Comparison of developmentally regulated lectins from three species of cellular slime mold

Extracts of the cohesive forms of the cellular slime molds Dictyostelium discoideum, Dictyostelium mucoroides and Dictyostelium purpureum contain lectin activity, assayed as hemagglutination activity. The lectin activity from each species binds quantitatively to Sepharose 4B and can be eluted with d-galactose. The resultant purified lectins are abundant proteins representing, in the case of D. purpureum, up to 5% of the total soluble protein of cohesive cells. The preparations from each species are similar but distinct in amino acid composition and other properties. Each purified preparation gives rise to two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the major band representing as little at 77% (D. purpureum) and as much as 96% (D. mucoroides) of the total protein in the two bands. The molecular weights of the pair of bands were different for each species, ranging between about 23 000 and 26 000. The two bands are believed to represent subunits of lectins made up of either one or a combination of these two proteins. The apparent molecular weights of the purified lectin activities determined by sucrose density gradient centrifugation were all in the range of 100 000. $\text{N-}\text{Acetyl-}\text{d}\text{-galactosamine}$ was a potent inhibitor of the hemagglutination activity of each preparation; but there were some differences in the relative inhibitory potency of a number of other saccharides. Antiserum raised against each preparation, as well as univalent antibody fragments derived from these antisera, reacted best with the antigens to which they were raised; but showed some cross reaction measured both by precipitin reactions and by inhibition of hemagglutination activity of the purified lectins. The differences between the lectins from the different species could be trivial; but they also could be important for defining specific properties of these three species which reliably segregate into colonies of a single species when grown in mixed culture.     

Apparent nuclear localization of unoccupied receptors for 1,25-dihydroxyvitamin D3

Previous studies have demonstrated that unoccupied 1,25-dihydroxyvitamin D₃ receptors are associated with crude chromatin under hypotonic conditions $\text{in}\text{vitro}$. The data presented herein show that unoccupied 1,25-dihydroxyvitamin D₃ receptors appear to be associated with chromatin prior to solubilization by dilution/homogenization in both high and low salt buffers. Additionally the unoccupied receptors are recovered nearly quantitatively from purified nuclei. These results suggest that unoccupied 1,25-dihydroxyitamin D₃ receptors may be localized within nuclei $\text{in}\text{vivo}$.     

Effect of some amphiphilic drugs on the membrane morphology and aggregation of rabbit platelets

Treatment of normal, disc-shaped rabbit platelets with lysophosphatidylcholine and chlorpromazine induced respectively spine formation and spherical transformation. In similar concentration ranges to those in which they induced these morphological changes, the drugs suppressed a series of events triggered by thrombin: pseudopod formation, arachidonate release from the membrane phospholipids, and aggregation. Washing the drug-treated platelets reversed the morphological changes and abolished the inhibitory effect on aggregation. These observations suggest that amphiphilic drugs perturb the plasma membrane structure of platelets, inducing the membrane shape change and inhibiting the stimulus-induced aggregation.     

Variants of a cloned synthetic lactose operator: I. A palindromic dimer lactose operator derived from one strand of the cloned 40-base pair operator

Starting with one strand of the 40-bp synthetic operator (Sadler et al., 1978), we have constructed and cloned a 66-bp, palindromic DNA segment with the following sequence

where the horizontal arrows indicate the locations of the two 21-bp “core? operator sequences in this segment and the vertical arrow designates the dyad axis of symmetry. Upon denaturation and rapid renaturation, each strand of this fragment forms a hairpin molecule still retaining an EcoRI cohesive end. Two hairpin molecules can be joined with T4 DNA ligase to form a duplex DNA molecule having no ends (dumbbell form A). Denaturation and rapid renaturation of dumbbell A yields a mixture of two dumbbell forms: dumbbell A which is a substrate for EcoKL, and a new form, dumbbell B, which is not a substrate. Each of the conformations of this DNA fragment have been purified and all are active in binding lactose repressor in vitro.     

Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis.

The intracellular distribution of ¹¹⁵cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling $\text{in}\text{vivo}$ - $\text{in}\text{vitro}$ experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.     

Protection of steroid hormone receptors by protease inhibitors

Steroid hormone receptors are proteolyzed by different types of enzymes present in target tissues. Effective protease inhibitors protecting steroid hormone receptors in various target tissues were investigated. Progesterone receptor (PR) in hen oviduct and estrogen receptor (ER) in cow uterus were specifically protected by relatively low concentrations (0.5 mM) of leupeptin or antipain (inhibitors of serine and thiol proteases). It was indicated that two different types of enzymes which modify native glucocorticoid receptor (GR) are present in rat liver. One was inhibited by 1 mM leupeptin or 1 mM antipain, while the other was inhibited by 1 mM phosphoramidon (inhibitor of thermolysin like proteases) or 10 mM sodium molybdate. Native PR, ER, and GR were shown to have similar Stokes radii (44 Å).     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca²⁺ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 μM free Ca²⁺. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca²⁺ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca²⁺-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca²⁺ and in the presence of Ca²⁺ appear to depend upon the level of protein phosphorylation.     

Involvement of calcium, calmodulin and phospholipase A in the alteration of membrane dynamics and superoxide production of human neutrophils stimulated by phorbol myristate acetate

We have reported an increased fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in the phorbol myristate acetate-stimulated plasma membrane of human neutrophils [FEBS Lett. (1982) 144, 199–203]. We now present evidence that both the increased fluorescence polarization and the production of super-oxide radicals by human neutrophils require calcium, calmodulin and phospholipase activity.     

GTPase from rod outer segments: characterization by photoaffinity labeling and tryptic peptide mapping

The photoaffinity label [γ-³²P]8-N₃GTP has been used to identify GTP-binding components in highly purified preparations of GTPase from bovine rod outer segments. These preparations contain two major polypeptides of 37,000 and 39,000 daltons. In the presence of photolyzing radiation, [γ-³²P]8-N₃GTP is covalently attached to the 37,000 dalton polypeptide. Tryptic peptide mapping of this polypeptide indicates that it is highly related to the 39,000 dalton species that has been previously identified as a GTP-binding component.     

Precursors of distinct size for chicken αA, αD and β globin mRNAs

The reconstitution of bovine cardiac troponin from its subunits has been investigated using hydrodynamic techniques. Gel filtration (Sephacryl S-300) and sedimentation velocity experiments indicate that troponin-C and troponin-I from a stable binary complex (1:1 mole ratio) with an apparent Stokes' radius of 36 Å (frictional ratio = 1.6). Troponin-C and troponin-T do not interact significantly while troponin-I and troponin-T undergo partial complex formation. The effect of subunit ratio on the reconstitution of whole troponin has been examined by SDS—polyacrylamide gel electrophoresis and gel filtration and the results suggest that native troponin contains the subunits in an equimolar ratio.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducer including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polycrylamide slab gel electrphoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with l-[¹⁴C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells.P180 was solubilized from ¹²⁵I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.