, to determine the effects of such a layer on mechanical material properties, and to identify a specific mechanism responsible for any strength increase observed. METHODS: Specimens were fabricated from dentin porcelain by a vibration blotting technique and were prepared to have either blunt or sharp surface flaws. Half of the specimens underwent accelerated aging. Specimens were fractured in three-point flexure to measure their strength, and fractographic analysis was used to determine fracture toughness and residual surface stress. Surface hardness and elastic modulus were measured using a microindentation method. Porcelain surface topography was examined using atomic force microscopy, and Fourier transform infrared spectroscopy was used to determine the composition of the surface layer. RESULTS: The aging treatment modified the porcelain surface topography but did not create a layer with increased hydroxyl ion content. Porcelain strength increased upon aging, and the increase was proportional to initial flaw severity. The apparent fracture toughness of sharp flaw specimens increased to match that for specimens containing blunt flaws upon aging. Surface hardness and elastic modulus decreased upon aging. SIGNIFICANCE: Previous studies on the strength increase of hydrothermal porcelain were contradictory because a variety of specimen preparation procedures were used. This study resolves the apparent contradiction by determining the effect of specimen preparation on material strength.     

Antiproliferative activity of taxol on human tumor and normal breast cells vs. effects on cardiac cells

The antiproliferative activity of the chemotherapeutic agent taxol was evaluated on 2 normal and 2 carcinoma human breast-cell lines and compared with its effects on newborn rat cardiac cells growing in vitro. Relatively little difference in ID₅₀ response (ranging from 0.6 to 2.0 ng/ml) to taxol was found between normal and tumorous breast epithelial cells. Arrhythmias and slowing of beat frequencies of cardiac cells were induced by taxol but at doses approximately 10 times higher than those necessary to inhibit proliferation in dividing cells. Microtubules assayed by immunostaining appeared to be similarly retracted around the nucleus in both breast and heart cells. Overall, our results suggest that taxol does not selectively inhibit the growth of tumor vs. normal human breast cells. They also support the hypothesis that effects on microtubule integrity are associated with effects on cardiac function and that the clinical cardiac activity of taxol already reported may be due, at least in part, to a direct effect of taxol on cardiac cells as demonstrated in these in vitro studies. Thus, caution is needed, in view of possible cardiac effects, when using taxol in future clinical protocols, especially when combined with other cardioactive agents such as Adriamycin.