Joel D. Oppenheim

Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Plasmodium falciparum: Assay in vitro for inhibitors of merozoite penetration of erythrocytes

Abstract Highly synchronous cultures of the erythrocytic stages of Plasmodium falciparum were used both to assay penetration of merozoites into human red blood cells, and to subsequently study the inhibitory effects of various substances on penetration. While several sugars exhibited no inhibitory effect, fucose, glucosamine-HCl, and N -acetyl glucosamine, when added to synchronous cultures at the schizont stage, inhibited invasion. On further testing fucose and glucosamine-HCl were found to be toxic to the intracellular growth and development of the parasite; only N -acetyl glucosamine had an inhibitory effect solely related to the inhibition of merozoite penetration. Glycophorin A, the major glycoprotein of the red blood cell surface, had no inhibitory effect at low concentrations, but had a slight effect at higher (500 μg/ml) levels.

Purification of a hemagglutinin from Limulus polyphemus by affinity chromatography.

Summary A. hemagglutinin has been isolated from the hemolymph of the common horseshoe crab ( Limulus polyphemus ) using affinity chromatography as the primary purification procedure. The hemagglutinin specifically binds to a bovine submaxillary mucin (BSM) affinity gel and is subsequently released upon the addition of Na citrate to the elution buffer. The purity of the final product was attested to by polyacrylamide gel electrophoresis, electron microscopic observation and by a greater than 1500-fold increase in specific activity as compared to the starting material. The purified hemagglutinin was able to give a precipitin reaction in immunodiffusion gels against purified BSM but not against desialated BSM.

Localization and distribution of Microccus lysodeikticus membrane ATPase determined by ferritin labeling

Antiserum to Ca2+-activated ATP phosphohydrolase (EC 3.6.1.3) isolated and purified from membranes of Micrococcus lysodeikicus was prepared in rabbits and guinea pigs. The γ-globulin fractions of these antisera reacted with and inhibited ATPase activity in isolated membranes but failed to absorb to intact protoplasts or purified mesosome fractions. ATPase activity was not detectable in the purified mesosomal preparations and trypsin treatment and sonication failed to release any activity. Ferritin conjugated to the γ-globulin fractions of the antiserum reacted with the ATPase particles on the membrane as visualized in negatively stained preparations examined in the electron microscope. Labeled membranes showed a distribution of ferritin very similar to the patterns observed for ATPase particles on untreated membranes. No significant labeling occurred when the ferritin conjugate was reacted with intact protoplasts or mesosome fractions. Thin sections of ferritin-labeled membranes established the asymmetric disposition of the ATPase, with the conjugate visible on only one side of the membrane. The results indicate that the ATPase protein occurs on the inner face of the membrane. All labeling experiments were verified immunologically. When ferritin-labeled membranes were subjected to the selective release procedure used in releasing the ATPase-like particles from the membranes, a complex of ferritin-conjugate associated with the ATPase particles was released. The selective release of ferritin-antibody-enzyme complexes from the membrane opens up a new way of studying the molecular architecture of cell membranes.

Prioritizing health disparities in medical education to improve care

Despite yearly advances in life‐saving and preventive medicine, as well as strategic approaches by governmental and social agencies and groups, significant disparities remain in health, health quality, and access to health care within the United States. The determinants of these disparities include baseline health status, race and ethnicity, culture, gender identity and expression, socioeconomic status, region or geography, sexual orientation, and age. In order to renew the commitment of the medical community to address health disparities, particularly at the medical school level, we must remind ourselves of the roles of doctors and medical schools as the gatekeepers and the value setters for medicine. Within those roles are responsibilities toward the social mission of working to eliminate health disparities. This effort will require partnerships with communities as well as with academic centers to actively develop and to implement diversity and inclusion strategies. Besides improving the diversity of trainees in the pipeline, access to health care can be improved, and awareness can be raised regarding population‐based health inequalities.

The use of lectins in the quantitation and analysis of macromolecules by affinoelectrophoresis.

The technique of rocket affinoelectrophoresis, initially introduced for the quantitation of a succinylated mannan by concanavalin A [Owen, P., and Salton, M. R. J. (1976) Anal. Biochem.73, 20–26] has been extended to (a) the quantitation of four other macromolecules: vz. streptococcal lipoteichoic acid, I blood group substance, desialylated bovine submaxillary mucin, and desialylated pig submaxillary mucin; and (b) the use of three other lectins: vz. wheat germ agglutinin, soybean agglutinin and peanut agglutinin. In all cases stable-affinity precipitin rockets were observed the heights of which bore an approximately linear relationships to the amount of sample analyzed. For all lectins, the detection limits were in the range of 15–25 ng. Furthermore, a new technique has been introduced called crossed affinoelectrophoresis which is basically a two-dimensional variant of rocket affinoelectrophoresis. This technique can be used with concanavalin A, wheat germ agglutinin, soybean agglutinin, or peanut agglutinin in the affinity gel and allows the examination of glycoprotein homogeneity. Modification of this technique, involving the use of other lectins or antiserum in intermediate gels, is also described and evaluated.

Plasmodium berghei and Plasmodium knowlesi: Serum binding to sporozoites

Abstract Plasmodium berghei salivary gland and oocyst sporozoites were examined with fluorescein isothiocyanate (FITC)-lectins to determine if sporozoites had carbohydrate-containing molecules on their surfaces. None of the eight fluorescein isothiocyanate-lectins bound to the sporozoites. However, incubation of sporozoites in mouse serum permitted subsequent binding of concanavalin A and Ricinus communis agglutinin I. In general, serum binding occurred when sporozoites were incubated in serum from hosts susceptible to sporozoite infection. Sporozoites of the rodent parasite, P. berghei, tended to bind rodent but not primate serum, while sporozoites of the monkey parasite, Plasmodium knowlesi, tended to bind primate but not rodent serum. The serum component(s) that bound to sporozoites were concentrated considerably by ammonium sulfate precipitation followed by concanavalin A—Sepharose affinity chromatography.

Some properties of flammutoxin from the edible mushroom Flammulina velutipes

A cytolytic toxin from the basidiocarps of the edible mushroom Flammulina velutipes was purified to homogeneity. The lysin, flammutoxin, is a single polypeptide chain of Mr 32,000 and pK about 5.4. It contains unusually large amounts of tryptophan, serine and glycine, and few or none of the sulfur-containing amino acids. Erythrocytes of rat, rabbit, guinea pig, man, mouse, cat and dog were sensitive to lysis, in that order, whereas erythrocytes of sheep, ox, goat, swine and horse were largely or completely resistant to lysis. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Hemolysis probably involves alteration of the erythrocyte membrane, with formation of submicroscopic ion channels, and it appears to be of the osmotic type. In some respects flammutoxin resembles phallolysin, a cytolytic toxin obtained from the mushroom Amanita phalloides.