Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or (13)C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay.     

Molecular characterization of a mouse heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue-specific expression

The human DNA- and RNA-binding protein JKTBP is a new member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis. We cloned and characterized a mouse homolog and studied its expression in mouse tissues. The cDNA encoded a 301-residue polypeptide. There is only a single amino acid difference between the mouse and human sequences. Northern blotting indicated ubiquitous but varied expressions of approximately 1.4 and 2.8kb mRNAs in various tissues. Immunoblotting indicated that the amounts of protein of about 38kDa were higher in the brain and testis than in other tissues. An additional protein of about 53kDa was found in the brain and testis. Germ cell-deficient W/W(v) mutant mice and aged mice had the reduced amounts of JKTBP in the testes. Immunohistochemical staining indicated cell type-specific expression of JKTBP in tissues: neurons and spermatocytes displayed strong signal intensities. The signals were confined to the nucleus. The amount of 38kDa JKTBP was estimated to be approximately 1.3x10(7) molecules per HL-60 cell. These results indicate that JKTBP is an abundant, highly conserved nuclear protein.     

Characterization of a novel member of the FGFR family, HrFGFR, in Halocynthia roretzi

The cDNA for a novel member of the FGFR family, named HrFGFR, was isolated from a Halocynthia roretzi cDNA library prepared at the mid-tailbud stage. This cDNA was 3507b long, and the deduced amino acid sequence contained a motif characteristic of the vertebrate FGFRs. The existence of a single copy of the FGFR homologue gene in H. roretzi was suggested by restriction site analysis of multiple clones. HrFGFR mRNA was expressed strongly in the posterior region in the epidermis from the middle neurula stage. By contrast, Xenopus FGFR homologues are expressed in the anterior region and are known to induce anterior neural formation. A transition of the region expressing FGFR might have induced the more complicated brain or head formation characteristic of vertebrates.     

Association between delayed bedtime and sleep-related problems among community-dwelling 2-year-old children in Japan

Background

Although delayed sleep timing causes many socio-psycho-biological problems such as sleep loss, excessive daytime sleepiness, obesity, and impaired daytime neurocognitive performance in adults, there are insufficient data showing the clinical significance of a ‘night owl lifestyle’ in early life. This study examined the association between habitual delayed bedtime and sleep-related problems among community-dwelling 2-year-old children in Japan.

Methods

Parents/caregivers of 708 community-dwelling 2-year-old children in Nishitokyo City, Tokyo, participated in the study. The participants answered a questionnaire to evaluate their child’s sleep habits and sleep-related problems for the past 1 month.

Results

Of the 425 children for whom complete data were collected, 90 (21.2%) went to bed at 22:00 or later. Children with delayed bedtime showed significantly more irregular bedtime, delayed wake time, shorter total sleep time, and difficulty in initiating and terminating sleep. Although this relationship indicated the presence of sleep debt in children with delayed bedtime, sleep onset latency did not differ between children with earlier bedtime and those with delayed bedtime. Rather, delayed bedtime was significantly associated with bedtime resistance and problems in the morning even when adjusting for nighttime and daytime sleep time.

Conclusions

Even in 2-year-old children, delayed bedtime was associated with various sleep-related problems. The causal factors may include diminished homeostatic sleep drive due to prolonged daytime nap as well as diurnal preference (morning or night type) regulated by the biological clock.     

An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/μL to 0.01 ng/μL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system’s potential to be applied to patient samples.     

Ameliorative effects of mulberry (Morus alba L.) leaves on hyperlipidemia in rats fed a high-fat diet: induction of fatty acid oxidation, inhibition of lipogenesis, and suppression of oxidative stress

To determine the effects of mulberry (Morus alba L.) leaves on hyperlipidemia, we performed gene expression profiling of the liver. Rats were fed a high-fat diet and administered mulberry leaves for 7 weeks. Plasma triglyceride and non-esterified fatty acid levels were significantly lower in the rats treated with mulberry leaves as compared with the untreated rats. DNA microarray analysis revealed that mulberry leaves upregulated expression of the genes involved in α-, β- and ω-oxidation of fatty acids, mainly related to the peroxisome proliferator-activated receptor signaling pathway, and downregulated the genes involved in lipogenesis. Furthermore, treatment with mulberry leaves upregulated expression of the genes involved in the response to oxidative stress. These results indicate that consumption of fatty acids and inhibition of lipogenesis are responsible for the reduction in plasma lipids caused by mulberry administration. In addition, mulberry treatment maintains the body's oxidative state at a low level despite enhancing fatty acid oxidation.     

A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome)

Early infantile epileptic encephalopathy with suppression-burst pattern (EIEE) is one of the most severe and earliest forms of epilepsy, often evolving into West syndrome; however, the pathogenesis of EIEE remains unclear. ARX is a crucial gene for the development of interneurons in the fetal brain, and a polyalanine expansion mutation of ARX causes mental retardation and seizures, including those of West syndrome, in males. We screened the ARX mutation and found a hemizygous, de novo, 33-bp duplication in exon 2, 298_330dupGCGGCA(GCG)9, in two of three unrelated male patients with EIEE. This mutation is thought to expand the original 16 alanine residues to 27 alanine residues (A110_A111insAAAAAAAAAAA) in the first polyalanine tract of the ARX protein. Although EIEE is mainly associated with brain malformations, ARX is the first gene found to be responsible for idiopathic EIEE. Our observation that EIEE had a longer expansion of the polyalanine tract than is seen in West syndrome is consistent with the findings of earlier onset and more-severe phenotypes in EIEE than in West syndrome.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.