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.     

Effect of Clostridium perfringens Beta Toxin on Blood Pressure of Rats

Guanethidine treatment or adrenal medullectomy significantly inhibited the elevation in blood pressure induced by Clostridium perfringens beta toxin, and the combination of the two drastically reduced the pressure rise, to less than 19% of that in control rats. When rats were pretreated with tetrodotoxin or hexamethonium, the toxin-evoked rise was significantly inhibited. Elevation in blood pressure induced by the toxin in spinal rats tended to be less than that in control rats. When investigated by a microscopical technique, arteriolar constriction in the mesenteric vasculature was observed after the blood pressure elevation induced by the toxin reached a maximum. Blood flow in the skin decreased with an increase in blood pressure following intravenous injection of the toxin. It is concluded that beta toxin acts on the autonomic nervous system and produces arterial constriction.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Ring chromosome 15 in a mother and her children

Summary A case of ring chromosome 15 passed on to the index patient's two children is reported, and possible reasons for the infrequent records of inheritance of ring chromosome are suggested.     

PISTIL DEVELOPMENT AND PARIETAL PLACENTATION IN THE PSEUDOMONOMEROUS OVARY OF ZELKOVA SERRATA (ULMACEAE)

Development of female flowers in Zelkova serrata was observed using epi-illuminated microscopy and scanning electron microscopy, with particular attention given to placentation. After the inception of staminodial primordia, the floral apex becomes flat, and the first and subsequently the second carpel primordia appear at opposite comers of the pistil primordium. Inside each carpel primordium a fossette forms. Through differential growth this depression becomes clear and the carpel wall encircles one side of the future placental region. The placental region is detectable even in early stages, but clear signs of ovule inception appear late when the placental region is elevated onto one side of the ovary wall by intercalary growth. Although the relative size of the two carpels varies among flowers, the placental position always appears to be the border between the two carpels and the floral apex. This suggests that the placentation of Zelkova is parietal. The ovule position in tricarpellate ovaries also suggests an evolutionary derivation from ovaries with parietal placentation. Parietal placentation appears to be the original condition in Urticales.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.