[Creation of Stereo Color Anatomical Charts Showing Nerve Fibers in the Cerebral Lobe and Gyrus of the Brain for Use in the MR Examination].

In anatomical charts in conventional books, the pathways of nerve fibers are drawn in illustrations. Conversely, with diffusion tensor tractography (DTT), we can visually understand the trajectory of nerve fibers through color. We created a stereo color anatomical chart of the nerve fibers that can be used for magnetic resonance (MR) examination to diagnose the pathway of nerve fibers and that can be used to explain the results of MR examination to visually understand how nerve fiber information is transmitted from the frontal lobe, parietal lobe, occipital lobe, temporal lobe, cerebellar lobe, and cerebral cortex.

Increased oxidative stress and coenzyme Q10 deficiency in centenarians.

Aging populations are expanding worldwide, and the increasing requirement for nursing care has become a serious problem. Furthermore, successful aging is one of the highest priorities for individuals and societies. Centenarians are an informative cohort to study and inflammation has been found to be a key factor in predicting cognition and physical capabilities. Inflammation scores have been determined based on the levels of cytokines and C-reactive protein, however, serum antioxidants and lipid profiles have not been carefully examined. We found that the redox balance of coenzyme Q10 significantly shifted to the oxidized form and levels of strong antioxidants, such as ascorbic acid and unconjugated bilirubin, decreased significantly compared to 76-year-old controls, indicating an increased oxidative stress in centenarians. Levels of uric acid, an endogenous peroxynitrite scavenger, remained unchanged, suggesting that centenarians were experiencing moderate, chronic inflammatory conditions. Centenarians exhibited a hypocholesterolemic condition, while an increase in the ratio of free cholesterol to cholesterol esters suggests some impairment of liver function. Serum free fatty acids and monoenoic acid composition, markers of tissue oxidative damage, were significantly decreased in centenarians, indicating an impairment in the tissue repair system. Despite an elevation of the coenzyme Q10 binding protein Psap, serum total coenzyme Q10 levels decreased in centenarians. This suggests a serious deficiency of coenzyme Q10 in tissues, since tissue levels of coenzyme Q10 significantly decrease with age. Therefore, coenzyme Q10 supplementation could be beneficial for centenarians.

Oxidative stress and abnormal cholesterol metabolism in patients with post-cardiac arrest syndrome.

Patients with post-cardiac arrest syndrome (PCAS) suffer from whole body ischemia/reperfusion injury similar to that experienced by newborn babies. Increased oxidative stress was confirmed in PCAS patients (n = 40) at the time of hospitalization by a significant increase in the percentage of the oxidized form of coenzyme Q10 in total coenzyme Q10 compared to age-matched healthy controls (n = 55). Tissue oxidative damage in patients was suggested by the significant increase in plasma levels of free fatty acids (FFA) and the significant decrease in polyunsaturated fatty acid contents in total FFA. A greater decrease in free cholesterol (FC) compared to cholesterol esters (CE) was observed. Therefore, the FC/CE ratio significantly increased, suggesting deficiency of lecithin-cholesterol acyltransferase secreted from the liver. Time course changes of the above parameters were compared among 6 groups of patients divided according to outcome severity. Rapid declines of FC and CE were observed in patients who died within a day, while levels remained unchanged in patients discharged in a week. These data suggest that liver function is one of the key factors determining the survival of patients. Interestingly, therapeutic hypothermia treatment enhanced the increment of plasma ratio of coenzyme Q10 to total cholesterol at the end of rewarming.

MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.

BACKGROUND: Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU. METHODS: An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay. RESULTS: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 µM. CONCLUSION: The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.

Kinetic analysis of superparamagnetic iron oxide nanoparticles in the liver of body-temperature-controlled mice using dynamic susceptibility contrast magnetic resonance imaging and an empirical mathematical model.

The purpose of this study was to develop a method for analyzing the kinetic behavior of superparamagnetic iron oxide nanoparticles (SPIONs) in the murine liver under control of body temperature using dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) and an empirical mathematical model (EMM). First, we investigated the influence of body temperature on the kinetic behavior of SPIONs in the liver by controlling body temperature using our temperature-control system. Second, we investigated the kinetic behavior of SPIONs in the liver when mice were injected with various doses of GdCl3, while keeping the body temperature at 36°C. Finally, we investigated it when mice were injected with various doses of zymosan, while keeping the body temperature at 36°C. We also investigated the effect of these substances on the number of Kupffer cells by immunohistochemical analysis using the specific surface antigen of Kupffer cells (CD68). To quantify the kinetic behavior of SPIONs in the liver, we calculated the upper limit of the relative enhancement (A), the rates of early contrast uptake (α) and washout or late contrast uptake (ß), the parameter related to the slope of early uptake (q), the area under the curve (AUC), the maximum change of transverse relaxation rate (ΔR2) (ΔR2(max)), the time to ΔR2(max) (Tmax), and ΔR2 at the last time point (ΔR2(last)) from the time courses of ΔR2 using the EMM. The ß and Tmax values significantly decreased and increased, respectively, with decreasing body temperature, suggesting that the phagocytic activity of Kupffer cells is significantly affected by body temperature. The AUC, ΔR2(max), and ΔR2(last) values decreased significantly with increasing dose of GdCl3, which was consistent with the change in the number of CD68-positive cells. They increased with increasing dose of zymosan, which was also consistent with the change in the number of CD68-positive cells. These results suggest that AUC, ΔR2(max), and ΔR2(last) reflect the number of Kupffer cells. In conclusion, we presented a method for analyzing the kinetic behavior of SPIONs in the liver using DSC-MRI and EMM, and investigated the influence of body temperature, GdCl3, and zymosan using body-temperature-controlled mice. The present study suggests that control of body temperature is essential for investigating the kinetic behavior of SPIONs in the liver and that our method will be applicable and useful for quantifying the responses of Kupffer cells to various drugs under control of body temperature.

