FOXO3 is essential for CD44 expression in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer and the 5-year survival rate is only 5%. Several studies have suggested that cancer stem cells (CSCs) are thought to be involved in recurrence and metastasis and so it is essential to establish an approach targeting CSCs. Here we have demonstrated that cyclic guanosine monophosphate (cGMP) suppressed CD44 expression and the properties of CSCs in PDAC. Microarray analysis suggested that cGMP inhibited Forkhead box O3 (FOXO3), which is known as a tumor suppressor. Surprisingly, our data demonstrated that FOXO3 is essential for CD44 expression and the properties of CSCs. Our data also indicated that patients with high FOXO3 activation signatures had poor prognoses. This evidence suggested that cGMP induction and FOXO3 inhibition could be ideal candidates for pancreatic CSC.

Successful treatment of nonunion with teriparatide after failed ankle arthrodesis for Charcot arthropathy.

We describe a case of successful treatment to nonunion after multiple arthrodesis operations for Charcot arthropathy with teriparatide. We describe the case of a 25-year-old woman with severe Type I diabetes mellitus that resulted in nonunion after multiple arthrodesis operations for Charcot arthropathy. The woman sustained a femoral shaft fracture for which she underwent surgery with intramedullary nail fixation. Immediately after surgery, an empiric course of teriparatide was initiated. Femoral shaft fracture healing was observed after 2 weeks, and the woman was able to walk 12 weeks after the surgery, at which point plain film and computed tomography images revealed complete union of the ankle.

TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

Cognitive and affective impairments of a novel SCA/MND crossroad mutation Asidan.

BACKGROUND: A variety of hereditary spinocerebellar ataxia (SCA) develops a broad spectrum of both ataxia and non-ataxia symptoms. Cognitive and affective changes are one such non-ataxia symptoms, but have been described only in hereditary SCAs with exonic CAG gene expansion. METHODS: We newly found intronic hexanucleotide GGCCTG gene expansion in NOP56 gene as the causative mutation (=SCA36) in nine unrelated Japanese familial SCA originating from Asida river area in the western part of Japan, thus nicknamed Asidan for this mutation. These patients show unique clinical balance of cerebellar ataxia and motor neuron disease (MND), locating on the crossroad of these two diseases. In the nine families, 14 patients were clinically examined and genetically confirmed to Asidan. In the present study, we examined cognitive and affective analyses on 12 patients (seven men and five women) who agreed to join the examination with average age at onset of 53.1 ± 3.2 years, average duration of 12.1 ± 5.2 years, and current average age at 65.1 ± 6.2 years. RESULTS: The 12 Asidan patients demonstrated a significant decrease in their frontal executive functions measured by frontal assessment battery (FAB) and Montreal cognitive assessment (MoCA) compared with age- and gender-matched controls, whilst mini-mental state examination (MMSE) and Hasegawa dementia score-revised (HDS-R) were within normal range. The decline of frontal executive function was related to their disease duration and scale for the assessment and rating of ataxias (SARA). They also demonstrated mild depression and apathy. Single-photon emission tomography (SPECT) analysis showed that these Asidan patients showed decline of regional cerebral blood flow (rCBF) in a particular areas of cerebral cortices such as Brodmann areas 24 and 44-46. CONCLUSION: These data suggest that the patients with Asidan mutation show unique cognitive and affective characteristics different from other hereditary SCAs with exonal CAG expansion or MND.

Reversed lateral upper arm flap with a vascularised fragment of the humerus for reconstruction of ulna shaft fracture after resection of malignant tumour: a case report.

The lateral upper arm flap (LAF) is used for a pedicled flap with the vascular arcade at the elbow. There has been no report, however, with regard to the reverse lateral upper arm flap (R-LAF) with a vascularised humerus as an osteocutaneous flap. We describe the case of a 72-year-old woman with a fracture of the ulna after wide resection of a malignant tumour at the proximal half of the forearm, which was reconstructed with an R-LAF with a vascularised fragment of the distal humerus. The flap has successfully survived and the fracture was healed without any adverse event. The R-LAF with vascularised bone is a useful option for the simultaneous treatment of soft tissue and bone defects.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid ß (Aß), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aß-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Lectin panning method: the prospective isolation of mouse neural progenitor cells by the attachment of cell surface N-glycans to Phaseolus vulgaris erythroagglutinating lectin-coated dishes.

Retrospective isolation of neural progenitor cells (NPCs) may cause deterioration of the phenotype during the long-term cultivation. Therefore, prospective isolation is essential for understanding the exact characteristics of intact NPCs in the brain. However, few suitable specific cell surface antigens on NPCs that could be used for their prospective isolation are available. The present study demonstrated that within 60 min after initial plating, embryonic day 12 (E12) brain cells firmly attach to several types of lectin-coated culture wells, including Phaseolus vulgaris erythroagglutinating lectin (E-PHA), concanavalin A (Con A) and wheat germ agglutinin (WGA). Approximately 80% of the cells isolated from E-PHA-coated wells expressed the nestin antigen, which is a specific intracellular marker for NPCs and the ratio of 5-bromo-2'-deoxyuridine (BrdU)-positive/nestin-positive cells to the cells attached on the E-PHA-coated wells was significantly higher than that of the cells attached on the wells coated with other adhesive substrates. The cells that were isolated from the E-PHA-coated wells continued to attach to the well for 1 week, while those isolated from Con A- and WGA-coated wells lost their attachment after 6 days and 1 day, respectively. Furthermore, the cells isolated from the E-PHA-coated wells grew quite satisfactorily and formed numerous attached neurospheres. Their growth rate was almost equal to that observed in suspension cultures. These results indicate that the lectin panning method enables the prospective, quick and easy isolation of mouse NPCs without requiring a fluorescence-activated cell sorter (FACS) system.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion.

