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.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Synthesis of desmosterol and epidesmosterol from hyodeoxycholic acid

A new synthesis of desmosterol was described using hyodeoxycholic acid (3,6-dihydroxy-5β-cholanic acid) as a starting material. Epidesmosterol (3-hydroxycholesta-5,24-diene) was also synthesized for the first time from the same starting material.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.