Occurrence of Chaperonin 60 and Chaperonin 10 in primary and secondary bacterial symbionts of aphids: Implications for the evolution of an endosymbiotic system in aphids

Summary All aphids harbor symbiotrophic prokaryotes (primary symbionts) in a specialized-abdominal cell, the bacteriocyte. Chaperonin 60 (Cpn60, symbionin) and chaperonin 10 (Cpn10), which are high and low molecular weight heatshock proteins, were sought in tissues of more than 60 aphid species. The endosymbionts were compared immunologically and histologically. It was demonstrated that (1) there are two types of aphids in terms of the endosymbiotic system: some with only primary symbionts and others with, in addition, secondary symbionts; (2) the primary symbionts of various aphids are quite similar in morphology whereas the secondary symbionts vary; and (3) irrespective of the aphid species, Cpn60 is abundant in both the primary and secondary symbionts, while Cpn10 is abundant in the secondary symbionts but present in small amounts in the primary ones. Based on these results, we suggest that the primary symbionts have been derived from a prokaryote that was acquired by the common ancestor of aphids whereas the secondary symbionts have been acquired by various aphids independently after divergence of the aphid species. In addition, we point out the possibility that the prokaryotes under intracellular conditions have been subject to some common evolutionary pressures, and as a result, have come to resemble cell organelles.     

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data

Journal of Plant Research - A group of temperate grassland plant species termed the “Mansen elements” occurs in Japan and is widely distributed in the grasslands of continental East...     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.     

Compilation of All Genes Encoding Bacterial Two-component Signal Transducers in the Genome of the Cyanobacterium, Synechocystis sp. Strain PCC 6803

Bacteria have devised sophisticated signaling systems for elicitinga variety of adaptive responses to their environment, whichare generally referred to as the "two-component regulatory system."The widespread occurrence of the two-component systems in bothprokaryotes and eukaryotes implies that it is a powerful devicefor a wide variety of adaptive responses of cells to their environment.The two-component signal transducers contain one or more ofthree conserved and characteristic phosphotransfer signalingdomains, named the "transmitter, receiver, and alternative transmitter."The recently determined entire genomic sequence of Synechocystissp. strain PCC 6803 allowed us to compile systematically a completelist of genes encoding such two-component signal transductionproteins. The results of such an effort, made in this study,revealed that at least 80 ORFs were identified as members ofthe two-component signal transducers in this single speciesof cyanobacteria.     

Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A.

Two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural region 5A (NS5A). Recently, we found that both proteins are phosphorylated at serine residues and that p58 is a hyperphosphorylated form of p56. Furthermore, hyper-phosphorylation depends on the production of an intact form of the HCV NS4A protein. To clarify the nature of NS5A phosphorylation, pulse-chase analysis was performed with a transient protein production system in cultured cells. The study indicated that basal and hyperphosphorylation of NS5A occurred after proteolytic production of NS5A was complete. In an attempt to identify the location of the hyperphosphorylation sites in p58, proteins with sequential deletions from the C-terminal region of NS5A and with mutations of possible phosphorylated serine residues to a neutral amino acid, alanine, were constructed. The deleted or mutated proteins were then tested for hyperphosphorylation in the presence of the NS4A product. Here, we report that serine residues 2197, 2201, and/or 2204 are important for hyper-phosphorylation. Important sites for basal phosphorylation were identified in the region from residues 2200 to 2250 and in the C-terminal region of the NS5A product. A subcellular localization study showed that most of the NS5A products were localized in the nuclear periplasmic membrane fraction.     

Circular DNA Plasmid in the Phytopathogenic Fungus Alternaria Alternata: Its Temperature-Dependent Curing and Association with Pathogenicity

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an ~5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26° showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29°. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23°, most of which maintained the plasmid, and from mycelia grown at 29°, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (~1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.     

Hepatitis C virus envelope proteins bind lactoferrin.

Hepatitis C virus (HCV) has two envelope proteins, E1 and E2, which form a heterooligomer. During dissection of interacting regions of HCV E1 and E2, we found the presence of an interfering compound or compounds in skim milk. Here we report that human as well as bovine lactoferrin, a multifunctional immunomodulator, binds two HCV envelope proteins. As determined by far-Western blotting, the bacterially expressed E1 and E2 could bind lactoferrin in human milk directly separated or immunopurified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bindings of lactoferrin and HCV envelope proteins in vitro were confirmed by another method, the pull-down assay, with immunoprecipitated lactoferrin-bound protein A resin. By the same assay, mammal-expressed recombinant E1 and E2 were also demonstrated to bind human lactoferrin efficiently in vitro. Direct interaction between E2 and lactoferrin was proved in vivo, since anti-human lactoferrin antibody efficiently coimmunoprecipitated with secreted and intracellular forms of the E2 protein, but not glutathione S-transferase (GST), from lysates of HepG2 cells transiently cotransfected with the expression plasmids of human lactoferrin and gE2t-GST (the N-terminal two-thirds of E2 fused to GST) or GST. The N-terminal loop of lactoferrin, the region important for the antibacterial activity, has only a little role in the binding ability to HCV E2 but affected the secretion or stability of lactoferrin. Taken together, these results indicate the specific interaction between lactoferrin and HCV envelope proteins in vivo and in vitro.     

Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu archipelago (southwestern Pacific).

In studying the relationship between genetic abnormalities of red blood cells and malaria endemicity in the Vanuatu archipelago in the southwestern Pacific, we have found that of 1,442 males tested, 98 (6.8%) were G6PD deficient. The prevalence of GdPD deficiency varied widely (0%-39%), both from one island to another and in different parts of the same island, and generally correlated positively with the degree of malaria transmission. The properties of G6PD from GdPD-deficient subjects were analyzed in a subset of 53 samples. In all cases the residual red-blood-cell activity was < 10%. There were three phenotypic patterns. PCR amplification and sequencing of the entire coding region of the G6PD gene showed that the first of these patterns corresponded to G6PD Union (nucleotide 1360C-->T; amino acid 454Arg-->Cys), previously encountered elsewhere. Analysis of samples exhibiting the second pattern revealed two new mutants: G6PD Vanua Lava (nucleotide 383T-->C; amino acid 128Leu-->Pro) and G6PD Namoru (nucleotide 208T-->C; amino acid 70Tyr-->His); in three samples, the underlying mutation has not yet been identified. Analysis of the sample exhibiting the third pattern revealed another new mutant: G6PD Naone (nucleotide 497G-->A; amino acid 166Arg-->His). Of the four mutations, G6PD Union and G6PD Vanua Lava have a polymorphic frequency in more than one island; and G6PD Vanua Lava has also been detected in a sample from Papua New Guinea. G6PD deficiency is of clinical importance in Vanuatu because it is a cause of neonatal jaundice and is responsible for numerous episodes of drug-induced acute hemolytic anemia.