Comparison of conventional and liquid-based cytology, and human papillomavirus testing using SurePath preparation in Japan

We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

Design and synthesis of disubstituted (4-piperidinyl)-piperazine derivatives as potent acetyl-CoA carboxylase inhibitors

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the structure-based design and synthesis of a novel series of disubstituted (4-piperidinyl)-piperazine derivatives as ACC inhibitors. Our structure-based approach led to the discovery of the indole derivatives 13i and 13j, which exhibited potent in vitro ACC inhibitory activity.     

Similarities and differences between phytochrome-mediated growth inhibition of coleoptiles and seminal roots in rice seedlings

In rice, light is known to inhibit the growth of coleoptiles and seminal roots of seedlings through phytochrome. Here we investigated the light-induced growth inhibition of seminal roots and compared the results with those recently determined for coleoptiles. Although three rice phytochromes, phyA, phyB and phyC functioned in a similar manner in coleoptile and seminal root, the Bunsen-Roscoe law of reciprocity was not observed in the growth inhibition of seminal root. We also found coiling of the seminal root at the root tip which appeared to be associated with the photoinhibition of seminal root growth. This could be a new light-induced phenomenon in certain cultivars of rice.Key words: growth, hypocotyl, Oryza sativa, phytochrome, seminal rootPhytochrome-mediated growth inhibition was reported for both coleoptiles and seminal roots of rice seedlings in the same year by two research groups in Nagoya and Tohoku University in Japan, respectively.^1,2 Forty years after the findings, a detailed photobiological study was carried out for the coleoptile growth inhibition.³ In this study, we examined photoinhibition of seminal root growth, and found similarities and differences between light-induced growth inhibition of the two organs in rice seedlings. Although coleoptile growth was inhibited by pulses of light, growth inhibition of seminal roots required light irradiation longer than 6 h. The Bunsen-Roscoe law of reciprocity was not observed in the growth inhibition of seminal root. Action spectra were determined for the growth inhibition of coleoptiles, and the mode of inhibition was found to depend on the age of the coleoptiles. At the early stage of development [40 h after inducing germination (AIG)], photoinhibition was predominantly due to the phyB-mediated low-fluence response (LFR), but at the late developmental stage (80 h AIG), it consisted of the phyA-mediated very low-fluence response (VLFR) as well as the phyB-mediated LFR.^3,4 In the case of root growth, the sensitivity of photoinhibition also depended on age, and was most sensitive in the period of 48–96 h AIG when seedlings were irradiated for 24 h. Using rice phytochrome mutants,⁵ we found that far-red light for root growth inhibition was perceived exclusively by phyA, that red light was perceived by both phyA and phyB, and that phyC had little or no role in growth inhibition. Furthermore, the fluence rate required for phyB-mediated inhibition was more than 10,000-fold greater than that required for phyA-mediated inhibition. These characteristics of photoinhibition in seminal roots are similar to those found in coleoptiles at the late stage of development.³ In seminal roots, photoinhibition appeared to be mediated by photoreceptors in the root itself.Interestingly, coiling of the root tips always occurred when root growth was inhibited under the light condition (Fig. 1B). Under continuous light irradiation, rice seeds germinated ∼30 h AIG. Seminal roots formed a coil at the root tips during the 48–96 h period AIG, and stopped growing. When they were irradiated for only 24 h on the 3rd day AIG, coils started to form just after the end of irradiation. The roots continued to coil for ∼28 h and then began growing straight again (Fig. 1C). The coils were larger and looser than those formed under continuous light condition (Fig. 1, Open in a separate windowFigure 1Light irradiation induces coiling of root tips in rice seedlings (Oryza sativa cv. Nipponbare). A rice seedling was grown in the dark (A), or in continuous white light (55 µole m⁻² s⁻¹) (B) for 7 d at 28°C. In (C), it was irradiated by white light for 24 h during the 48–72 h period after inducing germination, and kept in the dark again until the 7th day. Arrows and arrowheads indicate the seminal and crown roots, respectively. Seedlings were grown in glass tubes of 3-cm diameter.

Table 1

The size of coil of root tips formed after white light irradiation

Gain of deleterious function causes an autoimmune response and Bateson–Dobzhansky–Muller incompatibility in rice

Reproductive isolation plays an important role in speciation as it restricts gene flow and accelerates genetic divergence between formerly interbreeding population. In rice, hybrid breakdown is a common reproductive isolation observed in both intra and inter-specific crosses. It is a type of post-zygotic reproductive isolation in which sterility and weakness are manifested in the F₂ and later generations. In this study, the physiological and molecular basis of hybrid breakdown caused by two recessive genes, hbd2 and hbd3, in a cross between japonica variety, Koshihikari, and indica variety, Habataki, were investigated. Fine mapping of hbd2 resulted in the identification of the causal gene as casein kinase I (CKI1). Further analysis revealed that hbd2-CKI1 allele gains its deleterious function that causes the weakness phenotype by a change of one amino acid. As for the other gene, hbd3 was mapped to the NBS-LRR gene cluster region. It is the most common class of R-gene that triggers the immune signal in response to pathogen attack. Expression analysis of pathogen response marker genes suggested that weakness phenotype in this hybrid breakdown can be attributed to an autoimmune response. So far, this is the first evidence linking autoimmune response to post-zygotic isolation in rice. This finding provides a new insight in understanding the molecular and evolutionary mechanisms establishing post-zygotic isolation in plants.     

