Cra (or FruR), a global transcription factor with both repression and activation activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra-binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra-binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hithertounidentified genes. All six known promoters are repressed by Cra, but none of the activation-type promoters were cloned after two cyles of SELEX, because the Cra-binding affinity to the repressiontype promoters is higher than the activation-type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra-binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the in vivo promoter strength assay using a newly developed TFP (two-fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein.

Bioactivity-directed fractionation of a hot H2O extract from a blue-green alga Spirulina platensis led to the isolation of a novel sulfated polysaccharide named calcium spirulan (Ca-SP) as an antiviral principle. This polysaccharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium. Ca-SP was found to inhibit the replication of several enveloped viruses, including Herpes simplex virus type 1, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1. It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells. Retention of molecular conformation by chelation of calcium ion with sulfate groups was suggested to be indispensable to its antiviral effect.

The structure of flow around unsteady cloud cavitation on a stationary two-dimensional hydrofoil was investigated experimentally using a conditional sampling technique. The unsteady flow velocity around the cloud cavitation was measured by a Laser Doppler Anemometry (LDA) and matched with the unsteady cavitation appearance photographed by a high-speed camera. This matching procedure was performed using data from pressure fluctuation measurements on the foil surface. The velocities were divided into two components using a digital filter, i.e., large-scale (low-frequency) and small-scale (high frequency) ones. The large-scale component corresponds with the large-scale unsteady cloud cavitation motion. In this manner, the unsteady structure of the cloud cavitation was successfully measured. The experimental result showed that the cloud cavitation observed at the present experiment had a vorticity extremum at its center and a cluster containing many small cavitation bubbles. The convection velocity of the cavitation cloud was much lower than the uniform velocity. The small-scale velocity fluctuation was not distributed uniformly in the cavitation cloud, but was concentrated near its boundary.

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When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300-to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.The RNA polymerase of Escherichia coli is composed of the core enzyme (subunit composition, ␣ 2 ␤␤Ј) with RNA polymerization catalytic activity and one of seven different species of the sigma subunit, each of which participates in transcription of a specific set of genes (10, 15). The intracellular concentration of RNA polymerase in the steady state of growing E. coli W3350 cells is maintained at a constant level characteristic of the rate of cell growth (16). The total number of core enzymes is not more than the total number of genes on the E. coli genome (1). Thus, the distribution pattern for RNA polymerase genes among about 4,000 genes in the genome should vary depending on the culture conditions (15). This finding accentuates the importance of the need for the RNA polymerase to choose which genes to transcribe and how often they are transcribed. The replacement of one core enzyme-associated sigma subunit by another sigma subunit is the most efficient way to alter the promoter recognition specificity of the transcription apparatus and is thus believed to be the major mechanism for switching of the transcription pattern. Thus, the competition between available sigma subunits should be a key determinant of which group genes are transcribed (8,33). In addition to sigma subunit replacement, the activity and specificity of RNA polymerase is also modulated by interaction with about 300 molecular species of transcription factors (14,15). Most of these accessory transcription factors are DNA-binding proteins and interact with RNA polymerase when ...

Comamonas testosteroni TAU1 was not able to grow on phenol as a sole carbon and energy source, but it gained the ability to utilize phenol after a 2-3-week incubation in a medium containing phenol. Phenol hydroxylase (PH) and catechol2,3-dioxygenase (C230) were highly induced by phenol in the adapted strain designated as strain P1, suggesting that phenol was degraded via the meta-pathway. Gene clusters for phenol degradation were isolated from both strains TAU1 and P1. The structural genes encoding multicomponent PH and C230 (aphKLMNOPQB), and a regulatory gene of the NtrC family (aphR), were located in a divergent transcriptional organization. The cloned aphKLMNOPQl3 genes from either strain TAU1 or strain P1 produced active PH and C230 enzymes in strain TA441. No difference was found between the strains in the sequences of aphR and the intergenic promoter region of aphK and aphR. However, the transcriptional activities of the aphK and aphR promoters were higher in strain P1 than in strain TA441. The aphK-promoter activity was not observed in aphR mutant strains and these strains could not grow on phenol. The aphR mutant of strain P1 was able to grow on phenol after transformation with a recombinant aphR gene but strain TAM1 was not, suggesting that the expression of the aph genes is silenced by an unidentified repressor in strain TAU1 and that this repressor is modified in strain P1.

An antiviral sulfated polysaccharide, calcium spirulan (Ca-SP), isolated from Spirulina platensis, was subjected to further purification. Ca-SP was found to be composed of rhamnose, 3-O-methylrhamnose (acofriose), 2,3-di-O-methylrhamnose, 3-O-methylxylose, uronic acids, and sulfate. The backbone of Ca-SP consisted of 1,3-linked rhamnose and 1,2-linked 3-O-methylrhamnose units with some sulfate substitution at the 4-position. The polymer was terminated at the nonreducing end by 2,3-di-O-methylrhamnose and 3-O-methylxylose residues.

In order to evaluate the potency of novel antiviral drugs, 11 natural sulfated polysaccharides (SPs) from 10 green algae ( Enteromorpha compressa, Monostroma nitidum, Caulerpa brachypus, C. okamurai, C. scapelliformis, Chaetomorpha crassa, C. spiralis, Codium adhaerens, C. fragille, and C. latum) and 4 synthetic sulfated xylans (SXs) prepared from the beta-(1,3)-xylan of C. brachypus, were assayed for anti-Herpes simplex virus type 1 (HSV-1) activity. Except for one from E. compressa, all SPs showed potent anti-HSV-1 activities with 50 % inhibitory concentrations (IC (50)) of 0.38 - 8.5 microg/mL, while having low cytotoxicities with 50 % inhibitory concentrations of >2900 microg/mL. Anti-HSV-1 activities of SXs were dependent on their degrees of sulfation. To delineate the drug-sensitive phase, 4 polysaccharides, which showed potent anti-HSV-1 activities, were applied to time-of-addition experiments. Among the polysaccharides tested, 3 polysaccharides (SX4, SP4 from C. brachypus, and SP11 from C. latum) showed strong anti-HSV-1 activities with IC (50) of 6.0, 7.5, and 6.9 microg/mL, respectively, even when added to the medium 8 h post-infection. These experiments demonstrated that some sulfated polysaccharides not only inhibited the early stages of HSV-1 replication, such as virus binding to and penetration into host cells, but also interfered with late steps of virus replication. These results revealed that some sulfated polysaccharides from green algae should be promising candidates of antiviral agents which might act on different stages in the virus replication cycle.