An acidic protein has been extracted from sarcoplasmic reticulum with KCI and deoxycholate. believe that it is a major binding site for calcium in sarcoplasmic reticulum. We propose that the protein be designated Calsequestrin. MATERIALS AND METHODSPreparation of the calcium-sequestering proteinStep 1. Sarcoplasmic reticulum was prepared from two 14.4-kg albino rabbits and was extracted with deoxycholate as described (8), except that dithiothreitol (1 mM) was present during the extraction and the supernatant, after centrifugation at 165,000 X g, was retained as the source of the binding protein. The deoxycholate extract (about 80 ml) was dialyzed, first for 4 hr and then for 18 hr at 12'C, against 3 liters of 5 mM Tris * HCl, pH 7.5. At 4 and 22 hr, insoluble protein was removed by centrifugation at 165,000 X g for 30 min.Step 2. The supernatant solution (35-to 40-ml aliquot) was passed through a 1.5 X 18 cm column of DEAE-cellulose and the column was eluted with 400 ml of 5 mM Tris -HCl, pH 7.5, that contained a linear gradient of KCl from 0 to 0.7 M. The last peak to be eluted, at a KCl concentration of about 0.48 M, was collected, concentrated by ultrafiltration under pressure (Amicon Diaflo apparatus) to 8 ml, and dialyzed overnight against a solution of 0.15 M KCl-0.01 M Tris -HCl, pH 8.0.Step 3. The dialyzed sample was passed through a 2.5 X 40 cm column of Sephadex G-200 equilibrated with 0.15 M KCl-0.01 AM Tris HCl, pH 8.0. Material absorbing at 280 nm was eluted in a sharp peak at the void volume, in a second symmetrical peak, and in a tail following the second peak. Fractions making up the second symmetrical peak were collected, concentrated by Diaflo filtration to 10 ml, and dialyzed overnight against 10 mM potassium phosphate, pH 7.0.Step 4. The dialyzed sample was applied to a column (7 g) of hydroxylapatite (Biogel HTP) equilibrated with 10 mM potassium phosphate pH 7.0. The column was eluted with 400 ml of a linear gradient of potassium phosphate from 10 to 600 mM. The last peak to be eluted, at a phosphate concentration of about 330 mM, was concentrated to 5 ml and dialyzed overnight against a solution of 5 mM Tris .HC1, pH 7.5. The solution, containing purified protein (about 1 mg per ml), was stored at -20°C. Analytical methodsCalcium transport was measured (10) at a protein concentration of 200 ug/ml. After 10 min at 24°C, 0.5 ml was filtered

Background: In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.

We have found that a variety of clonogenic hemopoietic cells can be obtained in a viable state from mouse conceptuses as early as day 7 of gestation when tissues are disaggregated in a crude collagenase solution containing fetal bovine serum. Examination of the time course of colony formation, and the ultimate size and lineages represented in colonies produced in semisolid medium containing methylceflulose, together with analysis of individual erythroid colonies stained with rabbit antisera specific for adult (HbA) and embryonic (HbE) mouse hemoglobins, revealed the presence on days 7 and 8 of gestation (but not later) of erythropoietic progenitors that give rise to mature erythroid colonies containing up to 100 HbE-containing erythroblasts after 4-6 days of growth in culture. These progenitors are highly sensitive to the disaggregation conditions used. Clonogenic progenitors of exclusively HbA-positive erythroblasts can also be detected in the day-7 conceptus. Assays of progenitors from separately disaggregated yolk sacs and embryos from day-8 conceptuses yielded colonies only from yolk sac suspensions, and again these contained either HbE-and HbA-positive erythroblasts or only HbA-positive erythroblasts. These findings demonstrate the very early appearance in the yolk sac of a population of erythroid progenitors with a number of unique properties. Although most of these yield HbE-positive erythroblasts in vitro, some produce erythroblasts containing HbA only. Such a developmental pattern is consistent with the hypothesis that definitive erythropoiesis in the mammalian fetal liver is derived from stem cells that originate in the yolk sac blood islands.In the developing mouse embryo, hemopoiesis begins with the formation of the yolk sac blood islands on the seventh day of gestation (1). Two days later the circulatory system is complete, and "primitive" erythroblasts are the predominant cell type found in the embryonic blood at that time (1-4). Unlike adult erythroid cells, these circulating primitive erythroid cells remain nucleated until they disappear around the 15th day of gestation (1-5). In addition, they are unique in their ability to synthesize embryonic hemoglobin (HbE), although with time they also produce some adult hemoglobin (HbA) (6). Erythrocytes containing exclusively HbA first appear in the fetal liver, which is formed shortly after day 10 of gestation (7,8).The restriction of HbE production to a gestationally early cohort of erythroid cells that show other unique differentiation features has led to the concept of a developmentally separate lineage of primitive hemopoietic cells. In previous studies, we showed that progenitors of exclusively HbApositive erythroid cells were already present in the 9-day-old conceptus, at a time when the fetal liver is not yet formed (9). In addition these studies revealed an unusual sensitivity of gestationally early precursors to conventional methods for obtaining single-cell suspensions, although organ cultures of day-8 conceptuses did suggest the existe...

No abstract

No abstract

C3H͞HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran͞TC4 GTPase. We now show that this gene is mutated in

No abstract

A recombinant retrovirus containing the interleukin-3 (IL3) coding sequence and the neomycin-resistance gene (Neo') has been generated. Infection of fetal liver cells with the IL3 retrovirus, but not with the N2 parental virus, resulted in the formation of factor-independent, Neo R colonies containing various types of differentiated hemopoietic cells. Established cell lines could be generated from these mixed hemopoietic colonies. These cell lines contained the unrearranged viral genome, produced viral IL3, and secreted the growth factor; however, they were not tumorigenic. Identical results were obtained from infection of two factor-dependent cell lines with the IL3 virus, except that these clones all became tumorigenic. These data indicate that endogenous IL3 production can support normal differentiation and immortalization of primary hemopoietic cells, or, in previously immortalized cells, can lead to tumorigenicity.