Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

A method for accurate analysis of intermembrane space in organelles enclosed by double envelope membranes.

Centrifugal filtration through a double layer of silicone oil was applied to determine the intermembrane space of organelles enclosed by double envelope membranes, i.e. proplastids, chloroplasts, mitochondria and amyloplasts. The organelles, capable of transporting adenylates by an adenylate translocator located in the inner envelope membrane, were incubated with increasing concentrations of adenylates while maintaining their specific radioactivities constant. Intermembrane spaces were estimated by extrapolation of radioactivities recovered after filtration of the organelles. The values estimated were compared to those obtained employing the classical method measuring the intake of [14C]-sucrose and [14C]-sorbitol which are impermeable to the inner membranes of organelles. The intermembrane space determined by the present method was shown to be uniformly smaller than the sucrose-permeable space which was always smaller than the sorbitol-permeable space.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Differential Responsiveness in Zonal Growth of Cocklebur Seed Axes to CO2, C2H4, O2 and Respiration Inhibitors

The axial growth of de-coated cocklebur (Xanthium pennsylvanicumWallr.) seeds, whose axes were divided into 4 zones, was examinedin relation to the temperature-dependent shift of the effectof C₂H₄ on germination. At 23?C, where both C₂H₄ and CO₂ stimulatedgermination, CO₂ promoted the axial growth at the radicle tipzone, whereas C₂H₄ promoted growth in the proximal portion ofthe axis. At 33?C, C₂H₄ inhibited germination, and stronglysuppressed the growth at the radicle tip, whereas the effectof CO₂ did not change. The inhibition of growth at the radicletip zone was alleviated by O₂ enrichment, which also reversedthe inhibition of germination. It is thus apparent that thetemperature-dependent shift of the action of C₂H₄ is associatedwith a temperature-dependent responsiveness of the radicle tipzone to C₂H₄. Growth of the radicle tip zone was sensitive toNaN₃, whereas the proximal portion was sensitive to benzohydroxamicacid, an inhibitor of alternative respiration, suggesting thatthere may be an increase in the operation of the alternativerespiration path along a gradient of axial tissue from the tiptowards the cotyledonary side. The effects of CO₂ and C₂H₄ arediscussed in relation to the different respiratory activitiesin each axial zone of cocklebur seeds. (Received May 9, 1986; Accepted November 6, 1986)     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Porcine polymorphonuclear leukocyte NADPH-cytochrome c reductase generates superoxide in the presence of cytochrome b559 and phospholipid

NADPH-cytochrome c reductase and cytochrome b559 were purified from the membrane fraction of phorbol myristate acetate-stimulated porcine polymorphonuclear leukocytes. The highly purified reductase oxidized NADPH and generated superoxide when combined with partially purified cytochrome b559 in the presence of phosphatidylcholine. In the same system, but under the anaerobic condition, the reductase was found to reduce cytochrome b559.