Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha-/gamma-chain cross-linking that is promoted by the gamma-chain cross-linking.

Cross-linking of human fibrin by fibrin stabilizing factor (factor XIIIa) and tissue transglutaminase (ti-TG) was examined by immunoprobing electrophoregrams for positive identification of the cross-linked chains. The immunoprobing was carried out by a new, direct staining technique employing composite gels of a porous protein immobilizing matrix (glyoxyl agarose) blended with a removable polyacrylamide filler that eliminates need for Western blotting. We find that the known rapid cross-linking of gamma-chains into gamma 2-dyads by XIIIa is accompanied by co-cross-linking of the gamma 2-dyads with alpha-chains to form hybrid alpha gamma 2-triads. Little or no cross-linking of relatively abundant alpha- and gamma-chain monads into hybrid alpha gamma-dydads accompanies formation of the alpha gamma 2-triads. Thus, formation of the gamma 2-dyads accelerates the hybrid cross-linking. This acceleration is viewed as demonstrating a previously unknown mode of cooperative interaction between alpha- and gamma-chains arising from cross-linking of the D-domains of the molecules. This strengthened interaction is not critically dependent on fibrinopeptide-release, because alpha gamma 2-triads are similarly formed when fibrinogen is cross-linked by XIIIa. Also observed in the study with XIIIa was the formation of small amounts of homologous gamma 3 and gamma 4 oligomers which had been predicted by others to contribute to branching of fibrin strands. Unlike XIIIa, ti-TG acts preferentially on alpha-chains rather than gamma-chains as known. As alpha gamma-dyad, not seen in reactions with XIIIa, is produced concurrent with the homologous alpha-chain cross-linking. Also, three different species of alpha 2-dyads were produced by ti-TG, two of which were not seen in reactions with XIIIa. The differences in product formation revealed by the specific staining are viewed as providing criteria for distinguishing products of XIIIa and ti-TG in biologic specimens.     

Identification of a novel residue within the second transmembrane domain that confers use-facilitated block by picrotoxin in glycine alpha 1 receptors.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA(A) and glutamate-gated Cl(minus sign) channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA(C) receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine alpha1 receptors from non-use-facilitated to use-facilitated. In wild type alpha1 receptors, PTX inhibited glycine-gated Cl(minus sign) current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15'-serine to glutamine (alpha1(S15'Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in alpha1(S15'Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in alpha1(S15'N) receptors. The reciprocal mutation in the beta2 subunit of alpha1beta2 GABA(A) receptors (alpha1beta2(N15'S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome

Using a generally applicable assay for specific DNA-binding proteins in crude extracts, we have detected and purified an HMG-like nuclear protein from African green monkey cells that preferentially binds to the 172 bp repeat of alpha-satellite DNA (alpha-DNA). DNAase I footprinting with the purified protein detects three specific binding sites (I-III) per alpha-DNA repeat. Site II is 145 bp (one core nucleosome length) from site III on the adjacent alpha-DNA repeat, while site I lies midway between sites II and III. In the alpha-nucleosome phasing frame corresponding with this arrangement, sites I-III would be brought into mutual proximity by DNA folding in the nucleosome. This phasing frame is identical with the preferred frame detected previously in isolated chromatin. Our results suggest that this new and abundant protein recognizes a family of short, related nucleotide sequences found not only in alpha-DNA but also throughout the genome, and that functions of this protein are mediated through its nucleosome-positioning activity. Such nucleosome-positioning proteins may underlie the sequence specificity of both nucleosome arrangements and higher order chromatin structures.     

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.     

In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.     

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far.     

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells.

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells.     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.