Changes in the excitability of anterior horn cells in a mental rotation task of body parts.

INTRODUCTION/AIMS: Mental rotation (MR), a tool of implicit motor imagery, is the ability to rotate mental representations of two- or three-dimensional objects. Although many reports have described changes in brain activity during MR tasks, it is not clear whether the excitability of anterior horn cells in the spinal cord can be changed. In this study, we examined whether MR tasks of hand images affect the excitability of anterior horn cells using F-wave analysis. METHODS: Right-handed, healthy participants were recruited for this study. F-waves of the right abductor pollicis brevis were recorded after stimulation of the right median nerve at rest, during a non-MR task, and during an MR task. The F-wave persistence and the F/M amplitude ratio were calculated and analyzed. RESULTS: Twenty participants (11 men and 9 women; mean age, 29.2 ± 4.4 years) were initially recruited, and data from the 18 that met the inclusion criteria were analyzed. The F-wave persistence was significantly higher in the MR task than in the resting condition (p = .001) or the non-MR task (p = .012). The F/M amplitude ratio was significantly higher in the MR task than in the resting condition (p = .019). DISCUSSION: The MR task increases the excitability of anterior horn cells corresponding to the same body part. MR tasks may have the potential for improving motor function in patients with reduced excitability of the anterior horn cells, although this methodology must be further verified in a clinical setting.

Immediate Reduction in Spasticity of Ankle Plantar Flexors in a Stroke Patient after Treatment with a Spinning Permanent Magnet Device.

Background: Magnetic stimulation devices can be large because of the need for cooling systems. We developed a compact and lightweight Spinning Permanent Magnet (SPM) device that generates magnetic fields with intensities below the motor threshold. In this report, we present the case of a post-stroke patient in which an immediate reduction in spasticity of the ankle plantar flexors was achieved after SPM treatment. Case: A 37-year-old man was admitted to our hospital with a right putamen hemorrhage. The patient underwent conservative therapy and exhibited residual left hemiplegia and spasticity. Three months after stroke onset, he was able to walk with supervision while using a left ankle-foot orthosis and a T-cane. The Modified Ashworth Scale (MAS) score of the left ankle plantar flexors was 1+. The plantar flexors were stimulated by SPM treatment. The outcomes were the Hmax/Mmax of the tibial nerve (soleus muscle) and the MAS score. On the first day, SPM stimulation was applied for 30 min. On the second day, a sham stimulation of the same duration was performed. On the third day, the SPM stimulation was repeated. Hmax/Mmax decreased from 41.5% to 37.7% on the first day, and from 46.9% to 31.6% on the third day after SPM stimulation. The MAS score decreased from 1+ to 1 on both days. In contrast, after sham stimulation, Hmax/Mmax increased from 39.2% to 44.2%, whereas the MAS score remained unchanged at 1+. Discussion: Stimulation below the motor threshold using SPM treatment can effectively reduce spasticity.

NMR determination of the 2:1 binding complex of naphthyridine carbamate dimer (NCD) and CGG/CGG triad in double-stranded DNA.

Trinucleotide repeat (TNR) diseases are caused by the aberrant expansion of CXG (X = C, A, G and T) sequences in genomes. We have reported two small molecules binding to TNR, NCD, and NA, which strongly bind to CGG repeat (responsible sequence of fragile X syndrome) and CAG repeat (Huntington's disease). The NMR structure of NA binding to the CAG/CAG triad has been clarified, but the structure of NCD bound to the CGG/CGG triad remained to be addressed. We here report the structural determination of the NCD-CGG/CGG complex by NMR spectroscopy and the comparison with the NA-CAG/CAG complex. While the NCD-CGG/CGG structure shares the binding characteristics with that of the NA-CAG/CAG complex, a significant difference was found in the overall structure caused by the structural fluctuation at the ligand-bound site. The NCD-CGG/CGG complex was suggested in the equilibrium between stacked and kinked structures, although NA-CAG/CAG complex has only the stacked structures. The dynamic fluctuation of the NCD-CGG/CGG structure at the NCD-binding site suggested room for optimization in the linker structure of NCD to gain improved affinity to the CGG/CGG triad.

