A red fluorophore comprising a borinate-containing xanthene analogue as a polyol sensor.

A xanthene derivative containing a borinate moiety emitted red fluorescence with a high quantum yield. The interaction between the borinate and a sugar molecule induced a fluorescence change based on the change in the HOMO-LUMO gap. The response was pH-resistant in a wide range. In addition, catechol quenched through photoinduced electron transfer. The red fluorescence and polyol binding ability of dyes will pave the way for new biological applications of chemical sensors.

Support of Joint Function, Range of Motion, and Physical Activity Levels by Consumption of a Water-Soluble Egg Membrane Hydrolyzate.

This study evaluated the effects of consumption of hydrolyzed water-soluble egg membrane (WSEM) on joint function in an otherwise healthy population experiencing chronic pain. A randomized, double-blind, placebo-controlled crossover study included two 4-week periods of placebo and WSEM consumption, separated by a 4-week washout period. Twenty-five study participants were randomized to either the "placebo-first" or "WSEM first" sequence in the crossover trial, and 22 participants completed the study requirements. Range of motion (ROM) was assessed using digital inclinometry for joints associated with vertical weight bearing from neck to knees and for shoulders. Pain at rest and when physically active was scored for the same anatomical areas using visual analog scales (VAS). Physical functioning was tracked using questionnaires with VAS. Consumption of WSEM was associated with improved ROM for neck, spine, hips, and knees, with ROM for the neck and right knee being significantly improved during WSEM consumption compared to placebo (P < .05). ROM improvement for the dominant shoulder was highly significant during WSEM consumption (P < .01). Physical activity levels were significantly higher after WSEM than after placebo consumption (P < .05). Many aspects of physical functioning as part of daily living improved. Subgroup analysis showed rapid improvement of lower back pain after 5 days of WSEM consumption compared to placebo consumption (P < .05) in subjects who participated in the study during the winter season. Daily consumption of 450 mg WSEM was associated with improved joint function, comfort during daily activities, and increased physical activity.

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury.

Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H2) has been reported to have a suppressive effect on oxidative stress and inflammation. The aim of this study was to evaluate the effects of H2 on fetal lung injury caused by maternal inflammation. Cell viability and the production of interleukin-6 (IL-6) and reactive oxygen species (ROS) were examined by treatment with lipopolysaccharide (LPS) contained in ordinal or H2-rich medium (HM) using a human lung epithelial cell line, A549. Pregnant Sprague Dawley rats were divided into three groups: Control, LPS, and HW + LPS groups. Rats were injected with phosphate-buffered saline (Control) or LPS intraperitoneally (LPS) on gestational day 19 and provided H2 water (HW) ad libitum for 24 h before LPS injection (HW + LPS). Fetal lung samples were collected on day 20, and the levels of apoptosis, oxidative damage, IL-6, and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry. The number of apoptotic cells, and levels of ROS and IL-6 were significantly increased by LPS treatment, and repressed following cultured with HM in A549 cells. In the rat models, the population positive for cleaved caspase-3, 8-hydroxy-2'-deoxyguanosine, IL-6, and VEGF was significantly increased in the LPS group compared with that observed in the Control group and significantly decreased in the HW + LPS group. In this study, LPS administration induced apoptosis and oxidative damage in fetal lung cells that was ameliorated by maternal H2 intake. Antenatal H2 administration may decrease the pulmonary mobility associated with inflammation in premature infants.

Oral intake of a liquid high-molecular-weight hyaluronan associated with relief of chronic pain and reduced use of pain medication: results of a randomized, placebo-controlled double-blind pilot study.

The goal for this study was to evaluate the effects of daily oral intake of a consumable liquid fermentate containing high-molecular-weight hyaluronan, as well as to perform a basic evaluation of safety and tolerability. A randomized, double-blind placebo-controlled study design was used to examine the effects of oral intake of hyaluronan on chronic pain conditions. Safety assessment included a complete blood count with differential, blood chemistry and electrocardiogram. The study duration was 4 weeks, where three tablespoons (45 mL) product or placebo was ingested during the first 2 weeks, and two tablespoons (30 mL) was consumed during the last 2 weeks. Seventy-eight people between the age of 19 and 71 years enrolled, and 72 people completed the study. Statistical analysis was performed using the two-tailed independent t-test for between-group significance and using the paired t-test for within-group significance. A reduction in pain scores was seen after 2 weeks of consumption of both placebo (P<.1) and active (P<.065) product; the reduction was more pronounced in the group consuming the active test product. Using "within-subject" analysis, a highly significant reduction in chronic pain scores was seen after 2 weeks of consumption of three tablespoons of active product (P<.001), whereas only a mild nonsignificant reduction in pain scores was seen in the placebo group. During the reduced intake for the last 2 weeks of study participation, pain scores showed a slight increase. During the last 2 weeks, a significant increase in the quality of sleep (P<.005) and level of physical energy (P<.05) was seen. The pain reduction during the initial 2 weeks was associated with significant reduction in the use of pain medication (P<.05). Consumption of an oral liquid formula containing high-molecular-weight hyaluronan was associated with relief of chronic pain.

