1α, 25(OH)2D3 regulates agrin-induced acetylcholine receptor clustering through upregulation of rapsyn expression in C2C12 myotubes.

The active form of vitamin D (1α, 25-dihydroxyvitamin D3 [1α, 25(OH)2D3], referred to as 1,25D) has been suggested to play a pivotal role in skeletal muscle function and metabolism. However, the mechanisms through which 1,25D functions in this tissue remain to be elucidated. Recent studies have shown that vitamin D signaling regulates neuromuscular maintenance and improves locomotion in mice. In the present study, we examined the effects of 1,25D on neuromuscular synaptogenesis by measuring clustering of acetylcholine receptors (AChRs) in C2C12 myotubes. 1,25D treatment enhanced the agrin-induced AChR clustering in myotubes compared to treatment with agrin alone. Furthermore, siRNA-mediated knockdown of the vitamin D receptor (VDR) decreased the agrin-induced AChR clustering. 1,25D increased the expression of rapsyn, which is necessary for AChR clustering, while demonstrating no effect on other neuromuscular junction-related genes. In addition, rapsyn expression was dependent on 1,25D-VDR signaling. These results suggest that 1,25D-VDR signaling may regulate rapsin expression, resulting in the up-regulation of agrin-induced AChR clustering.

Cobalt chloride, a chemical hypoxia-mimicking agent, suppresses myoblast differentiation by downregulating myogenin expression.

Cobalt chloride can create hypoxia-like state in vitro (referred to as chemical hypoxia). Several studies have suggested that chemical hypoxia may cause deleterious effects on myogenesis. The intrinsic underlying mechanisms of myoblast differentiation, however, are not fully understood. Here, we show that cobalt chloride strongly suppresses myoblast differentiation in a dose-dependent manner. The impaired myoblast differentiation is accompanied by downregulation of myogenic regulatory factor myogenin. Under chemical hypoxia, myogenin stability is decreased at mRNA and protein levels. A muscle-specific E3 ubiquitin ligase MAFbx, which can target myogenin protein for proteasomal degradation, is upregulated along with changes in Akt/Foxo and AMPK/Foxo signaling pathways. A proteasome inhibitor completely prevents cobalt chloride-mediated decrease in myogenin protein. These results suggest that cobalt chloride might modulate myogenin expression at post-transcriptional and post-translational levels, resulting in the failure of the myoblasts to differentiate into myotubes.

A novel evaluation method of survival motor neuron protein as a biomarker of spinal muscular atrophy by imaging flow cytometry.

Spinal muscular atrophy (SMA) is caused by mutations within the survival motor neuron 1 (SMN1) gene. These mutations result in the reduction of survival motor neuron (SMN) protein expression and SMN complex in spinal motor neurons and other tissues. SMN protein has been used as a therapeutic biomarker in recent SMA clinical studies using enzyme-linked immunosorbent assay (ELISA). Here, we investigated whether imaging flow cytometry can be a viable source of quantitative information on the SMN protein. Using a FlowSight imaging flow cytometer (Merck-Millipore, Germany), we demonstrated that imaging flow cytometry could successfully identify different expression patterns and subcellular localization of SMN protein in healthy human fibroblasts and SMA patient-derived fibroblasts. In addition, we could also evaluate the therapeutic effects of SMN protein expression by valproic acid treatment of SMA patient-derived cells in vitro. Therefore, we suggest that imaging flow cytometry technology has the potential for identifying SMN protein expression level and pattern as an evaluation tool of clinical studies.

Human first-trimester chorionic villi have a myogenic potential.

First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

Amycolamicin: a novel broad-spectrum antibiotic inhibiting bacterial topoisomerase.

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury.

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.

IRS-2 deficiency in macrophages promotes their accumulation in the vascular wall.

The aim of this study was to investigate the role of insulin receptor substrate-2 (IRS-2) mediated signal in macrophages on the accumulation of macrophages in the vascular wall. Mice transplanted with IRS-2(-/-) bone marrow, a model of myeloid cell restricted defect of IRS-2, showed accumulation of monocyte chemoattractant protein-1-expressing macrophages in the vascular wall. Experiments using cultured peritoneal macrophages showed that IRS-2-mediated signal pathway stimulated by physiological concentrations of insulin, not by IL-4, contributed to the suppression of monocyte chemoattractant protein-1 expression induced by lipopolysaccharide. Our data indicated that IRS-2 deficiency in macrophages enhanced their accumulation in the vascular wall accompanied by increased expression of proinflammatory mediators in macrophages. These results suggest a role for insulin resistance in macrophages in early atherosclerogenesis.

In vivo evaluation method of the effect of nattokinase on carrageenan-induced tail thrombosis in a rat model.