The safety and effectiveness profile of eldecalcitol in a prospective, post-marketing observational study in Japanese male patients with osteoporosis.

We conducted a post-marketing observational study to investigate the safety and effectiveness of eldecalcitol for the treatment of osteoporosis in a Japanese clinical setting. The observation period was 12 months for women and 36 months for men. The final results for the female patients have already been published. In this article, the final results for the male patients are reported. A total of 470 male osteoporosis patients were enrolled. The safety analysis set included 431 patients (mean age, 76.8 years; mean ± SD follow-up period, 631.0 ± 450.3 days), and 175 patients continued treatment throughout the 3-year observational period. Adverse drug reactions (ADRs) were reported in 28 patients (6.49%); the most common ADRs were hypercalcemia (1.16%) and renal impairment (1.16%). Serious ADRs were reported in 5 patients (1.16%). Mean serum calcium was within the normal range throughout the observation period. The cumulative incidence of new vertebral and nonvertebral fractures at 36 months, estimated by Kaplan-Meier analysis, was 10.23 and 4.06%, respectively. At the last observation, mean lumbar spine bone mineral density was 3.49% higher (P < 0.0001) than at baseline, and levels of the bone turnover markers BAP and TRACP-5b were reduced (-14.64%; P = 0.0009, and - 29.51%; P < 0.0001, respectively). In conclusion, the safety and effectiveness of eldecalcitol for the treatment of Japanese male osteoporosis patients was confirmed in clinical practice. Careful monitoring of serum calcium and estimated glomerular filtration rate, both before and during treatment, is necessary to minimize the risk of hypercalcemia and renal impairment while maximizing the effectiveness of eldecalcitol.

Lower-limb muscle strength according to bodyweight and muscle mass among middle age patients with type 2 diabetes without diabetic neuropathy.

[Purpose] This study assessed the effects of type 2 diabetes without diabetic polyneuropathy on muscle strength according to body composition in middle age patients. [Subjects and Methods] This study included 45 non-diabetic individuals (control group) and 50 patients with type 2 diabetes (DM group), 40 to 64 years of age. The body composition was examined, including the leg muscle volume (LMV), which was the sum of the lower-limb muscle mass. The muscle strength was also examined, and the knee extension force (KEF), ankle dorsiflexion force (ADF). The KEF and ADF were normalized to the bodyweight, and the total leg muscle force (TLMF) were calculated by combining the KEF and ADF. The leg muscle quality (LMQ) was calculated as the TLMF normalized with the LMV. [Results] While no significant differences were found in the LMV between groups, the body mass index were higher in the DM group than in the control group. Significant differences were observed in %KEF, %ADF, and the LMQ in the DM group, with 15.8%, 18.7%, and 11.5% lower values than those in the control group, respectively. [Conclusion] The results of this study may demonstrate that muscle weakness occurs before diabetes progresses to a severe condition.

The safety and effectiveness profile of eldecalcitol in a prospective, post-marketing observational study in Japanese patients with osteoporosis: interim report.

This large-scale post-marketing surveillance study was conducted to assess the safety and effectiveness of eldecalcitol treatment in patients with osteoporosis in a Japanese clinical setting. A total of 3567 patients with osteoporosis were enrolled and received eldecalcitol 0.75 µg/day for 12 months. For this interim report, 3285 patients were eligible for analysis. Mean age was 74.9 ± 8.7 years; 86.8 % (2854/3285) were women. There were 142 reported adverse drug reactions (ADRs) in 129 patients (3.92 % of the total 3285 patients): the most common were hypercalcemia and increased blood calcium (0.88 %), renal impairment (0.27 %), abdominal discomfort (0.24 %), constipation (0.24 %), and pruritus (0.24 %). The incidence of ADRs was 5.10 % in men and 3.74 % in women. Although 10 serious ADRs were reported in 9 patients (0.27 %), no clinically significant safety issues were identified. Incidence of hypercalcemia or increased blood calcium was 8.47 % in patients with renal impairment and only 0.74 % in patients without renal impairment. At last observation, the incidence of new vertebral and nonvertebral fractures was 2.44 % and 1.70 %, respectively. There was a significant increase in bone mineral density at the lumbar spine and distal radius. The bone turnover markers BAP, serum NTX, urinary NTX, and TRACP-5b were suppressed by eldecalcitol treatment in both sexes. In conclusion, consistent with the findings of the phase III pivotal clinical trial, eldecalcitol was shown to have a favorable safety profile and effectiveness in Japanese patients with osteoporosis. However, periodic measurements of serum calcium were required to prevent occurrence of hypercalcemia during eldecalcitol treatment, especially in patients with renal impairment.