Cost-effective development of highly polymorphic microsatellite in Japanese quail facilitated by next-generation sequencing.

Next-generation sequencing technologies permit rapid and cost-effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild-derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3-4.1 and 0.11-0.53 respectively. Compared with the wild-derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (FST ) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.

Age-associated changes in bovine oocytes and granulosa cell complexes collected from early antral follicles.

PURPOSE: To assess the age-associated changes in oocytes and granulosa cells derived from early antral follicles (EAFs). METHOD: Gene expression analysis of granulosa cells of the EAFs using a genome analyzer (Illumina) and in vitro culture of oocyte-granulosa cell complexes (OGCs) of EAFs (400-700 µm in diameter) collected from ovaries of aged (>120 months) and young (<50 months) cows. RESULTS: Gene expression profiles in granulosa cells of EAFs of aged cows, which included changes in genes that encode chaperone proteins and antioxidants. In vivo development of EAFs, as determined by oocyte diameter of EAFs and AFs (3-6 mm in diameter), appeared to be impaired in aged cows and the OGCs of aged cows contained low GSH compared to younger counterparts. When the OGCs were cultured in a medium containing low estradiol (E2, 0.1 µg/mL), the ratio of antrum formation was higher for OGCs from aged animals than that from young animals, while higher abnormal fertilization rate and lower total cell number of the blastocysts were observed in the OGCs of aged cows compared with those of young cows. On the contrary, when the OGCs were cultured in a medium containing 10 µg/mL E2, the ratio of antrum formation and fertilization outcome was comparable between the two age groups, whereas the total cell number of the blastocysts was still low in the aged group. CONCLUSION: Aging affects the gene expression profiles of the granulosa cells, and impairs in vitro developmental ability of OGCs collected from EAFs.

Age-associated changes in gene expression and developmental competence of bovine oocytes, and a possible countermeasure against age-associated events.

In general, maternal age affects the quality of oocytes and embryos. The present study aimed to examine the features and age-associated gene expression profiles of bovine oocytes and embryos as well as to discover possible countermeasures against age-associated events. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and 8- to 16-cell-stage embryos were conducted using next-generation sequencing technology. The gene expression profiles of aged cows showed high expression of genes related to oxidative phosphorylation, eIF4 and p70S6K signaling, and mitochondrial dysfunction in MII-stage oocytes. Oocytes derived from aged cows, compared with those derived from their younger counterparts, exhibited high levels of abnormal fertilization and blastocysts with low total cell numbers. Levels of reactive oxygen species (ROS) and SIRT1 were higher in in vitro-matured oocytes derived from aged cows than in those derived from their younger counterparts. Supplementation of maturation medium with N-acetyl-cysteine (NAC), but not resveratrol, reduced the levels of ROS in the oocytes derived from cows of both age groups; however, resveratrol, but not NAC, improved the fertilization ratio. Conversely, EX 527, an inhibitor of SIRT1, increased the ratio of abnormal fertilization. In conclusion, gene expression profiles of oocytes and embryos derived from aged cows differ from those of oocytes and embryos derived from young cows; in particular, oocytes derived from aged cows show protein and mitochondrial dysfunction. In addition, activation of SIRT1 in oocytes may be a potential countermeasure against age-associated events in oocytes derived from aged cows.

Estradiol supports in vitro development of bovine early antral follicles.

Antrum formation and estradiol (E(2)) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E(2) on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E(2) or androstenedione (A(4)) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A(4), developmentally competent OGCs secreted more E(2) than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E(2) and A(4) on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E(2) (1 µg/ml; E(2)(+)), ii) GCs of OGCs cultured for 4 days without E(2) (E(2)(-)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E(2) (1 µg/ml; AF group). GCs of the E(2)(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E(2) biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E(2) impacts the gene expression profile of GCs to support the in vitro development of OGCs.