Thrombosis is characterized by congenital and acquired procatarxis. Nattokinase inhibits thrombus formation in vitro. However, in vivo evaluation of the therapeutic efficacy of nattokinase against thrombosis remains to be conducted. Subcutaneous nattokinase injections of 1 or 2 mg/ml were administered to the tails of rats. Subsequently, κ-carrageenan was intravenously administered to the tails at 12 h after nattokinase injections. The mean length of the infarcted regions in the tails of rats was significantly shorter in rats administered 2 mg/ml of nattokinase than those in control rats. Nattokinase exhibited significant prophylactic antithrombotic effects. Previously, the in vitro efficacy of nattokinase against thrombosis had been reported; now our study has revealed the in vivo efficacy of nattokinase against thrombosis.

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents.

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

Improving frequency of thrombosis by altering blood flow in the carrageenan-induced rat tail thrombosis model.

The carrageenan-induced tail thrombosis model in rats and mouse is useful for evaluating compounds having antithrombotic effects in drug discovery projects as an in vivo model. However, the frequency of thrombosis is low, and the signs of thrombosis are not constant under the standard conditions. In this study, we modified this model on the basis of hypercoagulability, endothelial injury, and alteration in the normal blood flow. The simple ligation to alter blood flow in rats significantly improved the frequency and signs of thrombosis after intravenous kappa-carrageenan injection. In particular, 1mg/kg kappa-carrageenan injection in combination with 10 min ligation at the tail increased the frequency of thrombosis to almost 100% in rats. These results suggest that this modified model has great advantages over the previously used conditions for evaluating the effects of antithrombotic and thrombolytic agents.

The relationship between the efficacy of epidural anesthesia and the concentration of lidocaine in cerebrospinal fluid.

BACKGROUND: Addition of bicarbonate to local anesthetics improves the efficacy of epidural anesthesia. We evaluated whether the addition of bicarbonate to lidocaine enhanced pain threshold in cesarean section. We speculated that bicarbonate would increase the concentration of lidocaine in cerebrospinal fluid (CSF). To examine this possibility, we evaluated the relationship between the lidocaine concentration in the CSF and the pain threshold. MATERIAL/METHODS: Twenty eight full-term parturients undergoing cesarean section under epidural anesthesia at L2-3 were divided into two groups: the first group (lidocaine group) received 17 mL of 2% lidocaine-epinephrine (1:200,000) and the second group (lidocaine-bicarbonate group) received the same concentration of lidocaine-epinephrine supplemented bicarbonate. Twenty min after administration of local anesthetics, we collected 1 mL samples of the CSF. The pain threshold after the repeated electrical stimulation was used to assess sensory blockade at the L2, S1, and S3 dermatomes. RESULTS: Demographic data were comparable between the groups. There were no differences in the pain threshold at all dermatomes and the lidocaine concentration between the groups. There was a significant correlation between the pain threshold and the lidocaine concentration at the combined S1 and S3 dermatomes in the lidocaine-bicarbonate group. CONCLUSIONS: We find neither bicarbonate caused a significant difference in the efficacy of epidural anesthesia nor it caused an increase of lidocaine concentration in the CSF. The result that we found a significant correlation between the lidocaine concentration in the CSF and the pain threshold at the sacral region in the lidocaine bicarbonate group suggests that, bicarbonate enhances the efficacy of anesthesia at outside of spinal canal.

Preparation of griseofulvin nanoparticle suspension by high-pressure homogenization and preservation of the suspension with saccharides and sugar alcohols.

AIM: We have attempted to micronize drug particles with a particle size of less than 100 nm and maintain the particle size of their suspension to improve the solubility and bioavailability of poorly water-soluble drugs. Furthermore, the method of freeze-drying nanoparticles was applied to maintain particulate nature of nanoparticles containing various saccharides and sugar alcohols for a long time. METHOD: Griseofulvin (GF)-lipid nanoparticle suspension is prepared using GF and a lipid by high-pressure homogenization. The particle size of the obtained GF-lipid nanoparticle suspension is maintained constant by freeze-drying. RESULT: The mean particle size of GF-lipid nanoparticles prepared by high-pressure homogenization is approximately 60 nm. The mean particle size remains less than 100 nm for 1 month. The GF-lipid nanoparticle suspension containing xylitol, trehalose, or sucrose is freeze-dried to maintain the particulate nature. The mean particle size of the rehydrated suspension is lower than that of the rehydrated suspension containing erythritol or lactose. In particular, it is new knowledge to have found that an aggregation is minimized by adding xylitol which is sugar alcohol. The minimum concentration of xylitol, trehalose, and sucrose required to maintain a constant particle size by rehydration is 3%, 3%, and 5% (w/v), respectively.