1,2,3-Triazole-containing uracil derivatives with excellent pharmacokinetics as a novel class of potent human deoxyuridine triphosphatase inhibitors.

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing uracil derivatives. Compound 45a, which possesses 1,5-disubstituted 1,2,3-triazole moiety that mimics the amide bond of tert-amide-containing inhibitor 6b locked in a cis conformation showed potent inhibitory activity, and its structure-activity relationship studies led us to the discovery of highly potent inhibitors 48c and 50c (IC(50) = ~0.029 µM). These derivatives dramatically enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = ~0.05 µM). In addition, compound 50c exhibited a markedly improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that 50c is a promising candidate for combination cancer chemotherapies with TS inhibitors.

Discovery of highly potent human deoxyuridine triphosphatase inhibitors based on the conformation restriction strategy.

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound 7, we designed and synthesized two conformation restricted analogues, i.e., compounds 8 and 9. These compounds exhibited increased in vitro potency compared with the parent compound 7. Further structure-activity relationship (SAR) studies identified a compound 43 with the highest in vitro potency (IC(50) = 39 nM, EC(50) = 66 nM). Furthermore, compound 43 had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.

Synthesis and discovery of N-carbonylpyrrolidine- or N-sulfonylpyrrolidine-containing uracil derivatives as potent human deoxyuridine triphosphatase inhibitors.

Recently, deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure-activity relationship (SAR) studies of uracil derivatives. N-Carbonylpyrrolidine- and N-sulfonylpyrrolidine-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (12k) having excellent potencies (IC(50) = 0.15 µM). The X-ray structure of a complex of 16a and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = 0.27-0.30 µM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors.

Discovery of a novel class of potent human deoxyuridine triphosphatase inhibitors remarkably enhancing the antitumor activity of thymidylate synthase inhibitors.

Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure-activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor 7 (IC(50) = 100 µM), we developed compound 26, which is the most potent human dUTPase inhibitor (IC(50) = 0.021 µM) reported to date. Not only does compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2'-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC(50) = 0.075 µM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising candidate for clinical development.

Familial amyotrophic lateral sclerosis with Cys111Tyr mutation in Cu/Zn superoxide dismutase showing widespread Lewy body-like hyaline inclusions.

We described a 43-year-old Japanese man with familial amyotrophic lateral sclerosis (FALS) in whom we identified a missense mutation (Cys111→Tyr) in exon 4 of the Cu/Zn superoxidase dismutase-1 (SOD1) gene in which no pathological data have been reported. The disease duration was 5 years, and he died of respiratory failure. The initial sign was weakness of the right leg. He had no clear upper motor involvement. Neuropathological examinations showed neuronal intracytoplasmic Lewy body-like hyaline inclusions (LBHIs) not only in the anterior horn cells of the spinal cord, but also in many other affected neurons. LBHIs were seen in the anterior horn cells, Onufrowicz nucleus, Clarke's nucleus, intermediolateral nucleus, and posterior gray horn of the spinal cord. In addition, LBHIs were observed in the periaqueductal gray matter, nucleus raphe dorsalis, locus ceruleus, trigeminal motor nucleus, vestibular nucleus, dorsal vagal nucleus, hypoglossal nucleus, and reticular formation of the brain stem. These are very specific findings that neuronal LBHIs in our case are for more widespread reported cases, and similar cases to ours have never reported in FALS.

Adult dermatomyositis with severe polyneuropathy: does neuromyositis exist?

