Rhododendrol biosynthesis and metabolism in Acer nikoense callus

Cell suspensions derived from Acer nikoense callus, not containing (S)-rhododendrol, converted 4-(p-hydroxyphenyl)-2- butanone into (R)-, (S)-rhododendrol and their glycosides. (R)- and (S)-rhododendrol formed was only detected in the culture medium and their glycosides only in the cells. The former compound disappeared within a short time and the latter one also tended to decrease during prolonged culture. Quantitative analysis of rhododendrol glycosides in the callus showed that most of them were (S)-rhododendrol 2-O--D-glucopyranoside and its content was much lower than that of the original plants. © Rapid Science Ltd. 1998     

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring (GC–MS–EI-SIM). Deuterated lidocaine (d₁₀-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d₁₀-lidocaine (I.S.) was heated at 120°C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS system. The calibration curves showed linearity in the range of 0.1–20 μg/g for lidocaine and mepivacaine, 0.5–20 μg/g for bupivacaine and 1–20 μg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico–legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 μg/g, respectively.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(-L-guluronate)lyase, which was active on poly(-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the -form of the enzyme molecule and resembled poly(-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The -sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Gait Variability before Surgery and at Discharge in Patients Who Undergo Total Knee Arthroplasty: A Cohort Study

This study aimed to determine gait ability at hospital discharge in patients undergoing total knee arthroplasty (TKA) as an indicator of the risk of falling. Fifty-seven patients undergoing primary TKA for knee osteoarthritis participated in this study. Gait variability measured with accelerometers and physical function including knee range of motion (ROM), quadriceps strength, walking speed, and the Timed Up and Go (TUG) test were evaluated preoperatively and at discharge from the hospital (1 month before and 5 days after surgery). All patients were discharged directly home at 5 days after surgery. Knee flexion of ROM, quadriceps strength, walking speed, and the TUG test results were significantly worse at hospital discharge than preoperatively (p < 0.001). However, gait variability was not significantly different before and after TKA. This result indicated that patients following TKA surgery could walk at hospital discharge as stably as preoperatively regardless of the decrease in physical function, including knee ROM, quadriceps strength, and gait speed after surgery.     

Selection of Orthologous Genes for Construction of a Highly Resolved Phylogenetic Tree and Clarification of the Phylogeny of Trichosporonales Species

The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of Trichosporon asahii and T. faecale were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of Cryptococcus neoformans (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of T. asahii, T. faecale and C. neoformans, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase TOR1 and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.     

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrP^Sc. Real-time quaking-induced conversion can amplify very small amounts of PrP^Sc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD₅₀) is reached approximately 10¹⁰/g brain (values varies 10^8.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrP^Sc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD₅₀ unit was equivalent to 0.06–0.27 fg of PrP^Sc. The SD₅₀ values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.     

No Viral Association Found in a Set of Differentiated Vulvar Intraepithelial Neoplasia Cases by Human Papillomavirus and Pan-Viral Microarray Testing

Vulvar Intraepithelial Neoplasia (VIN) is the precursor lesion of Vulvar Squamous Cell Carcinoma (VSCC), and the differentiated type (dVIN) is more frequently observed in relation to VSCC. In contrast to usual-type VIN (uVIN), which is related to infection by human papillomavirus (HPV), a germline mutation in the p53 gene is thought to be associated with ~90% of dVIN cases. To date, no infectious agent has been identified in association with dVIN, and studies investigating this possibility have been hindered by the difficulty in accurately diagnosing dVIN from small biopsies. Here, we used immunostaining for p16^ink4a, a biomarker for HPV infection, to study 14 uVIN high-grade VIN and 14 dVIN cases, and to select 10 dVIN cases to broadly screen for all known viruses using a pan-viral microarray platform (ViroChip). All of the uVIN tissue samples, including 8 warty and 6 basaloid cases, showed positivity with the p16^ink4a immunostain. The staining pattern was full-thickness for all except two cases in which positive staining was localized in the lower 1/3 of the epidermis. In contrast, immunostaining for p16^ink4a was negative in all dVIN cases. ViroChip analysis of 10 pure dVIN samples confirmed the absence of human papillomavirus subtypes or any other virus with the exception of a single sample that showed a weak microarray signature to a porcine herpesvirus. Follow-up PCR testing of the sample was negative for herpesvirus, and in-depth metagenomic next-generation sequencing revealed only sequences corresponding to non-pathogenic viral flora and bacterial contamination. In this study, we demonstrated lack of a virus association in 10 dVIN cases. Alternative pathways for carcinogenesis such as the p53 mutation should be considered for investigation of potential treatment options in dVIN.     

Influence of olive-derived hydroxytyrosol on the toll-like receptor 4-dependent inflammatory response of mouse peritoneal macrophages

Macrophages play important roles in the host innate immune response and are involved in the onset of diseases caused by inflammation. Toll-like receptor 4 (TLR4)-mediated inflammatory responses of macrophages may be associated with diseases such as diabetes and diseases of the cardiovascular system. Hydroxytyrosol (HT) exerts strong antioxidant and anti-inflammatory effects and may be applied in the treatment of inflammatory diseases. In the present study conducted in vitro, we investigated the effects of the TLR4-dependent anti-inflammatory effect of HT on peritoneal macrophage of BALB/c mice. We show here that the elevated levels of iNOS gene expression and nitric oxide production induced by lipopolysaccharide (LPS) (0.25 μg/ml) were suppressed by HT (12.5 μg/ml). LPS-dependent NF-κB gene expression and phosphorylation of NF-κB were not affected by HT under these conditions. In contrast, the expression of TNF-α was significantly increased in the presence of LPS and HT. These results suggest that HT suppressed nitric oxide production by decreasing iNOS gene expression through a mechanism independent of the NF-κB signaling pathway. These novel findings suggest that the modulation by HT of the expression of genes involved in inflammation may involve multiple mechanisms.     

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.