Identification of Two Forms of Qβ Replicase with Different Thermal Stabilities but Identical RNA Replication Activity

The enzyme Qβ replicase is an RNA-dependent RNA polymerase, which plays a central role in infection by the simple single-stranded RNA virus bacteriophage Qβ. This enzyme has been used in a number of applications because of its unique activity in amplifying RNA from an RNA template. Determination of the thermal stability of Qβ replicase is important to gain an understanding of its function and potential applications, but data reported to date have been contradictory. Here, we provide evidence that these previous inconsistencies were due to the heterogeneous forms of the replicase with different stabilities. We purified two forms of replicase expressed in Escherichia coli, which differed in their thermal stability but showed identical RNA replication activity. Furthermore, we found that the replicase undergoes conversion between these forms due to oxidation, and the Cys-533 residue in the catalytic β subunit and Cys-82 residue in the EF-Tu subunit of the replicase are essential prerequisites for this conversion to occur. These results strongly suggest that the thermal stable replicase contains the intersubunit disulfide bond between these cysteines. The established strategies for isolating and purifying a thermally stable replicase should increase the usefulness of Qβ replicase in various applications, and the data regarding thermal stability obtained in this study may yield insight into the precise mechanism of infection by bacteriophage Qβ.     

Real-time observation of a single DNA digestion by λ exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA–protein interactions, such as the DNA digestion reaction by λ exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual λ exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of λ exonuclease, a stained λ DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by λ exonuclease revealed that the DNA digestion rate was ~1000 bases/s and also demonstrated high processivity.     

Ecology of seaweed beds on two types of artificial reef

Artificial reefs for the development of valuable fishery resources wereplaced on sandy substrates at 8, 10 and 13 m depths in Muronohana,Ikata, Japan, and observed monthly or bimonthly from February 1999 to June2001.Enteromorpha intestinalis and Colpomeniasinuosa were the primary dominants in spring. The number of seaweedspecies at all the sites gradually increased in winter. Each reef reached aclimax stage of Sargassum spp., Eckloniakurome and Padina arborescens within 18 months.Overall, 38 seaweed species were found during the study.The settlement of kelp, such as E. kurome, was promoted byreduced sand cover as a result of turbulence. Large-scale surface roughnesscould be important in maintaining the communities after initial establishment.     

A field test of porous carbonated blocks used as artificial reef in seaweed beds of <Emphasis Type="Italic">Ecklonia cava</Emphasis>

Marine forests are the main primary producer in coastal waters, supplying food to fish and shellfish as well as providing their spawning and growing sites. It is important to conserve marine forest in order to protect coastal marine environments. A “Marine Block” comprised of steelmaking slag particles combined by CaCO₃ has several merits: (1) solidification of CO₂, (2) stability and safety, (3) porous materials covered with CaCO₃, (4) harmony with the seabed environment, (5) recycling of iron by-products, and (6) the ability to be mass produced in coastal ironworks. The objectives of this study were to observe the algal succession, the growth of Ecklonia cava and other attached organisms on Marine Blocks and concrete blocks compared to natural seaweed beds. In November 2001, five 1 m³ Marine Blocks and five 1 m³ concrete blocks were installed close to E. cava beds around the coastal frontage of Jogashima at the mouth of Tokyo Bay. Scuba-diving observations were continued until April 2006. In the field, a large number of seaweed and animal species were observed on the Marine Blocks, and it was found that the speed of succession, the plant length and the wet weight of E. cava were greater on Marine Blocks than on concrete blocks. The main advantages of Marine Blocks are considered to be the recycling of steelmaking slag, the absorption and solidification of atmospheric CO₂, and the conservation of coastal environments.     

Ca(2+)- and glycoconjugates-dependent prey capture in the heliozoon Actinophrys sol

Exocytosis of extrusomes, secretory granules found in protozoa, is involved in prey capture by the heliozoon Actinophrys sol. Here, we show that extracellular Ca(2+) is necessary for exocytosis and prey capture in A. sol. We found that A. sol could not capture prey cells in a Ca(2+)-free solution. L-type Ca(2+) channel blockers and a calmodulin antagonist also inhibited the capture of prey. These results suggest that Ca(2+) influx via L-type Ca(2+) channels plays a crucial role in exocytosis in A. sol. Concanavalin A (Con A) also inhibited prey capture, and the inhibition was relieved by the addition of its hapten sugar, alpha-mannoside, suggesting that Con A-binding glycoconjugates are implicated in exocytosis of extrusomes and the adhesion of prey cells.     

Hepatitis C virus core protein: Its coordinate roles with PA28γ in metabolic abnormality and carcinogenicity in the liver

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and epidemiological studies indicate that HCV is also associated with insulin resistance and type 2 diabetes mellitus. The HCV core protein is not only a viral structural component but also a pathogenic factor, since its expression leads to the development of liver steatosis, insulin resistance and hepatocellular carcinoma in mice. The nuclear proteasome activator PA28γ/REGγ, which specifically binds to the core protein, is required for the virulence of the core protein. Elucidation of the mechanisms by which HCV core protein participates in the above conditions may provide clues toward the development of novel therapeutic measures for chronic hepatitis C.     

Self-assembly of nanofiber with uniform width from wheel-type trigonal-beta-sheet-forming peptide

A novel C3 symmetric peptide conjugate "Wheel-FKFE" consisting of three beta-sheet-forming peptides with wheel-like arrangement is developed, and the morphology of self-assembled peptide conjugates in aqueous solutions is observed at various pH. The CD spectra of Wheel-FKFE show the formation of beta-sheet structures in pH 6.9 phosphate buffer, whereas random structures are formed in aqueous HCl (pH 3.3) and NaOH (pH 11) solutions. In transmission electron microscopy, nanofibers with a uniform width of 3-4 nm and lengths of several micrometers are observed in pH 6.9 phosphate buffer, whereas nanorods with the width of several nanometers and the length of several tens of nanometers are observed for that of aqueous HCl (pH 3.3) and NaOH (pH 11) solutions. The uniform width (3-4 nm) of the fibers observed in neutral solution indicates formation of columnar self-assembly of Wheel-FKFEs. The fluorescence spectrum of polarity sensitive dye, sodium 8-anilino-1-naphthalenesulfonate (ANS), in the presence of Wheel-FKFE fibers revealed that the polarity inside the fibers corresponds to that of acetone, indicating that the internal space of the fibers possesses medium hydrophobic environment.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.