Peripheral nerve involvement in dermatomyositis (DM) has been known as neuromyositis. However, the pathogenic mechanism is not clear, and the association between DM and peripheral neuropathy is still controversial. Our patient exhibited symptomatic polyneuropathy that was documented electrophysiologically in addition to typical features of DM. The sural nerve biopsy showed evidence of a continuing neuropathic process of axonal type. There was no finding of inflammatory cells infiltrating the vessels. Neither methylprednisolone nor intravenous immunoglobulin (IVIg) improved neurological symptoms including muscle weakness and sensory disturbance. Clinical, electrophysiological, and neuropathological features in our case demonstrate the association of DM and polyneuropathy. The possibility that the same pathological process affecting skin and skeletal muscles also affected peripheral nerves in our patient should be considered.

Cerebral microvascular disease predicts renal failure in type 2 diabetes.

Abnormalities in small renal vessels may increase the risk of developing impaired renal function, but methods to assess these vessels are extremely limited. We hypothesized that the presence of small vessel disease in the brain, which manifests as silent cerebral infarction (SCI), may predict the progression of kidney disease in patients with type 2 diabetes. We recruited 608 patients with type 2 diabetes without apparent cerebrovascular or cardiovascular disease or overt nephropathy and followed them for a mean of 7.5 years. At baseline, 177 of 608 patients had SCI, diagnosed by cerebral magnetic resonance imaging. The risk for the primary outcome of ESRD or death was significantly higher for patients with SCI than for patients without SCI [hazard ratio, 2.44; 95% confidence interval (CI) 1.36 to 4.38]. The risk for the secondary renal end point of any dialysis or doubling of the serum creatinine concentration was also significantly higher for patients with SCI (hazard ratio, 4.79; 95% CI 2.72 to 8.46). The estimated GFR declined more in patients with SCI than in those without SCI; however, the presence of SCI did not increase the risk for progression of albuminuria. In conclusion, independent of microalbuminuria, cerebral microvascular disease predicted renal morbidity among patients with type 2 diabetes.

Immunohistochemical studies of vascular endothelial growth factor in skin of patients with amyotrophic lateral sclerosis.

Vascular endothelial growth factor (VEGF) is a disulfide-linked dimeric glycoprotein that enhances vascular permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes growth of vascular endothelial cells. Furthermore, VEGF is a multifunctional cytokine, which influences neural cells directly, enhancing neuronal survival, axonal outgrowth, and Schwann cell proliferation. So far studies of the skin of amyotrophic lateral sclerosis (ALS) have shown unique pathological and biochemical abnormalities in collagen, elastic fibers, and the ground substance. However, the expression of VEGF in ALS skin has not previously been studied. We made a quantitative immunohistochemical study of the expression of VEGF in the skin from 15 patients with ALS and 15 control subjects. VEGF immunoreactivity was markedly positive in the epidermis and moderately positive in some dermal blood vessels and glands in ALS patients. These findings became more conspicuous as ALS progressed. The optical densities for VEGF immunoreactivity of the epidermis in ALS patients were significantly higher (p<0.001) than in control subjects. In addition, there was an appreciable positive correlation (r=0.85, p<0.001) in ALS patients between the densities for VEGF immunoreactivity and duration of illness, but there was no such correlation in control subjects. These data suggest that changes of VEGF in ALS skin are likely to be related to the disease process and that metabolic alterations of VEGF may take place in the skin of patients with ALS.

Development of an antitumor adenosine analog, 3''-ethynyladenosine.

3'-ethynyladenosine (EAdo) was an adenosine analog with potent antitumor activity against various human tumor cells in vitro. However, EAdo was enzymatically inactivated by adenosine deaminase (ADA) in vitro and in vivo. Therefore, we synthesized two ADA-resistant EAdo derivatives (2-F-EAdo and EAdo-5'-monophosphate, EAMP) and examined their antitumor activities.

Polymorphisms in the 3' UTR in the neurocalcin delta gene affect mRNA stability, and confer susceptibility to diabetic nephropathy.

