A rapid and simple method for plasmid copy number comparison

Summary A rapid, simple, and sensitive method for plasmid copy number comparison was developed. The extracted plasmids from the same amount of cells were subjected to agarose gel electrophoresis and the gels photographed. The photographs were processed by a Macintosh image analyser to enumerate the densities of plasmid bands. As a size reference, λ-DNA digested with a restriction enzyme was used. The densities divided by size of plasmids (base pair) would represent relative values of their copy numbers.     

Oxidation and reduction of plastoquinone by photosynthetic and respiratory electron transport in a cyanobacterium Synechococcus sp.

The I-D dip, an early transient of the fluorescence induction, was examined as a means to monitor redox changes of plastoquinone in cells of a cyanobacterium, Synechococcus sp. That the occurrence of the dip depends upon the reduced state of the plastoquinone pool was indicated by observations that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not affect the initial rise to I but abolished the subsequent decline from I to D and that illumination of the cells with light 1, prior to fluorescence measurements, eliminated the transient. The I-D dip was prominent in freshly harvested cells containing abundant endogenous substrates, disappeared slowly as the cells were starved by aeration but reappeared on addition of fructose to the starved cells in the dark. The dip that had been induced by a brief illumination of the starved cells with light 2 was rapidly diminished in the dark and KCN inhibited the dark decay of the transient. The results indicate that plastoquinone is reduced with endogenous as well as exogenous substrates and oxidized by a KCN-sensitive oxidase in the dark, thus providing strong support for the view that plastoquinone of photosynthetic electron transport also functions in respiration. In addition, the occurrence of a cyclic pathway of electrons from Photosystem I to plastoquinone, possibly via ferredoxin or NADP, was suggested. Several lines of evidence indicate that, under a strong light 2, Photosystem I-dependent oxidation of plastoquinone predominates over Photosystem II-dependent reduction of the quinone in the cyanobacterium which contains Photosystem I more abundantly than Photosystem II.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

Aspolin, a novel extremely aspartic acid-rich protein in fish muscle, promotes iron-mediated demethylation of trimethylamine-N-oxide

Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acid-rich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala42 and Asp43. Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5' part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca2+-binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe2+, whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat.     

Clinical usefulness of urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori: a collaborative study in nine medical institutions in Japan

Background. A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit.
Materials and Methods. A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests.
Results. Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA.
Conclusions. The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.     

Characteristics of a Japanese adult twin database of high school graduates.

This paper profiles a unique cohort of adult Japanese twins. The database contains more than 700 twin pairs, aged 18 to 66 years, who are all graduates of the secondary school attached to the faculty of education of the University of Tokyo. This school was established in 1948, when the study of twins was burgeoning in Japan, and about 10 to 20 pairs of twins have been admitted there every year to participate in studies on twins in education and in related projects. The zygosity of all twins was determined carefully on the basis of various sources. Data from the perinatal period to adulthood were linkable using ID numbers. Follow-up surveys in the field of medical genetics were performed in 1985, 1989 and 1999. For the third survey, which was sent and received exclusively by mail, the distribution and collection process was also assessed in detail. The response rate was around 40%, which statistically was influenced mainly by previous participation and sex. The limitation of this cohort is its selection bias concerning socioeconomic status and its imbalance in favor of monozygotic pairs.     

Interactions of phage Mu enhancer and termini that specify the assembly of a topologically unique interwrapped transpososome

The higher-order DNA-protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end-enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1-E interaction is essential to promote transpososome assembly, R3-E interaction contributes to the native topology of the transpososome, and L1-E and R2-E interactions are not required for assembly. The data on L2-E and L3-E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R-E and perhaps one L-E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions.