Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solutions

The dehydration condensation of glycine with trimetaphosphate in aqueous solution has been reinvestigated. Although it has been reported that the condensation of glycine under the alkaline conditions was brought about through the formation of cyclic acylphosphoramidate and hence the condensation of polyglycines could not occur, we found that the condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solution are possible through the formation of their acylphosphates under the neutral or weak acidic conditions.Aqueous solutions of 1.0 M glycylglycine and 1.0 M trimetaphosphate in the various pH from 4.0 to 9.0 were incubated at 38 °C. The solutions were analyzed by HPLC with ninhydrin reaction system. Tetraglycine and hexaglycine were detected and their maximum yields were given in the reaction carried out around pH 7. They are approximately 15% and 4% after 30 days, respectively. Analogous experiments were performed with tetrametaphosphate. The results showed a similar pH dependence for the condensation, but the yields were about one-tenth of those of corresponding experiments with trimetaphosphate.Relative rates of dimerization of glycine, diglycine and triglycine in the equimolar concentration were also investigated at pH 6.0 at 38 °C. The rates for digylcine and triglycine were approximately twice and four times as large as that for glycine.Relevance of the experiments to chemical evolution is discussed.     

Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.     

Demonstration and ontogenesis in the rat of a serum protein analogous to human thyroxine binding globulin

It has been reported evidence based on equilibrium binding, electrophoretic, immunoelectrophoretic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). It is shown that in the sera of postnatal developing animals, between 3 and 21 days, the thyroxine (T4) and the triiodothyronine (T3) binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as T4 carrier to the thyroid binding prealbumin (TBPA), but retains the major role as binder of T3, i.e. of the biologically active thyroid hormone.     

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Ultrastructure of spermatogenesis in the sea star,Asterina minor

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron-dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi-derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron-dense material is deposited under the electron-dense disk; and (6) the nucleus forms a hollow opposite the electron-dense material.