Preprocessing implementation for microarray (PRIM): an efficient method for processing cDNA microarray data.

cDNA microarray technology is useful for systematically analyzing the expression profiles of thousands of genes at once. Although many useful results inferred by using this technology and a hierarchical clustering method for statistical analysis have been confirmed using other methods, there are still questions about the reproducibility of the data. We have therefore developed a data processing method that very efficiently extracts reproducible data from the result of duplicate experiments. It is designed to automatically filter the raw results obtained from cDNA microarray image-analysis software. We optimize the threshold value for filtering the data by using the product of N and R, where N is the ratio of the number of spots that passed the filtering vs. the total number of spots, and R is the correlation coefficient for results obtained in the duplicate experiments. Using this method to process mouse tissue expression profile data that contain 1,881,600 points of analysis, we obtained clustered results more reasonable than those obtained using previously reported filtering methods.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Cloning of rabbit TR4 and its bone cell-specific activity to suppress estrogen receptor-mediated transactivation.

To clone a new nuclear receptor, we screened a rabbit heart complementary DNA (cDNA) library with degenerate oligonucleotide probes corresponding to the DNA-binding domain of nuclear receptors, which is highly conserved among receptors. One of the cDNA clones, clone 23, encodes a novel protein of 596 amino acids, and predicted molecular mass is 66 kDa. Homology search analysis identified this protein as rabbit TR4 (TR4-0). We also cloned the cDNA encoding a rabbit TR4 isoform (TR4-1), which lacks the putative C-terminal ligand-binding domain (350 amino acids) caused by a 23-bp exon deletion, which probably occurred during messenger RNA (mRNA) splicing. Northern blot analysis showed that TR4s are expressed with two kinds of mRNAs (9.0 kb and 2.8 kb), both of which are relatively abundant in brain, testis, and bone. RT-PCR analysis, using pairs of primers specific for each TR4, showed that both types of receptor express in various tissues. Furthermore, both are present in primary osteoblasts and bone marrow cells, though the mRNA levels of TR4-0 were much higher than those of TR4-1. A functional study, using a transient transfection assay, showed that both receptors suppressed retinoid X receptor (RXR)-retinoid acid receptor, RXR-TR, and RXR-VDR-mediated transactivation significantly in COS-1 and osteosarcoma cells (UMR-106, ROS17/2.8) and that TR4-0 was much more effective than TR4-1. Unexpectedly, we found that the TR4s effectively suppressed estrogen receptor-mediated transactivation in bone cells, but neither in kidney (COS-1) nor breast cancer cells (MCF-7, one of the major target cells of the estrogen action). Thus, the present study shows a novel property of the TR4 orphan receptor, acting as a bone cell-specific repressor in the estrogen receptor-mediated signaling pathway.

Gene expression of retinoic acid receptors, retinoid-X receptors, and cellular retinol-binding protein I in bone and its regulation by vitamin A.

We investigated the gene expression of retinoic acid (RA) receptors (RARs) and retinoid-binding proteins, and the effect of vitamin A on gene expression in the rat tibia to understand the actions of vitamin A on bone tissue. The transcripts of all three subtypes of all-trans RAR (alpha, beta, and gamma) and two of three subtypes of retinoid-X receptor (alpha and beta) were detected by Northern blotting. Among cellular retinol-binding protein I (CRBP-I) and CRBP-II and cellular retinoic acid-binding protein I and II, only the CRBP-I gene was expressed. These results indicated that in bone, the actions of vitamin A are exerted through these nuclear receptors by regulating target gene expression, and through CRBP-I by modulating the intracellular transport of vitamin A. Moreover, using rats of various retinoid status, we investigated whether the expression of target genes for vitamin A (RAR beta and CRBP-I) is regulated by retinoic acid (RA) in the adult rat tibia. The messenger RNA levels of these genes in vitamin A-deficient rats decreased to half of those in normal rats and were quickly restored (4 h) by either all-trans-RA or 9-cis-RA. Excess RA given to normal rats doubled the messenger RNA levels of these two genes. These results verified that, like other target tissues, bone is a target for vitamin A in terms of gene expression. In addition, we examined the effect of RAs on the expression of the target genes for vitamin D, because it is possible that 9-cis-RA is involved in the transcriptional control of vitamin D receptor by forming a heterodimer complex with retinoid-X receptor. The vitamin D-regulated osteopontin gene was induced 4 h after the administration of RA regardless of retinoid or vitamin D status. RA also induced osteopontin gene expression in concert with vitamin D in normal rats. Specific inhibitors of transcription showed that gene expression may be regulated by RA at the transcriptional level. Thus, the results presented here clarified at the molecular level that bone is a target organ for vitamin A in terms of gene expression.