Involvement of a spinal brain-derived neurotrophic factor/full-length TrkB pathway in the development of nerve injury-induced thermal hyperalgesia in mice.

Partial sciatic nerve ligation in mice caused a marked and persistent decrease in the latency of paw withdrawal from a thermal stimulus only on the ipsilateral side. This thermal hyperalgesia was abolished by repeated intrathecal pretreatment with a specific antibody to brain-derived neurotrophic factor (BDNF), but not neurotrophin-4, just before and after the nerve ligation. These results provide direct evidence that BDNF within the spinal cord may contribute to the development of thermal hyperalgesia caused by nerve injury in mice. We previously reported that protein level of full-length TrkB, which contains the cytoplasmic protein tyrosine kinase domain, were clearly increased on the ipsilateral side of spinal cord membranes obtained from sciatic nerve-ligated mice. In the present study, we further demonstrated that the increased in the protein level of full-length TrkB is completely reversed by concomitant intrathecal injection of BDNF antibody. Furthermore, thermal hyperalgesia induced by nerve ligation was completely suppressed by repeated intrathecal injection of a specific antibody to full-length TrkB and an inhibitor of the protein tyrosine kinase activity for the neurotrophin receptor, K-252a. However, repeated intrathecal injection of a specific antibody to truncated TrkB, which lacks the cytoplasmic protein tyrosine kinase domain, failed to reverse thermal hyperalgesia observed in nerve-ligated mice. These findings suggest the possibility that the binding of BDNF to full-length TrkB and subsequent its activation may play a critical role in the development of neuropathic pain-like thermal hyperalgesia induced by nerve injury in mice.

Investigation of chromatographic performances and binding characteristics of BSA-encapsulated capillary column prepared by the sol-gel method.

We have developed a novel protein-encapsulation technique using the sol-gel method for the preparation of monolithic capillary columns for capillary electrochromatography. Bovine serum albumin (BSA) was encapsulated in tetramethoxysilane-based hydrogel and the enantioselectivity was evaluated. The present work examined the effect of various factors such as running buffer pH and concentration or organic modifier on the chromatographic performances as well as binding characteristics of BSA for D,L-tryptophan (Trp). The retention and enantioseparation dramatically changed depending on the buffer pH and concentrations. These factors influenced EOF and especially binding characteristics of BSA for L-Trp. Under the optimized conditions, the BSA-encapsulated columns revealed the adequate repeatability concerning column-to-column and run-to-run.

Role of methotrexate polyglutamation and reduced folate carrier 1 (RFC1) gene polymorphisms in clinical assessment indexes.

The aims of the present study were to define inter-individual differences in response to methotrexate (MTX) through MTX polyglutamate (MTX-PG) levels in red blood cells (RBC) and MTX-related gene polymorphisms. A total of 145 rheumatoid arthritis patients were recruited. MTX-PG1-5 concentrations in RBC were measured, and 11 single nucleotide polymorphisms, all in MTX-related genes involved in the folate pathway, were analyzed. Disease activity was also assessed. There was no direct relationship between any MTX-PG concentration and the patient's disease condition, but detectability of MTX-PG5 was extracted as a candidate marker for response to MTX. When disease activity was compared between patients in which MTX-PG5 was detectable and undetectable, all indexes except the visual analog scale (VAS) and C-reactive protein (CRP) were found to be significantly lower in the former patients. Reduced folate carrier 1 (RFC1) 80G>A was significantly associated with the detectability of MTX-PG5; detectability of MTX-PG5 was lower in patients with the A mutant allele. The present study suggests that detectability of MTX-PG5 in RBC is a possible biomarker for response to MTX, and the RFC1 80G>A mutation is associated with low detectability of MTX-PG5. Prospective studies with a sufficient number of patients are needed to confirm the present findings.

A protein-encapsulation technique by the sol-gel method for the preparation of monolithic columns for capillary electrochromatography.

A novel protein-encapsulation technique using sol-gels was developed for the preparation of monolithic capillary columns for capillary electrochromatography. Two chiral compounds, bovine serum albumin (BSA) and ovomucoid (OVM) from chicken egg white, were encapsulated in tetramethoxysilane-based hydrogel and their chiral selectivity was evaluated for the separation of some selected enantiomers (tryptophan, benzoin, eperisone, chlorpheniramine). The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. Two different proteins, BSA and OVM, were successfully encapsulated into the gel matrixes by changing the alkoxysilane compositions of the gel. Run-to-run repeatability was quite satisfactory. The consecutive analysis of the neutral compound, benzoin, by the OVM-encapsulated column showed good repeatability in the retention time (RSD = 1.23% for the first peak, N = 10). Under optimized conditions, the theoretical plate number for the first peak of benzoin reached 72,000 plates/m.