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.     

Promotion of Rooting in Azukia Cuttings by Possible Glycoproteins Extracted from Lentinus edodes Culture

Macromolecules purified from Lentinus edodes mycelia cultureenhanced adventitious root formation in Azukia epicotyl cuttings.Partial purification by sequential column chromatographies gavematerial composed of 71% polysaccharides and 29% proteins. Thesugar moiety consisted of mainly xylose, glucose and arabinose,the sum of their contents being more than 76% of the total carbohydrates.The protein moiety consisted of mainly glycine and serine, whichaccounted for more than 40% of the total amino acid residues. (Received June 9, 1984; Accepted November 12, 1984)     

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.     

Effect of dietary tin deficiency on growth and mineral status in rats

To clarify the influence of dietary tin deficiency on growth and mineral status, the following two different synthetic diets were fed to male Wistar rats: group 1—a diet containing 1.99 μg tin/g; group 2—a diet containing 17 ng tin/g. The rats in group 2 showed poor growth, lowered response to sound, and alopecia, with decreased food efficiency compared with rats in group 1. The changes of mineral concentrations in tissues observed in group 2, compared with group 1, are summarized as follows: calcium concentration in lung increased; magnesium concentration in lung decreased; iron concentrations in spleen and kidney increased; iron concentration in femoral muscle decreased; zinc concentration in heart decreased; copper concentrations in heart and tibia decreased; manganese concentrations in femoral muscle and tibia decreased. These results suggest that tin may be essential for rat growth.     

Neurological disorder and excessive accumulation of calcium in brain of clinically vitamin A-deficient rats

Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological disorders in vitamin A-deficient rats.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Effect of selenium and protein deficiency on selenium and glutathione peroxidase in rats

Twenty-four weanling male Wistar rats were divided into four groups fed diets containing adequate or deficient levels of selenium (0.5 ppm [+ Se] or <0.02 ppm [−Se] and protein (15% [+Pro] or 5% [−Pro]), but adequate levels of all other nutrients for 4 wk to determine the effects of Se deficiency and protein deficiency on tissue Se and glutathione peroxidase (GSHPx) activity in rats. Plasma, heart, liver, and kidney Se and GSHPx were significantly lower in Se-deficient groups in relation to Se-sufficient groups. In Se-deficient groups, Se and GSHPx were significantly higher in −Se−Pro rats in heart, liver, and kidney. Data analysis showed that there were significant interaction effects between dietary Se and protein on Se and GSHPx of rats. It is assumed that under the condition of Se deficiency. a low level of protein may decrease Se and GSHPx utilization, increase GSHPx synthesis, and result in Se redistribution. This could account for high levels of Se and GSHPx in the −Se−Pro rats compared to −Se+Pro rats.