Transforming growth factor-beta1 modulates tumor-stromal cell interactions of prostate cancer through insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) secreted from the prostate stroma mediates tumor-stromal cell interactions in prostate cancer development. We have recently reported that human prostate stromal cells (PrSC) stimulate human prostate cancer DU-145 cell growth via IGF-I. Transforming growth factor-beta1 (TGF-beta1) also plays a critical role in tumor-stromal cell interactions, but TGF-beta1 has pleiotropic effects as it can modulate growth of prostate cancer either positively or negatively. To elucidate the complex behavior of TGF-beta1, the effect of TGF-beta1 on the IGF axis in PrSC was studied. TGF-beta1 increased the expression of IGF-I and IGF binding protein-3 (IGFBP-3). RT-PCR experiments revealed that TGF-beta1 upregulated the mRNA expression of IGF-I and IGFBP-3. However, while TGF-beta1 significantly increased IGFBP-3 secretion by PrSC, it conversely reduced the amounts of biologically active IGF-I unbound to IGFBP-3. Immunohistochemical analyses of 49 human prostate cancer tissues showed that IGF-I expression in the stroma correlated positively with TGF-beta1 expression in the stroma (r = 0.551, p = 0.0001). Furthermore, TGF-beta1 actually suppressed the growth of DU-145 cells in coculture with PrSC. But, IGFBP-3 proteinases, such as prostate specific antigen (PSA) and matrix metalloproteinase-7 (MMP-7), restored the DU-145 cell growth, suggesting that degradation of IGFBP-3 potentiates cancer growth. Taken together, these results indicated that TGF-beta1 modulates the growth of prostate cancer, either positively or negatively, through the balance between the amounts of IGF-I and IGFBP-3. This complex behavior of TGF-beta1 on the IGF axis is one explanation for the pleiotropic activities of TGF-beta1 on the growth of prostate cancer.

Preparation and stabilization of nifedipine lipid nanoparticles.

Design methods of nanoparticle formulations are divided into a break-down method and a build-up method. Furthermore, the former is further divided into dry and wet processes. For drug nanoparticle preparations, the wet process is generally employed, and organic solvents are used in most formulations. In this study, we investigated the preparation of nifedipine (NI) nanoparticles without using any organic solvent. NI nanoparticles with a mean particle size of approximately 50 nm could be prepared without organic solvent by a combination of roll mixing and high-pressure homogenization. The X-ray diffraction peak of the sample prepared by roll mixing was present at an identical position (2theta) to that of NI crystals, showing that no peak shift was induced by interaction with lipid. These findings clarified that most NI remained as crystals in lipid. To maintain the particle size of the nanoparticles in suspension for a long time, a method of adding gelatin powder to the NI-lipid nanoparticle suspension, dissolving the mixture by heating, and then solidifying by cooling was investigated. The mean particle size of the sample was about 55 nm, and that after heat-liquefaction of the NI-lipid nanoparticle suspension gelated at 5 degrees C for 24h was also about 55 nm, showing that the nanoparticle condition was retained.

A new terrein glucoside, a novel inhibitor of angiogenin secretion in tumor angiogenesis.

Angiogenesis is a critical step for the tumor therapy. Many angiogenic factors are involved in the tumor angiogenesis. In the course of our screening for inhibitors of angiogenin secretion, one of angiogenic factors, we have isolated a new terrein glucoside (1) and terrein (2) from the fermentation broth of fungal strain Aspergillus sp. PF1381. The structure and absolute stereochemistry of 1 was determined to be (4S,5R)-5-[(alpha-D-glucopyranosyl)oxy]-4-hydroxy-3-(E-1-propenyl)-2-cyclopenten-1-one on the basis of spectral and enzymatic analyses. Compounds 1 and 2 equally inhibited angiogenin secretion from androgen-dependent prostate cancer cells, LNCaP-CR, with IC50 values of 13 microM. However, both compounds did not affect VEGF secretion, another angiogenic factor. Furthermore, both compounds inhibited tube formation of human umbilical vein endothelial cells (HUVEC). These results suggested that 1 and 2 act as angiogenesis inhibitors through the inhibition of angiogenin secretion.

A new biomarker candidate for spinal muscular atrophy: Identification of a peripheral blood cell population capable of monitoring the level of survival motor neuron protein.

Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder caused by insufficiency of functional survival motor neuron (SMN) protein. Several clinical trials have been conducted with the aim of upregulating the expression of the SMN protein in SMA patients. In order to evaluate the efficiency of these SMN-targeted approaches, it has become necessary to verify SMN protein levels in the cells of SMA patients. Accordingly, we have developed a method allowing the evaluation of the functional SMN protein with < 1.5 mL of peripheral blood using imaging flow cytometry. The expression of SMN protein in CD3+, CD19+, and CD33++ cells obtained from SMA patients, was significantly reduced compared with that in cells from control subjects. In spot analysis of CD33++ cells, the intensities of SMN spots were significantly reduced in SMA subjects, when compared with that in controls. Therefore, SMN spots implied the presence of functional SMN protein in the cell nucleus. To our knowledge, our results are the first to demonstrate the presence of functional SMN protein in freshly isolated peripheral blood cells. We anticipate that SMN spot analysis will become the primary endpoint assay for the evaluation and monitoring of therapeutic intervention, with SMN serving as a reliable biomarker of therapeutic efficacy in SMA patients.

Transduction of anti-cell death protein FNK protects isolated rat hearts from myocardial infarction induced by ischemia/reperfusion.

Artificial anti-cell death protein FNK, a Bcl-x(L) derivative with three amino acid-substitutions (Y22F, Q26N, and R165K) has enhanced anti-apoptotic and anti-necrotic activity and facilitates cell survival in many species and cell types. The objectives of this study were (i) to investigate whether the protein conjugated with a protein transduction domain (PTD-FNK) reduces myocardial infarct size and improves post-ischemic cardiac function in ischemic/reperfused rat hearts, and (ii) to understand the mechanism(s) by which PTD-FNK exerts a protective effect. Isolated rat hearts were subjected to 35-min global ischemia, followed by 120-min reperfusion using the Langendorff methods. PTD-FNK (a total of 30 microl) was injected intramuscularly into the anterior wall of the left ventricle either at 1 min after induction of global ischemia (group A) or at 30 min after induction of global ischemia (at 5 min before reperfusion) (group B). In group A, infarct size was significantly reduced from 47.8+/-6.8% in the control to 30.4+/-5.2, 28.7+/-3.8, and 30.4+/-6.8% with PTD-FNK at 5, 50, and 500 nmol/l, respectively (p<0.05). Temporal recovery of left ventricular developed pressure at 60 min and 120 min after reperfusion was significantly better in PTD-FNK (50 and 500 nmol/l)-treated groups than in the control (p<0.05). In contrast, PTD-FNK treatment had no effect on group B. Western blot analysis showed that PTD-FNK markedly inhibited procaspase-3 cleavage (activation of caspase-3) and reduced the number of nuclei stained by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphoshate nick-end labeling (TUNEL) assay. These findings suggest that PTD-FNK reduces the volume of myocardial infarction with corresponding functional recovery, at least in part, through the suppression of myocardial apoptosis following ischemia/reperfusion.

Glucose homeostasis in a diabetic patient during liver transplantation: a case report.

A 66-year-old woman with type C hepatitis had been treated for hepatocellular carcinoma (HCC) with transcatheter arterial embolization and radiofrequency ablation. Liver function worsened gradually to decompensated liver cirrhosis. She had recurrence of HCC and was later admitted to Juntendo University Hospital for living-donor liver transplantation. Although blood glucose was high, she had never been diagnosed with diabetes mellitus. No diabetes-related complications were detected at that time. We started treatment with multiple insulin injections. There is a unique time called the anhepatic phase during liver transplantation during which the liver does not exist in the body. Recent reports show that it is not necessary to administer glucose for patients with normal glucose tolerance during the anhepatic phase since plasma glucose could be maintained at normoglycemia to hyperglycemia (100-150 mg/dl). In our patient, plasma glucose concentration was rather high during the anhepatic phase without glucose administration. We analyzed the levels of blood glucose, insulin and various other hormones during the anhepatic phase. This could be the first report on glucose homeostasis during the anhepatic phase in a diabetic patient.

[Glinide(s), sulfonylurea(s)].

Diabetic macroangiopathy has already developed before diagnosis of diabetes mellitus. Postprandial hyperglycemia has been known as a risk factor for diabetic macroangiopathy and may be more powerful than fasting hyperglycemia. To intervene in hyperglycemia, insulin secretagogues, glinides which selectively stimulate early meal-induced insulin secretion and improve postprandial hyperglycemia, and sulfonylureas which enhance total daily insulin secretion and improve fasting hyperglycemia, have been prescribed as major oral antidiabetic agents. Few evidences that amelioration of glycemic control with insulin secretagogues lower the risk of cardiovascular diseases have been reported. But current studies have shown that intervention in postprandial hyperglycemia with drugs including glinides decreased thickness of carotid IMT as a surrogate marker of atherosclerosis. Results from on-going large scale intervention study with glinides may clarify whether amelioration of hyperglycemia lower the risk of atherosclerotic events.

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. METHODS: After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. RESULTS: Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. CONCLUSIONS: The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials.