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.     

Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA.

Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4. The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha. It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs. There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA. It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA.     

Bacillus subtilis inositol dehydrogenase-encoding gene (idh): sequence and expression in Escherichia coli.

The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho 11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an Mr of 38,351, was determined. E. coli, bearing pIOL05d15, in which expression of the idh gene is under the control of the lac promoter of pUC118, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-beta-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same Mr and substrate specificity as those of the B. subtilis enzyme.     

Involvement of the histidine residues in the pH-induced conformational change of histone H5

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.     

Distinction and similarity in the structure of histones H1 and H5 as indicated by 13C nuclear-magnetic-resonance spectroscopy

The 13C nuclear magnetic resonance studies have been carried out on histones H1 and H5, by focusing our interest on possible formation of specific salt bridges between acidic and basic amino acid residues in the proteins and also on the structural difference between the two proteins. The 13C chemical shift and pKa values of the carboxyl group of glutamic acid residues in the histones coincided with those of free glutamic acid. Based on this result and another experiment using completely modified lysine residues in the histones, no evidence for a specific interaction between acidic and basic residues has been found. It has also been shown that the pH-effects of aliphatic and aromatic resonances are quite different between H1 and H5, suggesting that the globular domain of H5 is more stable than that of H1. The correlation time (1.5 ns) for the alpha-carbons of H5 estimated from 13C nuclear Overhauser enhancement was twice as long as that of H1 (0.9 ns), indicating that the backbone in the N-terminal and C-terminal domains of H5 is less mobile than that of H1.     

Membrane Viscosity Correlates with α1-Adrenergic Signal Transduction of the Aged Rat Cerebral Cortex

We investigated, using adult (2-month-old) and senescent (12- and 24-month-old) rats, the effects of aging on the relationship between the alpha 1-adrenergic coupling system and the membrane viscosity of the cerebral cortex. There was no age-related difference in the KD values of [3H]prazosin binding on the membranes. The Bmax values of [3H]prazosin binding were reduced with advanced age. Norepinephrine-induced formation of 3H-labeled inositol phosphates (3H-IPs) in the slices increased with advanced age. The EC50 values for norepinephrine to stimulate the formation of 3H-IPs at advanced age were lower than that at adult age. The cholesterol content in membranes increased with advanced age. No changes in the phospholipid content in membranes were observed with advanced age. Concomitantly, an increase of the molar ratio of cholesterol to phospholipids was observed with advanced age. The membrane viscosity as measured by 1,6-diphenyl-1,3,5-hexatriene increased with advanced age. These results indicate that the altered cholesterol content and/or viscosity in cortical membranes of the aged rat may account for the loss of alpha 1-adrenergic receptor density and/or compensatory changes in the receptor-phospholipase C coupling system.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Karyotyping by PFGE of clinical isolates of Sporothrix schenckii

Abstract From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.