The Association of the Cholesterol Efflux Capacity with the Paraoxonase 1 Q192R Genotype and the Paraoxonase Activity.

AIMS: Paraoxonase 1 (PON1) binds to high-density lipoprotein (HDL) and protects against atherosclerosis. However, the relationship between functional PON1 Q192R polymorphism, which is associated with the hydrolysis of paraoxon (POXase activity) and atherosclerotic cardiovascular disease (ASCVD), remains controversial. As the effect of PON1 Q192R polymorphism on the HDL function is unclear, we investigated the relationship between this polymorphism and the cholesterol efflux capacity (CEC), one of the biological functions of HDL, in association with the PON1 activity. METHODS: The relationship between PON1 Q192R polymorphisms and CEC was investigated retrospectively in 150 subjects without ASCVD (50 with the PON1 Q/Q genotype, 50 with the Q/R genotype, and 50 with the R/R genotype) who participated in a health screening program. The POXase and arylesterase (AREase: hydrolysis of aromatic esters) activities were used as measures of the PON1 activity. RESULTS: The AREase activity was positively correlated with CEC independent of the HDL cholesterol levels. When stratified by the PON1 Q192R genotype, the POXase activity was also positively correlated with CEC independent of HDL cholesterol. PON1 Q192R R/R genotype carriers had a lower CEC than Q/Q or Q/R genotype carriers, despite having a higher POXase activity. Moreover, in a multiple regression analysis, the PON1 Q192R genotype was associated with the degree of CEC, independent of the HDL cholesterol and POXase activity. CONCLUSIONS: The PON1 Q192R R allele is associated with reduced CEC in Japanese people without ASCVD. Further studies on the impact of this association on the severity of atherosclerosis and ASCVD development are thus called for.

Impaired Cholesterol Efflux Capacity rather than Low HDL-C Reflects Oxidative Stress under Acute Myocardial Infarction.

AIMS: Acute myocardial infarction (AMI) causes irreversible damage to cardiomyocytes due to the discontinuation of oxygen supply and leads to systemic oxidative stress. It has been reported that high-density lipoprotein (HDL) particles have antioxidant capacity, and reduced antioxidant capacity is associated with decreased cholesterol efflux capacity (CEC). The purpose of this study was to clarify the usefulness of CEC measurement in patients with AMI. METHODS: We investigated the association between CEC and oxidative stress status in a case-control study. This study included 193 AMI cases and 445 age- and sex-matched controls. We examined the associations of CEC with HDL-cholesterol (HDL-C) and oxidized human serum albumin (HSA), an index of systemic oxidative stress status, and the effect of aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism, which has been reported to affect HDL-C level and risk for MI, on these associations. RESULTS: Both bivariable and multivariable analyses showed that CEC was positively correlated with HDL-C levels in both AMI cases and controls, with a weaker correlation in AMI cases than in controls. In AMI cases, oxidized HSA levels were associated with CEC in both bivariable and multivariable analyses, but not with HDL-C. These associations did not differ among the ALDH2 genotypes. CONCLUSIONS: CEC, but not HDL-C level, reflects systemic oxidative stress status in patients with AMI. CEC measurement for patients with AMI may be useful in that it provides information on systemic oxidative stress status as well as atherosclerosis risk.

Association between tongue pressure and oral status and activities of daily living in stroke patients admitted to a convalescent rehabilitation unit.

BACKGROUND: Clarifying how tongue pressure in convalescent stroke patients affects oral condition and activities of daily living (ADL) is important for developing oral rehabilitation programs and for rehabilitation care to reacquire ADL. OBJECTIVE: To clarify how tongue pressure is associated with oral status, ADL, and nutritional status in stroke patients. METHODS: Sixty-eight patients aged 77.9 ± 10.0 years participated. The Japanese version of the Oral Health Assessment Tool was used to evaluate oral status. Data such as the ADL index functional independence measure (FIM), nutritional intake method, serum albumin, and body mass index were extracted from medical records. To examine factors associated with tongue pressure, multiple regression analysis was performed adjusting for confounding factors. The level of statistical significance was set at p < .05. RESULTS: In recovery phase stroke patients, tongue pressure was significantly lower in the total assistance group than in the partial assistance/independent group. In addition, tongue pressure was significantly lower in tube feeding patients than in oral feeding patients. FIM cognition score was an independent factor that had a significant effect on tongue pressure. CONCLUSION: These findings suggest that ADL status also affects tongue pressure, thus patients' ADL including the FIM cognition subscale should also be evaluated while measuring tongue pressure.

Serum Fatty Acid Composition Balance by Fuzzy C-Means Method in Individuals with or without Metabolic Dysfunction-Associated Fatty Liver Disease.