[Detection and identification of mycobacterial DNA with sputa and AIDS samples by nested-polymerase chain reaction (nested-PCR)].

We have demonstrated the nested-polymerase chain reaction (nested-PCR) assay detected from 14 mycobacterial species. We have further demonstrated the species specific restriction sites within amplified dnaJ gene, which allowed us to differentiate the mycobacterial DNA by combination of the PCR with the restriction fragment length polymorphism (PCR-RFLP). Nested-PCR-RFLP was used to detect and identified mycobacterial DNA in the sputa samples and HIV related samples. The target DNA was a 196-base pair segment of dnaJ gene. Of 68 sputum samples tested, 7 were smear positive for acid-fast bacilli and culture. The 7 samples were also identified with Mycobacterium tuberculosis by PCR-RFLP. We tested 69 HIV related samples (19 frozen samples, 22 paraffin embedded samples and 28 lymphocyte from HIV infected people). Of 38 samples were positive for nested-PCR, 8 M. tuberculosis complex, 7 M. avium, 8 M. intracellulare, and 16 others were detected by PCR-RFLP. The PCR method is useful for the rapid diagnosis of mycobacterial infection.

Chronic melioidosis: a report of the first case in Japan.

A 41-year-old Japanese male with uncontrolled diabetes mellitus and alcoholic liver dysfunction developed melioidosis after his business trip to Indonesia and Singapore in 1988. His disease started with spiked fever on the following day after extraction of a tooth, and a liver abscess developed, followed by abscesses in the spleen and in the subphrenic space. In spite of splenectomy and intensive antimicrobial treatments for three months, he developed parotitis, prostatitis, and abscess of the right submandibular gland at 5 to 16-month interval. Pseudomonas pseudomallei was isolated from the blood and pus from each abscess. The lung was not involved. At present, he has returned to work, with continued intravenous instillation of imipenem/cilastatin.

[Detection of mycobacteria by DNA amplification].

Polymerase Chain Reaction (PCR) was used to detect and to identify Mycobacterium species. In this study, 13 out of 14 Mycobacterium species were detected by using six pairs of oligonucleotide primers. The PCR product was detected by non-isotopic southern blot hybridization even when as little as 10 fg of purified M. tuberculosis DNA was used. And 8 mycobacterial species were identified by PCR-Restriction Fragment Length Polymorphism (RFLP) using two kinds of endonuclease.

[Zygomycosis caused by Cunninghamella bertholletiae].

Cunninghamella bertholletiae, an uncommon cause of human fungal infection, has been reported with increasing frequency in recent years in Western countries. We report a case of acute myelogenous leukemia terminated by an uncommon complication of zygomycosis caused by C. bertholletiae, which seems to be the first human case reported in Japan. In this case, the fungus disseminated many organs, including the thyroid gland.

[Dosimetry of fast neutrons in 1W nuclear reactor with plastic nuclear-track detectors].

A nuclear reactor at Kinki University is operated at the maximum of 1W. It produces fission neutrons as much as gamma-rays. To facilitate its use for neutron radiobiology, fast neutrons inside the reactor were measured with nuclear-track detectors TS 16 N and a pair of ion chambers. The angular dependence of TS 16 N response, an anisotropy of fast neutron fluxes in the reactor and misuse of the kerma factor assumed for radiation protection business are the major causes of discrepancy is measured doses by the two methods. Correction factors for the three causes are proposed. After correction, neutron doses estimated with TS 16 N and chambers agree within 5%. The dose-rate at the reactor's center is about 20 tissue-cGy/h. This is the first in situ dosimetry of fast neutrons in a reactor with track detectors attached to biologic samples. Our routine usage has demonstrated that, if used with caution, TS 16 N elements are handy, reliable monitors for fast neutron dosimetry as they are insensitive to contaminated gamma-rays and small enough to be attached to biologic samples.