Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.     

Palaeoecological and palaeoethnological analysis of botanical macrofossils found at the Neolithic site of Rivaltella ca'Romensini, northern Italy

The botanical macrofossils (charcoals, seeds and fruits) found during the archaeological excavation of the middle Neolithic site of Rivaltella Ca'Romensini near Reggio Emilia, northern Italy, have been analyzed. Among the charcoal fragments 11 different taxa have been identified, with a clear predominance of oaks. The relative frequencies of the different taxa probably do not reflect the real frequencies in the forest, but suggest that firewood had been gathered selectively. Among the seeds and fruits, four different Gramineae, a small wild apple and some hazelnut shells have been identified. These results indicate that the food economy of the Neolithic Rivaltella inhabitants was partially based on agriculture and on fruit gathering.     

Parathyroid hormone binding to cultured avian osteoclasts.

Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with [125I]--bovine PTH (1-84). Specific binding of the hormone to the cells, which reached the equilibrium within 60 min, was observed. Half maximal binding was reached by 10 min. Binding was competitively inhibited by increasing doses of unlabeled PTH, and was about 55% displaced by adding, at the equilibrium, 10(-6) M unlabeled PTH. Autoradiography demonstrated specific label on the osteoclast. The cellular mechanism activated by the hormone remains to be elucidated.     

Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts.

The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase. The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes.     

Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type-2 Astrocyte Cell Lineage

Abstract: We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[³H]aminobutyric acid ([³H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μ M . Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μ M ). N -Methyl- d -aspartic acid and dihydrokainate (100 μ M ) did not stimulate [³H]GABA release from cultured astrocytes. l -Glutamic acid (20–100 μ M ) stimulated [³H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [³H]GABA release were completely Na⁺ dependent; that of kainate was also partially Ca²⁺ dependent. Kynurenic acid (50–200 μ M ) selectively antagonized the releasing effects of kainic acid and also that of l -glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μ M ). d -[³H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.     

Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates

A quenching technique for the study of rapid protein reactions is described which consists of injecting a small volume of aqueous solution of reactants into a large volume (× 10) of hydro-organic solvent cooled at subzero temperature and mechanically shaken. The protein reaction intermediates, stabilized at subzero temperature and brought into a hydro-organic solution, can then be separated by subzero temperature electrophoretic methods, such as isoelectric focusing, in the same solvent. The alkaline hydrolysis of 2,4-dinitrophenylacetate was studied by the use of this quenching technique in order to compare the quenching and the rate constants of the reaction with those obtained by normal rapid quenching methods. It was found that first-order reactions having rate constants up to about 5 s^?1 can be satisfactorily studied by this technique. The technique is not suitable for the study of faster reactions because of the high value of the quenching time (40–100 ms). The hybridization reaction of carboxyhemoglobins A and C in aqueous solution at 22°C was studied as an example of the application of this quenching technique and of the isoelectric focusing method at subzero temperature to the isolation of unstable intermediates in a protein reaction.     

Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence

Summary Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band.Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein, does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin.The possible meaning of the different distribution of the two calcium binding proteins is discussed.A preliminary report on this work was presented at the meeting of the Union of Swiss Societies for experimental Biology, Lausanne, 1975. This work was supported by the Swiss National Science Foundation, grants No. 3.725.72 and 3.0330.73     

Evidence for a similar compartmentation of recaptured and endogenously synthesized dopamine in striatal synaptosomes

The aim of the present study was to compare the release pattern of [³H]dopamine ([³H]DA) originated from [³H]tyrosine or by uptake in striatal synaptosomes. Synaptosomes prelabeled either with [³H]DA or with [³H]tyrosine were superfused in three conditions stimulating DA release by different mechanisms: (1) depolarization with high K⁺; (2) inversion of the Na⁺ gradient across the plasma membrane; (3) exposure tod-amphetamine. Since DA contained in different pools may exit from nerve endings by different processes, DA release was analyzed in the presence or in the absence of nomifensine which allows discrimination between carrier-mediated and carrier-independent processes. The pattern of DA release in the three conditions tested was identical, whether [³H]DA originated from synthesis or from uptake. Nomifensine did not affect the high-K⁺-induced release and inhibited that induced by the other two stimuli. The results suggest that newly synthesized and recaptured DA have a similar compartmentation in nerve endings.