Circulating fatty acid composition is assumed to play an important role in metabolic dysfunction-associated fatty liver disease (MAFLD) pathogenesis. This study aimed to investigate the association between the overall balance of serum fatty acid composition and MAFLD prevalence. This cross-sectional study involved 400 Japanese individuals recruited from a health-screening program. We measured fatty acids in serum lipids using gas chromatography-mass spectrometry. The serum fatty acid composition balance was evaluated using fuzzy c-means clustering, which assigns individual data points to multiple clusters and calculates the percentage of data points belonging to multiple clusters, and serum fatty acid mass%. The participants were classified into four characteristic subclasses (i.e., Clusters 1, 2, 3, and 4), and the specific serum fatty acid composition balance (i.e., Cluster 4) was associated with a higher MAFLD prevalence. We suggest that the fuzzy c-means method can be used to determine the circulating fatty acid composition balance and highlight the importance of focusing on this balance when examining the relationship between MAFLD and serum fatty acids.

Additive Effects of Drinking Habits and a Susceptible Genetic Polymorphism on Cholesterol Efflux Capacity.

AIMS: High levels of high-density lipoprotein cholesterol (HDL-C) are not necessarily effective in preventing atherosclerotic cardiovascular disease, and cholesterol efflux capacity (CEC) has attracted attention regarding HDL functionality. We aimed to elucidate whether drinking habits are associated with CEC levels, while also paying careful attention to confounding factors including serum HDL-C levels, other life style factors, and rs671 (＊2), a genetic polymorphism of the aldehyde dehydrogenase 2 (ALDH2) gene determining alcohol consumption habit. METHODS: A cross-sectional study was performed in 505 Japanese male subjects who were recruited from a health screening program. Associations of HDL-C and CEC levels with drinking habits and ALDH2 genotypes were examined. RESULTS: The genotype frequencies of ALDH2 ＊1/＊1 (homozygous wild-type genotype), ＊1/＊2 and ＊2/＊2 (homozygous mutant genotype) were 55%, 37% and 8%, respectively. Both HDL-C and CEC levels were higher in ALDH2 ＊1/＊1 genotype carriers than in ＊2 allele carriers. Although HDL-C levels were higher in subjects who had a drinking habit than in non-drinkers, CEC levels tended to be lower in subjects with ≥ 46 g/day of alcohol consumption than in non-drinkers. Furthermore, CEC levels tended to be lower in ALDH2 ＊1/＊1 genotype carriers with a drinking habit of ≥ 46 g/day than non-drinkers, while for ＊2 allele carriers, CEC levels tended to be lower with a drinking habit of 23-45.9 g/day compared to no drinking habit. CONCLUSIONS: Our results suggest that heavy drinking habits may tend to decrease CEC levels, and in the ALDH2 ＊2 allele carriers, even moderate drinking habits may tend to decrease CEC levels.

Transgenic Forsythia plants expressing sesame cytochrome P450 produce beneficial lignans.

Lignans are widely distributed plant secondary metabolites that have received attention for their benefits to human health. Sesamin is a furofran lignan that is conventionally extracted from Sesamum seeds and shows anti-oxidant and anti-inflammatory activities in the human liver. Sesamin is biosynthesized by the Sesamum-specific enzyme CYP81Q1, and the natural sources of sesamin are annual plants that are at risk from climate change. In contrast, Forsythia species are widely distributed perennial woody plants that highly accumulate the precursor lignan pinoresinol. To sustainably supply sesamin, we developed a transformation method for Forsythia leaf explants and generated transgenic Forsythia plants that heterologously expressed the CYP81Q1 gene. High-performance liquid chromatography (HPLC) and LC-mass spectrometry analyses detected sesamin and its intermediate piperitol in the leaves of two independent transgenic lines of F. intermedia and F. koreana. We also detected the accumulation of sesamin and piperitol in their vegetatively propagated descendants, demonstrating the stable and efficient production of these lignans. These results indicate that CYP81Q1-transgenic Forsythia plants are promising prototypes to produce diverse lignans and provide an important strategy for the cost-effective and scalable production of lignans.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene.

