Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.     

Breakdown of self-incompatibility in a natural population of Petunia axillaris caused by loss of pollen function

Although Petunia axillaris subsp. axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S-haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an S(C1)- or an S(C2)-haplotype. S(C1)S(C1) and S(C2)S(C2) homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S-homozygotes (S(1)S(1) through S(40)S(40)) generated from plants identified from three mixed populations, including U83. The S(C1)S(C1) homozygote was reciprocally compatible with all the genotypes examined. The S(C2)S(C2) homozygote accepted pollen from all but the S(17)S(17) homozygote (identified from the U1 population), but the S(17)S(17) homozygote accepted pollen from the S(C2)S(C2) homozygote. cDNAs encoding S(C2)- and S(17)-RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying S(C1) or S(C2) showed that the SI behavior of S(C1) and S(C2) was identical to that of S(C1) and S(C2) homozygotes, respectively. All these results taken together suggested that the S(C2)-haplotype was a mutant form of the S(17)-haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.     

ESR spin trapping of hydroxyl radicals in aqueous solution irradiated with high-LET carbon-ion beams

The aim of this study was to quantify the hydroxyl radicals (*OH) produced when aqueous solutions are decomposed by high-linear energy transfer (LET) 290 MeV/nucleon carbon-ion beams using an electron spin resonance (ESR) spectrometer. Aerated cell culture medium containing 200 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was irradiated with doses of 0 to 20 Gy with an LET of 20 to 90 keV/ micro m. We were able to obtain ESR spectra 10 min after irradiation, and the formation of *OH and hydrogen atoms was confirmed by radiolysis of deuterium oxide and ethanol containing DMPO. Our results showed that the yield of *OH by carbon-ion radiolysis increased in proportion to the absorbed dose over the range of 0 to 20 Gy. Furthermore, we discovered that the yield of *OH decreased linearity as LET increased logarithmically from 20 to 90 keV/ micro m. The generation of *OH by carbon-ion radiolysis at LETs of 20, 40, 60, 80 and 90 keV/ micro m was 64, 58, 52, 49 and 50%, respectively, of that for low-LET X radiolysis. These unique findings provide a further understanding of the indirect effect of high-LET radiation.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.     

Skin temperature changes induced by strong static magnetic field exposure

High intensity static magnetic fields, when applied to the whole body of the anesthetized rat, have previously been reported to decrease skin temperature. The hypothesis of the present study was that in diamagnetic water, molecules in the air play significant roles in the mechanism of skin temperature decrease. We used a horizontal cylindrical superconducting magnet. The magnet produced 8 T at its center. A thermistor probe was inserted in a subcutaneous pocket of the anesthetized rats to measure skin temperature. Animals (n=10) were placed in an open plastic holder in which the ambient air was free to move in any direction (group I). Animals (n=10) were placed in a closed holder in which the air circulation toward the direction of weak magnetic field was restricted (group II). Each holder was connected to a hydrometer to measure humidity around the animal in the holder. The data acquisition phase consisted of a 5 min baseline interval, followed by inserting the animal together with the holder into the center of the magnet bore for a 5 min exposure and a 5 min postexposure period outside the bore. In group I, skin temperature and humidity around the animal significantly decreased during exposure, followed by recovery after exposure. In group II, skin temperature and humidity did not decrease during the measurement. The skin temperature decrease was closely related to the decrease in humidity around the body of the animal in the holder, and the changes were completely blocked by restricting the air circulation in the direction of the bore entrance. Possible mechanisms responsible for the decrease in skin temperature may be associated with magnetically induced movement of water vapor at the skin surface, leading to skin temperature decrease.     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes

Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.     

Mushroom tyrosinase inhibitory activity of esculetin isolated from seeds of Euphorbia lathyris L

A tyrosinase inhibitor was isolated from the seeds of Euphorbia lathyris L. by bioassay-guided fractionation and purification, using silica gel column chromatography. It was identified as esculetin by comparing its physical properties and spectral data with those of an authentic sample. The IC50 value of esculetin in the mushroom tyrosinase activity test was 43 microM. The kinetic study indicates that esculetin exhibited competitive inhibition against the oxidation of 3-(3,4-dihydroxyphenyl)-alanine by mushroom tyrosinase. The structure-activity relationships among five esculetin analogs suggest that hydroxyl groups at the C6 and C7 positions of the coumarin skeleton played an important role in the expression of tyrosinase inhibitory activity.