Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

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.     

Specific effect of magnesium ion on 2′, 3′-cyclic amp synthesis from adenosine and trimeta phosphate in aqueous solution

Phosphorylation of adenosine by trimetaphosphate was investigated using various catalysts in aqueous solution under mild conditions at pH 7.0 and at 41 °C. The product was primarily 2,3-cyclic AMP together with smaller amounts of ATP. Magnesium ion was found to have a remarkable catalytic effect of approximately one hundred times greater than the other chemicals tested. The mechanism for the specific effect of magnesium ion is discussed.     

Oligo-glycine synthesis in an aqueous solution of glycine under oxidative conditions

Di-and tri-glycine were synthesized in 1M aqueous solution of glycine by bubbling for 90 hr with oxygen discharged in the path from an oxygen cylinder. The peptides were also produced by an incubation at 37°C of 2M glycine solution prepared with 75% hydrogen peroxide, and the yields were traced for 200 days. The final yields were about 0.25% and 0.01% for di-and tri-glycine, respectively. The solution at 166 days of incubation was applied to a Sephadex G 10 column, and the fractions around the top of the chromatogram were found to increase the intensity of ninhydrin color about 45 times after hydrolysis, indicating an existence of oligo-glycine. The solutions of 1M glycine and 0.5M diglycine prepared with 30% hydrogen peroxide were incubated at 37°C for 38 days, and di-and tetra-glycine were detected in the yields of 0.12% and 0.33%, respectively.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

The intermediary metabolite pyruvate attenuates stunning and reduces infarct size in in vivo porcine myocardium

The intermediary metabolite pyruvate has been shown to exert significant beneficial effects in in vitro models of myocardial oxidative stress and ischemia-reperfusion injury. However, there have been few reports of the ability of pyruvate to attenuate myocardial stunning or reduce infarct size in vivo. This study tested whether supraphysiological levels of pyruvate protect against reversible and irreversible in vivo myocardial ischemia-reperfusion injury. Anesthetized, open-chest pigs (n = 7/group) underwent 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion to induce stunning. Load-insensitive contractility measurements of regional preload recruitable stroke work (PRSW) and PRSW area (PRSWA) were generated. Vehicle or pyruvate (100 mg/kg i.v. bolus + 10 mg x kg(-1) x min(-1) intra-atrial infusion) was administered during ischemia and for the first hour of reperfusion. In infarct studies, pigs (n = 6/group) underwent 1 h of LAD ischemia and 3 h of reperfusion. Group I pigs received vehicle or pyruvate for 30 min before and throughout ischemia. In group II, the infusion was extended through 1 h of reperfusion. In the stunning protocol, pyruvate significantly improved the recovery of PRSWA at 1 h (50 +/- 4% vs. 23 +/- 3% in controls) and 3 h (69 +/- 5% vs. 39 +/- 3% in controls) reperfusion. Control pigs exhibited infarct sizes of 66 +/- 1% of the area at risk. The pyruvate I protocol was associated with an infarct size of 49 +/- 3% (P < 0.05), whereas the pyruvate II protocol was associated with an infarct size of 30 +/- 2% (P < 0.05 vs. control and pyruvate I). These findings suggest that pyruvate attenuates stunning and decreases myocardial infarction in vivo in part by reduction of reperfusion injury. Metabolic interventions such as pyruvate should be considered when designing the optimal therapeutic strategies for limiting myocardial ischemia-reperfusion injury.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Identification of ortho-hydroxy anilide as a novel scaffold for lysine demethylase 5 inhibitors

Fe(II)/α-ketoglutarate-dependent lysine demethylases (KDMs) are attractive drug targets for several diseases including cancer. In this study, we designed and screened ortho-substituted anilides that are expected to function as Fe(II) chelators, and identified ortho-hydroxy anilide as a novel scaffold for KDM5A inhibitors. Treatment of human lung cancer A549 cells with a prodrug form of 4-carboxy-2-hydroxy-formanilide (9c) increased trimethylated lysine 4 on histone H3 level, suggesting KDM5 inhibition in the cells.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.