Forsythia spp. are perennial woody plants which are one of the most extensively used medicinal sources of Chinese medicines and functional diets owing to their lignan contents. Lignans have received widespread attention as leading compounds in the development of antitumor drugs and healthy diets for reducing the risks of lifestyle-related diseases. However, the molecular basis of Forsythia has yet to be established. In this study, we have verified de novo deep transcriptome of Forsythia koreana leaf and callus using the Illumina HiSeq 1500 platform. A total of 89 million reads were assembled into 116,824 contigs using Trinity, and 1,576 of the contigs displayed the sequence similarity to the enzymes responsible for plant specialized metabolism including lignan biosynthesis. Notably, gene ontology (GO) analysis indicated the remarkable enrichment of lignan-biosynthetic enzyme genes in the callus transcriptome. Nevertheless, precise annotation and molecular phylogenetic analyses were hindered by partial sequences of open reading frames (ORFs) of the Trinity-based contigs. To obtain more numerous contigs harboring a full-length ORF, we developed a novel overlapping layout consensus-based procedure, virtual primer-based sequence reassembly (VP-seq). VP-seq elucidated 709 full-length ORFs, whereas only 146 full-length ORFs were assembled by Trinity. The comparison of expression profiles of leaf and callus using VP-seq-based full-length ORFs revealed 50-fold upregulation of secoisolariciresinol dehydrogenase (SIRD) in callus. Expression and phylogenetic cluster analyses predicted candidates for matairesinol-glucosylating enzymes. We also performed VP-seq analysis of lignan-biosynthetic enzyme genes in the transcriptome data of other lignan-rich plants, Linum flavum, Linum usitatissimum and Podophyllum hexandrum. The comparative analysis indicated both common gene clusters involved in biosynthesis upstream of matairesinol such as SIRD and plant lineage-specific gene clusters, in particular, genes responsible for biosynthetic pathways for production of podophyllotoxin; CYP71BE54, a key enzyme gene for podophyllotoxin biosynthesis in P. hexandrum, was not found in L. flavum, although both P. hexandrum. and L. flavum yield podophyllotoxin. Altogether, these data have established the fruitful molecular basis of Forsythia and provided insight into the molecular evolution and diversity of lignan biosynthetic pathways.

Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans.

Sesamin is a furofuran lignan biosynthesized from the precursor lignan pinoresinol specifically in sesame seeds. This lignan is shown to exhibit anti-hypertensive activity, protect the liver from damages by ethanol and lipid oxidation, and reduce lung tumor growth. Despite rapidly elevating demand, plant sources of lignans are frequently limited because of the high cost of locating and collecting plants. Indeed, the acquisition of sesamin exclusively depends on the conventional extraction of particular Sesamum seeds. In this study, we have created the efficient, stable and sustainable sesamin production system using triple-transgenic Forsythia koreana cell suspension cultures, U18i-CPi-Fk. These transgenic cell cultures were generated by stably introducing an RNAi sequence against the pinoresinol-glucosylating enzyme, UGT71A18, into existing CPi-Fk cells, which had been created by introducing Sesamum indicum sesamin synthase (CYP81Q1) and an RNA interference (RNAi) sequence against pinoresinol/lariciresinol reductase (PLR) into F. koreanna cells. Compared to its transgenic prototype, U18i-CPi-Fk displayed 5-fold higher production of pinoresinol aglycone and 1.4-fold higher production of sesamin, respectively, while the wildtype cannot produce sesamin due to a lack of any intrinsic sesamin synthase. Moreover, red LED irradiation of U18i-CPi-Fk specifically resulted in 3.0-fold greater production in both pinoresinol aglycone and sesamin than production of these lignans under the dark condition, whereas pinoresinol production was decreased in the wildtype under red LED. Moreover, we developed a procedure for sodium alginate-based long-term storage of U18i-CPi-Fk in liquid nitrogen. Production of sesamin in U18i-CPi-Fk re-thawed after six-month cryopreservation was equivalent to that of non-cryopreserved U18i-CPi-Fk. These data warrant on-demand production of sesamin anytime and anywhere. Collectively, the present study provides evidence that U18i-CP-Fk is an unprecedented platform for efficient, stable, and sustainable production of sesamin, and shows that a transgenic and specific light-regulated Forsythia cell-based metabolic engineering is a promising strategy for the acquisition of rare and beneficial lignans.

Essences in metabolic engineering of lignan biosynthesis.

Lignans are structurally and functionally diverse phytochemicals biosynthesized in diverse plant species and have received wide attentions as leading compounds of novel drugs for tumor treatment and healthy diets to reduce of the risks of lifestyle-related non-communicable diseases. However, the lineage-specific distribution and the low-amount of production in natural plants, some of which are endangered species, hinder the efficient and stable production of beneficial lignans. Accordingly, the development of new procedures for lignan production is of keen interest. Recent marked advances in the molecular and functional characterization of lignan biosynthetic enzymes and endogenous and exogenous factors for lignan biosynthesis have suggested new methods for the metabolic engineering of lignan biosynthesis cascades leading to the efficient, sustainable, and stable lignan production in plants, including plant cell/organ cultures. Optimization of light conditions, utilization of a wide range of elicitor treatments, and construction of transiently gene-transfected or transgenic lignan-biosynthesizing plants are mainly being attempted. This review will present the basic and latest knowledge regarding metabolic engineering of lignans based on their biosynthetic pathways and biological activities, and the perspectives in lignan production via metabolic engineering.

