Studies on complement-fixation reaction in equine infectious anemia. II. Identification of complement-fixing inhibitors.

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

Late domain-dependent inhibition of equine infectious anemia virus budding

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.     

Histochemical demonstration of pancreatic B-cells by means of the colloidal iron (Hale) reaction (author's transl)]

The stainability of B-cells in islets of Langerhans by means of colloidal iron reaction has been examined using two standard modifications (Graumann resp. Mowry) of the original Hale-reaction. After previous oxidation of the sections with performic acid a strong and selective staining of B-cells was obtained by the use of a colloidal iron reaction based upon Graumann's method. With Mowry's technique B-cells remained unstained. The demonstration of B-cells using a performic acid-colloidal iron reaction has been compared with methods of known selectivity (Aldehyde Fuchsin, Dichlorpseudoisocyanine). By restaining procedures it could be shown that with the three methods the same type of cell i.e. B-cell is stained.     

The anemia of thermal injury: mechanism of inhibition of erythropoiesis

The anemia of thermal injury is a multifactorial process and includes hemorrhage and hemolysis. Much evidence suggests that a reduced rate of erythropoiesis contributes to this anemia. Prior studies show that this anemia is temporally related to the appearance in burn patients sera of a substance(s) capable of inhibiting erythropoiesis in vitro. Four experiments were done to elucidate the mechanism of action of this inhibitor. In all experiments sera from burn patients previously shown to be inhibitory to erythropoiesis in vitro were studied. In the first, inhibitory sera were exposed to erythropoietin solutions without loss of erythropoietic activity. Second, mouse marrow cells were preincubated with serum without loss of their ability to form erythroid colonies. Third, the inhibitory effect could not be overcome with increasing amounts of erythropoietin. Finally, erythroid colony formation was effected only if the inhibitory serum was present during the first 8 to 12 hr of culture. The data suggest that the erythropoietic inhibitor in these sera acts directly on erythroid stem cells in vitro and not by inactivating or interference with erythropoietin.     

The genus anemia (Schizaeaceae) in Mexico

The fern genusAnemia is represented in Mexico by 21 species and hybrids, of which five are here described as new:A. familiaris, A. multiplex. A. ×paraphyllitidis. A.×recondita andA. semihirsuta. Hybridization is frequent, resulting in both sterile and fertile hybrids, and has probably led to much recent speciation in the genus, including presumably sexual polyploids up to possibly the tetrakaidecaploid level.     

Experimental demonstration of T:(G:G:G:G):T hexad and T:A:A:T tetrad alignments within a DNA quadruplex stem

A template-based approach was used to design unprecedented architectural motifs into a known DNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na(+) solution has been determined using molecular dynamics simulations constrained by distance and dihedral restraints derived from NMR experiments. The molecular topology has been previously observed for the sequence d(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymine into the double chain reversal formed by the segment GGTGG results in the unprecedented experimental demonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment of two G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of the thymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:T tetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick aligned A:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposed tetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. By employing a systematic approach analysis of sequence motifs appearing in double chain reversals, bridging tetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologically relevant genomic regions.     

Characteristics of inhibition of infectious pancreatic necrosis virus (IPNV) by normal rainbow trout Oncorhynchus mykiss serum

We studied the characteristics of rainbow trout serum (RTS) inhibitory activity against infectious pancreatic necrosis virus (IPNV). Serum inhibition was related to the serum source and host cell in which the virus had been propagated. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines, with inhibition highest in rainbow trout gonad (RTG)-2 cells. The RTS sensitivity of the virus was modified by the cell line through which the virus passed, with multiple passages through Chinook salmon embryo (CHSE)-214 cells producing a virus that was less sensitive to RTS. The RTS inhibition level was dependent on cell density: at a cell density of < or = 2 x 10(5) cells ml(-1), inhibition was insignificant (tissue culture infective dose 50% = 10(-1.1) TCID50 ml(-1) reduction); however, above a density of 3 x 10(5) cells ml(-1), the inhibition level was very high (> or = 10(-6.3) TCID50 ml(-1) reduction). The salmonid sera tested showed high inhibition, except for brook trout serum (BTS), while non-salmonid sera did not inhibit IPNV, replication on RTG-2 cells. Pretreatment of cultured cells with RTS prior to exposure did not affect inhibition of IPNV and thus did not mask a viral receptor. The RTS inhibition level was dependent on the time of serum addition, with inhibition being maintained for at least 16 h postinfection. Pretreatment of IPNV revealed that the virus is directly inhibited by RTS, and more strongly so when RTS is present during viral replication.