Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain.     

Nitrogen subsidies to pelagic food webs through profundal methane-oxidising bacteria in oligotrophic fresh water

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Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V

A gene for T4 endonuclease V was constructed by joining chemically synthesized oligodeoxyribonucleotides and expressed efficiently in Escherichia coli under the control of the E. coli tryptophan promoter. Overproduced T4 endonuclease V, which can cleave thymine photodimers as well as the corresponding phosphodiester linkage of DNA, was used to investigate the precise mode of the reaction with single- or double-stranded synthetic DNA fragments containing a thymine photodimer. The substrates, three oligodeoxyribonucleotides, d(GCGGTTGGCG) (10-mer), d(CGAAGGTTGGAAGC) (14-mer), and d(CACGAAGGTTGGAAGCAC) (18-mer), were prepared by UV irradiation of the nascent oligonucleotides. These single-stranded oligonucleotides were cleaved by the enzyme with a concentration 100 times higher than that required for the corresponding duplexes. The Km values for the TT duplex (14- and 18-mer) were found to be on the order of 10(-8) M. Dissociation constants for the 14- and 18-mer duplexes were measured by a binding assay on a nitrocellulose filter and found to be 10(-9).     

Gene flow and natural selection in oceanic human populations inferred from genome-wide SNP typing

It is suggested that the major prehistoric human colonizations of Oceania occurred twice, namely, about 50,000 and 4,000 years ago. The first settlers are considered as ancestors of indigenous people in New Guinea and Australia. The second settlers are Austronesian-speaking people who dispersed by voyaging in the Pacific Ocean. In this study, we performed genome-wide single-nucleotide polymorphism (SNP) typing on an indigenous Melanesian (Papuan) population, Gidra, and a Polynesian population, Tongans, by using the Affymetrix 500K assay. The SNP data were analyzed together with the data of the HapMap samples provided by Affymetrix. In agreement with previous studies, our phylogenetic analysis indicated that indigenous Melanesians are genetically closer to Asians than to Africans and European Americans. Population structure analyses revealed that the Tongan population is genetically originated from Asians at 70% and indigenous Melanesians at 30%, which thus supports the so-called Slow train model. We also applied the SNP data to genome-wide scans for positive selection by examining haplotypic variation and identified many candidates of locally selected genes. Providing a clue to understand human adaptation to environments, our approach based on evolutionary genetics must contribute to revealing unknown gene functions as well as functional differences between alleles. Conversely, this approach can also shed some light onto the invisible phenotypic differences between populations.     

Production of Host‐selective SV‐toxins by Stemphylium sp. Causing Brown Spot of European Pear in Japan

Recently, a new disease known as ‘brown spot of European pear’ caused by Stemphylium sp. appeared on the leaves, twigs and fruits of the cultivar Le Lectier in Niigata Prefecture, Japan. Because Stemphylium vesicarium (teleomorph: Pleospora allii), which causes a similar disease in Europe, has been shown to produce host‐selective SV‐toxins in culture filtrates (CFs), SV‐toxin production by Stemphylium sp. in Japan was investigated. In pathogenicity tests, the pathogen induced severe necrotic spots on the leaves of the European pear cultivar Le Lectier, slight spots on cultivar La France and slight or no spots on cultivar Bartlett. The Japanese pear cultivar Nijisseiki was not affected by the pathogen. Culture filtrates of the pathogen were tested for phytotoxicity on cultivars by a leaf necrosis assay. The sensitivity of cultivars to the CFs was consistent with the susceptibility of cultivars to the pathogen infection, indicating the presence of host‐selective toxins. The toxins were purified from the CFs according to the procedure reported for SV‐toxin purification in S. vesicarium. The results indicated that Stemphylium sp. in Japan produces the same SV‐toxins as S. vesicarium in Europe.     

In vivo and in vitro patch-clamp recording analysis of the process of sensory transmission in the spinal cord and sensory cortex

Following the integration and modification of the sensory inputs in the spinal cord, the information is transmitted to the primary sensory cortex where the integrated information is further processed and perceived. Processing of the sensory information in the spinal cord has been intensively investigated. However, the mechanisms of how the inputs are processed in the cortex are still unclear. To know the correlation of the sensory processing in the dorsal horn and cortex, in vivo and in vitro patch-clamp recordings were made from rat dorsal horn and sensory cortex. Although dorsal horn neurons showed spontaneous and evoked EPSCs by noxious and non-noxious stimuli, most somatosensory neurons located at 100 to 1000 microm from the surface of the cortex exhibited an oscillatory activity and received synaptic inputs from non-noxious but not noxious receptors. These observations suggest that the synaptic responses in cortical neurons are processed in a more complex manner; and this may be due to the reciprocal synaptic connection between thalamus and cortex.     

Streptomycin-resistant (rpsL) or rifampicin-resistant (rpoB) mutation in Pseudomonas putida KH146-2 confers enhanced tolerance to organic chemicals

We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA). The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene. The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12. The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit. Mutation points in Gen mutants still remain unknown. Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA. Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance. Uptake experiments using [14C]-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA. Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.     

Gender difference in daily time and space use among Bangladeshi villagers under arsenic hazard: application of the compact spot-check method

The compact spot-check method was applied to elucidate the daily time and space use patterns of 121 adults from a Bangladeshi village in which the authors had previously found more skin manifestations due to arsenic poisoning in males than females. The analysis of 2178 spot-check data, which were obtained over a net period of 2 days, highlighted that farming and cash-earning activities were almost exclusively conducted by males. This is probably due to the Muslim Bangladeshi norm for females to be 'sedentary' in or around their own dwellings. Discussion focuses on the advantages of the compact spot-check method and the possibility that males' larger energy expenditure and longer exposure to sunlight are responsible for their severer skin manifestations.     

Structure-activity relationships of a novel class of endothelin receptor selective antagonists; 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridines

The synthesis and structure-activity relationships of 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridine class of ET(A) receptor selective antagonists were described. These derivatives were prepared from the optically active key intermediates (3, 4, 10, and 13). Optimization of the substituent at the 2-position of the bottom 4-methoxyphenyl ring of the lead compound 1 led to identification of 2-hydroxy-1-methylethoxy (2g and h), hydroxyalkyl (2i, m, and p), 3-methoxy-2-methylpropyl (2t and u), N-acetyl-N-methylaminomethyl (2v), and 2-(dimethylcarbamoyl)propyl (2w) derivatives that showed greater than 1000-fold selectivity for the ET(A) receptor over the ET(B) receptor with excellent binding affinity (IC(50)<0.10 nM). Further screening of these compounds by assessing the plasma exposures at 1 h, 4 h, and 8 h after oral administration (3 or 10 mg/kg) in rats led to identification of the hydroxymethyl (2i) and 3-methoxy-2-methylpropyl (2u) derivatives exhibiting good oral bioavailability in rats.