Colchicine and cytochalasin B (CB) effects on random movement, spreading and adhesion of mouse macrophages

The role of microtubules and microfilaments in the control of random movement of mouse peritoneal macrophages was examined by studying the colchicine and cytochalasin B (CB) effects. Colchicine in the concentration range of 10^?8–10^?4 M enhances the random movement of these cells. Enhanced movement of macrophages is observed only at colchicine concentrations which cause inhibition of their spreading and adhesion. CB does not enhance random movement at any concentration; it inhibits movement at 10^?7 M and higher concentrations. Furthermore, though 10^?8 M CB alone has no effect on the migration of macrophages, when present along with colchicine, the two drugs act synergistically and enhance random movement to a greater extent than colchicine alone. These findings suggest a cooperative interaction between microtubules and microfilaments in the control of movement of macrophages. A model is presented to explain the nature of this interaction.     

Fluorescence studies of 1,N6-ethenoadenosine triphosphate bound to G-actin: the nucleotide base is inaccessible to water

When 1,N6-ethenoadenosine triphosphate (epsilon-ATP) is free in solution, its fluorescence is collisionally quenched by iodide ion, by methionine, by tryptophan, and by cysteine. None of these quenches the fluorescence of epsilon-ATP bound to G-actin. Thus, the ethenoadenine base is bound in a region of the protein which is inaccessible to collisions with these reagents. Since we have previously shown that the fluorescence of epsilon-ATP is quenched by water, the long lifetime of epsilon-ATP bound to G-actin (36 nsec, vs 27 nsec for epsilon-ATP in water) indicates that the bound nucleotide base is inaccessible to collisional quenching by water molecules.     

Adhesion of lymphoid cell lines to fibronectin-coated substratum: Biochemical and physiological characterization and the identification of a 140-kDa fibronectin receptor

Little information is available on the interaction between lymphocytes and fibronectin (fn). To gain a better understanding on this issue we examined the adhesion of 12 lymphoid cell lines, each exhibiting different phenotypic characteristics, to fn-coated substratum. Of the cell lines tested, five that adhered to fn possessed B-cell characteristics, while neither the T-cell lines nor the pre-B-cell line adhered. The physiology and biochemistry of adhesion of a B-cell line, MOPC 315, were examined in detail. Our results indicated that (1) the adhesion was a specific and time-dependent process, (2) the adhesion was temperature-dependent and inhibited by metabolic inhibitors, such as KCN and 2-deoxyglucose, (3) the presence of cycloheximide and pretreatment of cells with trypsin inhibited adhesion, (4) a 140-kDa surface protein was immunoprecipitated by anti-fn receptor antibodies, (5) the presence of divalent cations was essential for adhesion, (6) the presence of colchicine had no effect on adhesion, while cytochalasin B partially inhibited adhesion, and (7) the treatment of cells by both phorbol 12-myristate 13-acetate and calcium ionophore A23187 enhanced adhesion. In this study, we have established the interaction between lymphoid cell lines and fn. Such an interaction might play an important role in the behavior of lymphocytes in tissues.     

Homogeneous detection of cyanobacterial DNA via polymerase chain reaction

Aims: To design a primer set enabling the identification through PCR of high‐quality DNA for routine and high‐throughput genomic screening of a diverse range of cyanobacteria. Methods and Results: A codon‐equivalent multiple alignment of the phycocyanin alpha‐subunit coding sequence (cpcA) of 22 cyanobacteria was generated and analysed to produce a single degeneracy primer set with virtually uniform product size. Also, an 18S ribosomal RNA detection set is proposed for rejecting false positives. The primer sets were tested against five diverse cyanobacteria, Chlorella vulgaris, Saccharomyces cerevisiae, and Escherichia coli. All five cyanobacteria showed positive amplification of cpcA product with homogeneous fragment length, and no products were observed for any other organism. Additionally, the only product formation observed for the 18S rRNA set was in C. vulgaris and S. cerevisiae. Conclusions: The newly proposed primer set served as effective check primers for cyanobacteria. Cyanobacteria gDNA had a positive, homogenous result, while other bacteria, eukaryotes and alga tested were negative. Significance and Impact of the Study: These novel, broad‐spectrum primers will greatly increase the utility of PCR on newly discovered cyanobacterial species.     

The human cytomegalovirus Fc receptor gp68 binds the Fc CH2-CH3 interface of immunoglobulin G

Recognition of immunoglobulin G (IgG) by surface receptors for the Fc domain of immunoglobulin G (Fcgamma), FcgammaRs, can trigger both humoral and cellular immune responses. Two human cytomegalovirus (HCMV)-encoded type I transmembrane receptors with Fcgamma-binding properties (vFcgammaRs), gp34 and gp68, have been identified on the surface of HCMV-infected cells and are assumed to confer protection against IgG-mediated immunity. Here we show that Fcgamma recognition by both vFcgammaRs occurs independently of N-linked glycosylation of Fcgamma, in contrast with the properties of host FcgammaRs. To gain further insight into the interaction with Fcgamma, truncation mutants of the vFcgammaR gp68 ectodomain were probed for Fcgamma binding, resulting in localization of the Fcgamma binding site on gp68 to residues 71 to 289, a region including an immunoglobulin-like domain. Gel filtration and biosensor binding experiments revealed that, unlike host FcgammaRs but similar to the herpes simplex virus type 1 (HSV-1) Fc receptor gE-gI, gp68 binds to the C(H)2-C(H)3 interdomain interface of the Fcgamma dimer with a nanomolar affinity and a 2:1 stoichiometry. Unlike gE-gI, which binds Fcgamma at the slightly basic pH of the extracellular milieu but not at the acidic pH of endosomes, the gp68/Fcgamma complex is stable at pH values from 5.6 to pH 8.1. These data indicate that the mechanistic details of Fc binding by HCMV gp68 differ from those of host FcgammaRs and from that of HSV-1 gE-gI, suggesting distinct functional and recognition properties.     

Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones

During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.