Using a large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese type 2 diabetic patients, we have identified a gene encoding neurocalcin delta (NCALD) as a candidate for a susceptibility gene to diabetic nephropathy; the landmark SNP was found in the 3' UTR of NCALD (rs1131863: exon 4 +1340 A vs. G, P = 0.00004, odds ratio = 1.59, 95% CI 1.27-1.98). We also discovered two other SNPs in exon 4 of this gene (+999 T/A, +1307 A/G) that showed absolute linkage disequilibrium to the landmark SNP. Subsequent in vitro functional analysis revealed that synthetic mRNA corresponding to the disease susceptible haplotype (exon 4 +1340 G, +1307 G, +999 A) was degraded faster than mRNA corresponding to the major haplotype (exon 4 +1340 A, +1307 A, +999 T), and allelic mRNA expression of the disease susceptibility allele was significantly lower than that of the major allele in normal kidney tissues. In an experiment using a short interfering RNA targeting NCALD, we found that silencing of the NCALD led to a considerable enhancement of cell migration, accompanied by a significant reduction in E-cadherin expression, and by an elevation of alpha smooth muscle actin expression in cultured renal proximal tubular epithelial cells. We also identified the association of the landmark SNP with the progression of diabetic nephropathy in a 8-year prospective study (A vs. G, P = 0.03, odds ratio = 1.91, 95% CI 1.07-3.42). These results suggest that the NCALD gene is a likely candidate for conferring susceptibility to diabetic nephropathy.

Effects of three Chinese herbal medicines on plasma and liver lipids in mice fed a high-fat diet.

Chinese herbal medicines, Inchinko-to, Bofu-tsusho-san and Dai-saiko-to, containing 3, 18 and 8 components, respectively, have since long been used as an anti-inflammatory, antipyretic, choleretic and diuretic agent for liver disorders and jaundice, as an anti-obesity agent, a hypocholesterolemic agent for liver disorders and a therapeutic and/or preventive agent for cholesterol gallstone disease with hypertriglycerid-emia in China and Japan, respectively. In the present study, we investigated the effects of these three herbal medicines in young male mice fed a high-fat diet. Plasma levels of lipids and the numbers of the fatty droplets in the liver cytoplasm were markedly lowered by the diets supplemented with three herbal medicines. The liver weights and the body growth were reduced by the diet supplemented with Dai-saiko-to, which slightly affected the concentrations of total protein, albumin, creatinine or calcium, and the activity of lactate dehydrogenase. Thus, Dai-saiko-to, besides Bofu-tsusho-san, seems effective in the activities of anti-obesity, anti-hyperlipidemia and anti-hyperlipids in liver cytoplasm, when used carefully.

Studies of DNA recognition mechanism of transcription factor IRF-4.

Transcription factor IFN regulatory factor-4 (IRF-4) prefers a DNA sequence including CCGAAA, though the consensus DNA-binding sequence of the IRF family proteins is NNGAAA, and the crystal structure of PU.1/IRF-4/DNA (GTGAAA) ternary complex indicates the NN region of DNA does not interact with IRF-4 directly. This suggests that there is an indirect DNA recognition mechanism in IRF-4. In order to account for the sequence preference of IRF-4, we focused on structural properties of DNA duplexes recognized by IRF-4. Here, we performed solution NMR studies on DNA duplexes containing GGGAAA and CCGAAA sequences, and assigned most of proton resonances of DNA 17 mer with GGGAAA. (1)H-(1)H NOESY spectra indicated B-form like structure for GGGAAA. We also assigned imino proton resonances of DNA 17 mer with CCGAAA. For the imino proton region, the (1)H-(1)H NOESY spectra of these two DNA duplexes were similar.

NMR spectroscopic study of a DNA duplex with mercury-mediated T-T base pairs.

Recently, we reported that T-T mismatches can specifically recognize Hg(II) (T-Hg(II)-T pair formation). In order to understand the properties of the T-Hg(II)-T pair, we recorded NMR spectra for a DNA duplex, d(CGCGTTGTCC).d(GGACTTCGCG), with two successive T-T mismatches (Hg (II)-binding sites). We assigned 1H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg(II) by using 1D 1H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.