Characterization and application of a L-specific amino acid oxidase from Rhodococcus sp. AIU LAB-3.

An L-specific amino acid oxidase (L-AAO) suitable for assay of N-acyl-L-amino acid amidohydrolase (L-aminoacylase) activity was purified from Rhodococcus sp. AIU LAB-3. The enzyme exhibited broad substrate specificity and catalyzed an oxidative deamination of the a-amino group of L-amino acids. The optimal enzyme activities for L-amino acids tested were observed in the pH range from 6.0 to 8.5, and more than 80% of the maximum activity was obtained at pH 7.5. The enzyme was stable in the pH range from 7.0 to 8.5, and the apparent Km values for those L-amino acids were small. We, therefore, developed a new enzymatic method for assay of L-aminoacylase activity using the L-AAO at pH 7.5. The new enzymatic method had advantages that the L-aminoacylase reaction was spectrophotometrically followed by measuring absorbance at 555 nm. The L-aminoacylase activity was assayed within 10 min using a small reaction volume. Thus, the new enzymatic method was simple and sensitive compared to the ninhydrin method.

Role of osteoglycin in the linkage between muscle and bone.

The interaction between muscle tissues and bone metabolism is incompletely understood. We hypothesized that there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We, therefore, performed comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2 (R206H), the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, to search for muscle-derived bone anabolic factors. Twenty-five genes whose expression was decreased to <1/4, were identified; these included osteoglycin (OGN). Stable overexpression of OGN significantly decreased the levels of Runx2 and Osterix mRNA compared with those in cells transfected with vector alone in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of alkaline phosphatase (ALP), type I collagen (Col1), and osteocalcin (OCN) mRNA as well as ß-catenin and mineralization. A reduction in endogenous OGN level showed the opposite effects to those of OGN overexpression in MC3T3-E1 and mouse calvarial osteoblastic cells. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, ALP, Col1, and OCN mRNA induced by BMP-2 in C2C12 cells. The conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased, respectively, the levels of ALP, Col1, and ß-catenin in MC3T3-E1 cells. Moreover, OGN increased Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-ß in these cells. In conclusion, this study suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.

FAM5C is a soluble osteoblast differentiation factor linking muscle to bone.

Muscle mass is related to higher bone mass and a reduction in fracture risk. However, the interactions between muscle tissues and bone metabolism are incompletely understood and there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We therefore investigated the role of FAM5C in osteoblast differentiation and the interactions between muscle and bone. A reduction of endogenous FAM5C by siRNA reduced the levels of osterix, alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA as well as the levels of type 1 collagen and ß-catenin in mouse osteoblastic MC3T3-E1 cells and mouse calvarial osteoblasts, although FAM5C overexpression significantly antagonized the levels of osterix, ALP and OCN mRNA induced by bone morphogenetic protein-2 in C2C12 cells. The conditioned medium from FAM5C-overexpressed and -suppressed C2C12 cells increased and decreased the levels of osterix, ALP and OCN mRNA in MC3T3-E1 cells, respectively. In conclusion, the present study is the first to show that FAM5C enhances osteoblast differentiation in differentiated osteoblasts, and that the effects of the conditioned medium from FAM5C-modulated myoblastic cells were positively correlated with the effects of FAM5C on osteoblast phenotype in osteoblasts. FAM5C might be an important humoral bone anabolic factor produced from muscle cells.

An outbreak of measles in Saitama City in 2007. What is the vaccination strategy to eliminate measles in Japan?

In the present study, to elucidate an outbreak of measles in Saitama City, Japan, we analyzed the data for all notified subjects with measles. According to an active surveillance program, a total of 464 subjects were notified in 2007. The clinical criteria for the diagnosis of measles were defined as at least 3 days of a generalized maculopapular rash; a fever of 38.0 degrees C or more; and cough, mucus, or pharyngitis. Two peaks according to age group were recognized: namely, children less than 2 years of age and adolescents from 15 to 19 years of age. The latter peak was associated with the period of time when the measles-mumps-rubella vaccine had become a social problem (40.9% of vaccinees and 41.6% of non-vaccinees in this group). Japan is said to be a developing country regarding its measles vaccination strategy. In addition, no national program against measles has yet been established. Continuous efforts to increase immunization coverage are needed to interrupt indigenous measles transmission. The Japanese Ministry of Health, Labor and Welfare should therefore plan and implement a nationwide program to eliminate measles in Japan.