Isolation and identification of potent allelopathic substances in rattail fescue

Aqueous methanol extracts of rattail fescue (Vulpia myuros) inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum pratense), Digitaria sanguinalis and Lolium multiflorum. Increasing the extract concentration increased the inhibition, suggesting that rattail fescue may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of rattail fescue was purified and two main inhibitory substances were isolated and identified by spectral data as (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol. Both substances inhibited root and shoot growth of cress at concentrations greater than 0.3 μM. The concentrations required for 50% growth inhibition on root and shoot growth of cress, lettuce, alfalfa, timothy, D. sanguinalis and L. multiflorum were 2.7–19.7 μM for (−)-3-hydroxy-β-ionone, and 2.1–34.5 μM for (+)-3-oxo-α-ionol. The concentration of (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol, respectively, in rattail fescue was 7.8 and 3.7 μg g⁻¹ fresh weight. Considering the endogenous level and the inhibitory activity, (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol may work as allelopathic substances in rattail fescue through the growth inhibition of neighboring plant species.     

Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270) was obtained using sequence-based screening. From {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.Prolyl aminopeptidase (PAP) (EC 3.4.11.5), belonging to the S33 family, is an exopeptidase that catalyzes the hydrolysis of the N terminus prolyl residue of peptides or proteins. This family has catalytic Ser. To date, few applications of this enzyme for peptide synthesis have been reported. However, from the perspective of biotechnology, PAP might be a good tool for synthesizing proline-containing peptides by catalyzing aminolysis.Recently nutraceutical properties of peptides containing proline have received increasing attention. For example, prolyl hydroxyproline (Pro-Hyp) stimulates the growth of fibroblasts from mouse skin (11). Pro-Arg can protect against oxidative stress/damage and H₂O₂-induced human diploid fibroblast cell death (13). Furthermore, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro exhibit angiotensin I-converting enzyme-inhibiting activity (9). In addition to these dipeptides and tripeptides, a cyclic dipeptide (namely, diketopiperazine) containing proline shows several physiological functions. Cyclo[Pro-Pro] (cPP) exerts antibacterial activity against Micrococcus luteus and Pseudomonas aeruginosa (8). Caspase-3 activation by cyclo[Pro-Phe] in HT-29 cells has been described (3). However, its synthesis method has not been established. Enzymatic peptide synthesis presents a useful and desirable strategy because it can conduct specific reactions under milder conditions than those of chemical synthesis.Engineered endoserine proteases that have Cys substituted for catalytic Ser have also been applied for peptide synthesis since subtiligase was constructed by Abrahmsén et al. (1). Because of the weakened hydrolytic activity of the parent enzyme, it is considered that Ser/Cys-substituted protease can trap the substrate (acyl donor). Then, a nucleophilic reaction occurs between another substrate (acyl acceptor) and the trapped acyl donor (2). This is a so-called “aminolysis” reaction. Although aminolysis can conduct peptide synthesis in an aqueous solution, the problem of the necessity of using an N-protected amino acid as an acyl donor remains when using endoproteases.These problems would be solved using exoprotease as a catalyst, because N-terminal free amino groups of acyl donors are recognized by enzymes. It is rarely reported that exoprotease was applied for peptide synthesis, except in the report of Oshiro et al., in which Pro-Phe, Pro-Tyr, and Pro-Trp were synthesized (10). Recently our group reported that the Ser/Cys variant of exoprotease, aminolysin-S, has been constructed and has produced l-Phe-l-Phe ethyl ester and their derivatives from non-N-protected phenylalanine and phenylalanine ethyl ester as acyl donors in aqueous solution (12). However, aminolysin-S cannot produce proline-containing dipeptides.In this study, we describe a PAP from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270). Furthermore, synthesis of various proline peptides was attempted through catalysis by its Ser/Cys variant (scPAP14270) from proline ester and several amino acids and their esters in aqueous solution. A basic characterization to determine the effect of pH and the amount of substrate was conducted. Moreover, correlation was found between proline peptide synthesis and the log D value, which is the distribution coefficient between octanol and water, of acyl acceptors in aminolysis mediated by scPAP14270.     

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.