Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South‐East Asia

Tropical rainforests in South‐East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag‐based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south‐western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28–0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.     

Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

Construction of Transducing Phage ρ 11 Containing α-Amylase Structural Gene of Bacillus subtilis

We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 μM to 10 μM.     

Conformational Change of a Natto Mucin in Solution

The mucin obtained from a natto sample was found to be composed of 58 % of γ-polyglutamic acid and 40% of polysaccharide. The ratio of l- and d-glutamic acid was determined to be 58:42 using l-glutamic acid decarboxylase. The weight- and z-average molecular weight were 2.08 × 10⁵ and 2.22 × 10⁵, respectively. The distribution curve of the sedimentation coefficient showed a small heterogeneity. The mucin molecule was considered to be randomly coiled at pH 5.0 ~ 8.8 and to be a rod-like molecule in the lower pH region.     

Comparison of the Key Enzymes of Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

For Hyphomicrobium 53-49 capable of growing under various conditions, aerobic methanol, anaerobic methanol (with denitrification), autotrophic (H₂-O₂-CO₂), aerobic ethanol and aerobic acetate, investigation and comparison of the specific activities of the following enzymes were performed: alcohol dehydrogenase (NAD-ethanol linked and NAD-methanol linked), primary alcohol dehydrogenase, formaldehyde dehydrogenase (NAD-GSH linked and DCPIP linked), formate dehydrogenase, serine hydroxymethyl transferase, hydroxypyruvate reductase, isocitrate lyase (icl), malate lyase, malate dehydrogenase, ribulosebisphosphate (RuBP) carboxylase, phos-phoenolpyruvate (PEP) carboxykinase (ADP linked), PEP carboxylase (phosphorylating), pyruvate carboxylase (NADH linked and NADPH linked) and α-ketoglutarate carboxylase (NADH linked and NADPH linked). On the basis of the data obtained, it was concluded that during growth on methanol, aerobically and anaerobically, the icl+ serine pathway operated, while during autotrophic growth on H₂-O₂-CO₂, CO₂ was incorporated through the RuBP pathway and others, and during growth on ethanol or acetate, neither the serine pathway nor the RuBP pathway operated. The organism changed its metabolism through the regulation of the metabolic enzymes according to the growth conditions.