Immunoprecipitation of nucleosomal DNA is a novel procedure to improve the sensitivity of serum screening for the p16 hypermethylation associated with colon cancer

Background: We developed a novel method of methylation-specific PCR (MSP) using immunoprecipitation with anti-histone antibody (IP-MSP) to efficiently detect serum methylated DNA tightly bound to de-acetylated histones. Materials and methods: The detection limit of IP-MSP for p16 methylation was determined with a standard made by cell line (SKCO-1) lysate. p16 methylation of tumor and/or serum of 51 colorectal cancers and 10 adenoma patients, and 10 healthy volunteers was detected with conventional MSP or IP-MSP. Results: IP-MSP detected p16 methylation from 0.5 pg/μl of the cell lysate. The sensitivity of IP-MSP for detecting serum p16 methylation in 27 patients with tumors characterized by p16 methylation was significantly higher than that with conventional method (81% versus 59%), particularly in Stage II patients (91% versus 45%). IP-MSP detected no p16 hypermethylation in sera of adenoma patients and volunteers. Conclusions: IP-MSP is thus considered to be a promising procedure to detect serum methylated DNA in colorectal cancer patients.     

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography–mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.     

Nascentome analysis uncovers futile protein synthesis in Escherichia coli

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a "nascentome." We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.     

Simplification of the genetic code: restricted diversity of genetically encoded amino acids

At earlier stages in the evolution of the universal genetic code, fewer than 20 amino acids were considered to be used. Although this notion is supported by a wide range of data, the actual existence and function of the genetic codes with a limited set of canonical amino acids have not been addressed experimentally, in contrast to the successful development of the expanded codes. Here, we constructed artificial genetic codes involving a reduced alphabet. In one of the codes, a tRNA^Ala variant with the Trp anticodon reassigns alanine to an unassigned UGG codon in the Escherichia coli S30 cell-free translation system lacking tryptophan. We confirmed that the efficiency and accuracy of protein synthesis by this Trp-lacking code were comparable to those by the universal genetic code, by an amino acid composition analysis, green fluorescent protein fluorescence measurements and the crystal structure determination. We also showed that another code, in which UGU/UGC codons are assigned to Ser, synthesizes an active enzyme. This method will provide not only new insights into primordial genetic codes, but also an essential protein engineering tool for the assessment of the early stages of protein evolution and for the improvement of pharmaceuticals.     

ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl‐tRNA hydrolysis in Escherichia coli

The ribosomes stalled at the end of non‐stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non‐productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans‐translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome‐tethered peptidyl‐tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl‐tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl‐tRNAs located in the P‐site of the stalled ribosome. This reaction required the GGQ (Gly–Gly–Gln) hydrolysis motif, but not the SPF (Ser–Pro–Phe) codon–recognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A‐site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl‐tRNA in a GGQ motif‐dependent but codon‐independent manner. In support of this model, the ArfA‐RF2 pathway did not act on the SecM‐arrested ribosome, which contains an aminoacyl‐tRNA in the A‐site.     

Infection status of masu salmon Oncorhynchus masou by the parasite Salmonema cf. ephemeridarum (Nematoda,Cystidicolidae)

Nematodes that parasitize salmonids are found in both seawater and freshwater. Unlike seawater species such as those in family Anisakidae, freshwater species have not been well studied. In particular, the influences of these nematodes on the body condition of salmonids remain unclear. We studied the effects of Salmonema cf. ephemeridarum on the body condition of masu salmon Oncorhynchus masou. We found a positive relationship between the number of parasites and fish fork length. In contrast, we found a negative relationship between the body condition (condition factor) of fish and the number of parasites. These results suggest that nematode infection could affect host energy reserves for future growth.     

Prevalence of Metabolic Syndrome and Its Components among Japanese Workers by Clustered Business Category

The present study was a cross-sectional study conducted to reveal the prevalence of metabolic syndrome and its components and describe the features of such prevalence among Japanese workers by clustered business category using big data. The data of approximately 120,000 workers were obtained from a national representative insurance organization, and the study analyzed the health checkup and questionnaire results according to the field of business of each subject. Abnormalities found during the checkups such as excessive waist circumference, hypertension or glucose intolerance, and metabolic syndrome, were recorded. All subjects were classified by business field into 18 categories based on The North American Industry Classification System. Based on the criteria of the Japanese Committee for the Diagnostic Criteria of Metabolic Syndrome, the standardized prevalence ratio (SPR) of metabolic syndrome and its components by business category was calculated, and the 95% confidence interval of the SPR was computed. Hierarchical cluster analysis was then performed based on the SPR of metabolic syndrome components, and the 18 business categories were classified into three clusters for both males and females. The following business categories were at significantly high risk of metabolic syndrome: among males, Construction, Transportation, Professional Services, and Cooperative Association; and among females, Health Care and Cooperative Association. The results of the cluster analysis indicated one cluster for each gender with a higher prevalence of metabolic syndrome components; among males, a cluster consisting of Manufacturing, Transportation, Finance, and Cooperative Association, and among females, a cluster consisting of Mining, Transportation, Finance, Accommodation, and Cooperative Association. These findings reveal that, when providing health guidance and support regarding metabolic syndrome, consideration must be given to its components and the variety of its prevalence rates by business category and gender.     

The effect of temperature on growth characteristics and competitions of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Oscillatoria mougeotii</Emphasis> in a shallow,eutrophic lake simulator system

Blue-green algal blooms formed by Microcystis and Oscillatoria often occur in shallow eutrophic lakes, such as Lake Taihu (China) and Lake Kasumigaura (Japan). Growth characteristics and competitions between Microcystis aeruginosa and Oscillatoria mougeotii were investigated using lake simulator systems (microcosms) at various temperatures. Oscillatoria was the superior competitor, which suppressed Microcystis, when temperature was <20°C, whereas the opposite phenomenon occurred at 30°C. Oscillatoria had a long exponential phase (20 day) and a low growth rate of 0.22 day⁻¹ and 0.20 day⁻¹ at 15°C and 20°C, respectively, whereas Microcystis had a shorter exponential phase (2–3 days) at 30°C and a higher growth rate (0.86 day⁻¹). Interactions between the algae were stronger and more complex in the lake simulator system than flask systems. Algal growth in the lake simulator system was susceptible to light attenuation and pH change, and algae biomasses were lower than those in flasks. The outcome of competition between Microcystis and Oscillatoria at different temperatures agrees with field observations of algal communities in Lake Taihu, indicating that temperature is a significant factor affecting competition between Microcystis and Oscillatoria in shallow, eutrophic lakes.