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.

TSG-6, an arthritis-associated hyaluronan binding protein, forms a stable complex with the serum protein inter-alpha-inhibitor.

TSG-6 is a secreted 35-kDa glycoprotein, inducible by TNF and IL-1. The N-terminal portion of TSG-6 shows sequence homology to members of the cartilage link protein family of hyaluronan binding proteins. The C-terminal half of TSG-6 contains a so-called CUB domain, characteristic for developmentally regulated proteins. High levels of TSG-6 protein are found in the synovial fluid of patients with rheumatoid arthritis and some other arthritic diseases. Here we show that TSG-6 readily formed a complex with a protein present in human, bovine, rabbit, and mouse serum. This complex was stable during SDS-PAGE under reducing conditions, and in the presence of 8 M urea. The protein that binds TSG-6 was purified from human serum and identified as inter-alpha-inhibitor (I alpha I) by N-terminal microsequencing. Microsequencing of the complex itself revealed the presence of TSG-6 and two of the three polypeptide chains of I alpha I (bikunin and HC2). Experiments with recombinant TSG-6 and I alpha I purified from human serum showed that the TSG-6/I alpha I complex is rapidly formed even in the apparent absence of other proteins at 37 degrees C, but not at 4 degrees C. The TSG-6/I alpha I complex was cleaved by chondroitin sulfate ABC lyase, suggesting that cross-linking by chondroitin sulfate is required for the stability of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.

The human Ras-related nuclear protein Ran/TC4 (refs 1-4) is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1 (refs 12, 13), interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, we searched for a Ran-specific GTPase-activating protein (GAP), and for putative effectors (proteins that interact specifically with Ran/TC4-GTP). We report here the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran/TC4-GTP.

The immunoaugmenting properties of murine IgD reside in its C delta 1 and C delta 3 regions: potential role for IgD-associated glycans.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.

Specificity of the murine IgD receptor on T cells is for N-linked glycans on IgD molecules.

IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors.

A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD.

The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.

Seasonally occurring lectins from the bryozoan Bugula neritina.

Two different lectins (termed BnA-I and BnA-II) with distinct carbohydrate specificities were identified and subsequently isolated from the marine bryozoan Bugula neritina. BnA-I hemagglutinating activity was inhibited by N-acetylated hexosamines, their polymers, and glycoproteins rich in these moieties. BnA-II-induced hemagglutination was not blocked by any simple sugars but could be inhibited by several complex glycoproteins (e.g., thyroglobulin and orosomucoid). Both lectins required the presence of Ca(+)+ for reactivity and were purified by affinity chromatographic procedures. Purified BnA-I was determined to have a native molecular weight of 240 Kd and appeared to be a hexameric homopolymer while BnA-II was shown to be a 65-70 Kd monomer. Both lectins showed seasonality in expression, BnA-I appearing in animal extracts prepared in the spring and fall while BnA-II was expressed only during the summer and winter.

Expression of the cloned gene for enterotoxin STb of Escherichia coli.

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the BglII site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong pL promoter. Northern (RNA) blotting assays revealed a 10- to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-beta-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-beta-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to keyhole limpet hemocyanin.

Kinetics of hemolysis induced by a toxin from Bacillus thuringiensis israelensis.

The kinetics of hemolysis resulting from the action on rabbit erythrocytes of a highly purified cytolytic toxin (26,000 mol. wt) isolated from a spore-crystal mixture of Bacillus thuringiensis israelensis was studied. Course of hemolysis, as determined by release of hemoglobin, yielded sigmoid curves whose maximum slopes were taken as a measure of the rate of lysis. Hemolysis occurred without an induction period, and the rate of lysis was a linear function of toxin concentration. Rate of hemolysis as a function of temperature yielded an Arrhenius constant of 9300 calories per mole. The toxin was active between pH 4.5 and 8.0. Lysis was strongly inhibited by Cu2+, Fe2+ and Zn2+ in concentrations as low as 0.025 M. Phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin inhibited lysis, whereas phosphatidylethanolamine, cerebroside, cholesterol and major integral erythrocyte membrane proteins caused little or no inhibition. Inhibition of lysis by sucrose indicates that hemolysis is of the colloid-osmotic type.

Isolation of a hemolysin from a spore-crystal mixture of Bacillus thuringiensis israelensis (serotype H-14).

A hemolytic toxin from Bacillus thuringiensis israelensis was obtained by alkaline extraction and fast protein liquid chromatography (chromatofocusing followed by gel filtration). The toxin displayed a pI of 4.6-4.8 and an Mr of 26,000. Amino acid analysis demonstrated large amounts of serine, glycine and glutamic acid. The toxin was strongly lytic for rabbit, human and non-human primate erythrocytes, and was weakly lytic toward equine, feline, canine, bovine, amphibian and reptilian erythrocytes. It was weakly entomocidal as indicated by a lethal potency (LC50) of 25 micrograms/ml for second instar larvae of Anopheles stephansi. Direct micro-ELISA indicated that the toxin lacked antigenic identity with numerous eukaryotic and prokaryotic toxins and venoms.

Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor.

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes"). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.

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.

Lack of inhibitory effects of alpha 1-acid glycoprotein (orosomucoid) on Plasmodium falciparum invasion of human erythrocytes.

There has been controversy whether the plasma protein, alpha 1-acid glycoprotein (AGP), is able to inhibit invasion of erythrocytes by P. falciparum merozoites. Because AGP resembles a typical cell membrane sialoglycoprotein, it has been proposed that it can inhibit the parasite from interacting with its sialoglycoprotein receptor on the erythrocyte surface. We therefore isolated and tested samples of AGP obtained from a series of separate individuals. For comparative purposes, we also tested AGP prepared from the plasma of patients with elevated levels of AGP, as well as AGP obtained from two commercial sources. The authenticity and purity of the AGP samples was established by SDS-PAGE, radial immunodiffusion, and crossed immunoelectrophoresis. Our results indicated that none of the nine samples tested had any significant inhibitory effects in our P. falciparum invasion assay system.

Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

Rapid purification of anti-I and anti-i cold antibodies by affinity chromatography.

A method is described for the rapid purification of serologically active high titer anti-I and anti-i cold antibodies from the sera of patients with chronic cold agglutinin disease (CCAD). The purification procedure is based on thermal affinity chromatography, using desialated orosomucoid (alpha 1-acid glycoprotein)-Sepharose 4B conjugated beads. The nature of the interaction between the cold agglutinins (CA) and the desialated orosomucoid is unknown. Inhibition studies, however, revealed that the cold hemagglutinating activities of all the anti-i sera were inhibited by desialated orosomucoid while only 1 out of 4 of the anti-I sera was similarly affected. Anti-I or anti-i antibodies were separated from whole sera in 7 out of 7 samples with a recovery in most cases of 100% of the cold hemagglutinating activity. The resultant products were purified monoclonal IgM fractions which could react with anti-kappa and anti-mu but not with anti-lambda sera. The homogeneity, purity and specificity of all preparations were confirmed by immunodiffusion analysis against purified I and i blood group antigens isolated from human erythrocyte membranes, zonal and right-angle electrophoresis and hemagglutination or hemagglutination inhibition studies.

Role of the carbohydrate domains of glycophorins as erythrocyte receptors for invasion by Plasmodium falciparum merozoites.

Solubilized preparations of purified glycophorins and specific domains of these molecules were assessed for their effects as inhibitors of Plasmodium falciparum invasion of human erythrocytes in vitro. The ability of newly invaded merozoites to continue developing and incorporating [3H]hypoxanthine during a 24-h period after their invasion was used as an assay for merozoite invasion. Glycophorins A, B, and C were found to be equally effective as inhibitors. Previous studies had shown N-acetylglucosamine, a sugar component of glycophorins A and C but not B, to be an effective inhibitor. Accordingly, molecular domains common to all of the glycophorins were further assessed. Sialic acid was shown to act almost as effectively as N-acetylglucosamine, presumably because of the structural similarities between these sugars. The inhibitory ability of sialic acid is considerably enhanced when presented to the parasite in a clustered form, as in an oligosaccharide. The acetyl group of these sugars does not appear to play an essential role in this inhibition. How the P. falciparum merozoite recognizes and interacts with the sugar domains of the glycophorin molecule remains to be determined.

Assay of erythrocyte components as inhibitors of Plasmodium falciparum merozoite invasion of erythrocytes.

Three red blood cell membrane fractions (Band 3, macromolecules exhibiting I antigenic determinants, and a delipidated glycoprotein fraction) were separated from red blood cell membranes and tested for their ability to inhibit penetration of red blood cells by Plasmodium falciparum merozoites in an in vitro inhibition assay. The delipidated glycoprotein fraction (containing the major sialoglycoproteins and devoid of Band 3) was the only fraction that inhibited merozoite invasion. This fraction showed 73% and 70% inhibition at 1 mg/ml and 500 micrograms/ml, respectively, and slight inhibition below these levels.

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.