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammon's horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl-d-aspartate receptor agonist, into the hippocampal field CA1 of Ammon's horn (CA1) region in hippocalcin-knockout (-/-) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in -/- mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in -/- mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from -/- mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from -/- neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

Effect of ankle joint stiffness during eccentric phase in rebound jumps on ankle joint torque at midpoint.

The purposes of this study were to investigate firstly, the ankle joint stiffness during the eccentric phase in rebound jumps, and secondly, the effect of ankle joint stiffness during the eccentric phase on ankle joint torque at midpoint. Nine active males executed rebound drop jumps from a height of 30 cm (RDJ30) and 50 cm (RDJ50), and 5-repetition rebound jumps (5RJ). Force plate data, limb position and electromyogram (EMG) of the medial head of gastrocnemius (GAS), soleus (SOL), and tibialis anterior (TA) muscles were recorded simultaneously during all jumps. When compared with RDJ30 and RDJ50, 5RJ displayed significantly higher jumping height, ankle joint torque at midpoint, ankle joint stiffness during the eccentric phase, and integrated EMG (IEMG) of GAS and SOL. Furthermore, 5RJ displayed significantly lower contact time and IEMG of TA than RDJ30 and RDJ50 did. The ankle joint torque at midpoint showed a significant positive correlation with jumping height but a significant negative correlation with contact time in rebound jumps. There was a significant positive correlation between the ankle joint stiffness during the eccentric phase and ankle joint torque at midpoint in rebound jumps. These results suggested that for the rebound jumps to be effective, it may be important to enhance the ankle joint torque at midpoint by greater ankle joint stiffness during the eccentric phase. These results also suggested that compared to 5RJ, RDJ30 and RDJ50 may reduce ankle joint stiffness in order to protect the tendomuscular system from the stretch load. However, ankle joint stiffness is possibly increased in 5RJ because the restraint to defend the tendomuscular system is reduced by prediction or adaptation to the stretch load because it is a continuous jump.

Serum lipoprotein(a) dynamics before/after menopause and long-term effects of hormone replacement therapy on lipoprotein(a) levels in middle-aged and older Japanese women.

AIM: To assess lipoprotein(a) Lp(a) dynamics before and after menopause and to examine long-term changes during hormone replacement therapy (HRT) in middle-aged and older Japanese women. METHODS: (1) Serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and Lp(a) concentrations of 526 patients were compared. The patients were divided into 3 groups on the basis of menopausal status (premenopause, perimenopause, postmenopause). (2) Serum markers of lipid metabolism were measured at baseline and at 6-month intervals in 161 postmenopausal women who continuously received HRT with conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) for 4 years. (3) Changes in serum concentrations of markers were compared among 120 women with hypercholesterolemia who were randomly assigned to receive HRT (CEE plus MPA, or transdermal estradiol plus MPA) or pravastatin. RESULTS: (1) Lp(a) concentrations were significantly higher in the postmenopausal women than in the premenopausal or perimenopausal women. (2) The mean Lp(a) concentration after 6 months of HRT decreased by about 19%, and similar levels were maintained for 4 years (3). The mean Lp(a) concentration after 6 months of HRT decreased by 19.9% in the CEE plus MPA group, but did not change significantly in the transdermal estradiol plus MPA group or the pravastatin group. CONCLUSION: Our results suggest that HRT with CEE plus MPA is useful for the management of elevated serum Lp(a) concentrations in middle-aged and older women. However, follow-up studies are needed to determine whether this finding is related to the future prevention of coronary heart disease events.

A randomized comparison of locking and non-locking palmar plating for unstable Colles' fractures in the elderly.

This study compared the effectiveness of locking and non-locking palmar plating for unstable Colles' fractures in the elderly. The patients treated with locking plates included 4 men and 18 women with a mean age of 68 years (Group A) and those treated with non-locking plates included 3 men and 28 women with a mean age of 74 years (Group B). Radiographic parameters, including palmar tilt, radial inclination and radial length were measured before surgery, after surgery and at final followup. There were no significant differences in respect of any of the radiographic parameters between the two groups pre-operatively. After surgery, all of the radiographic parameters were improved in both groups and there were no significant differences between the two groups at final followup.

Hippocalcin-deficient mice display a defect in cAMP response element-binding protein activation associated with impaired spatial and associative memory.

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (-/-) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca(2+)-binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N-methyl-d-aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in -/- hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of -/- mice was comparable to that of wild-type littermates except in the probe test, where -/- mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca(2+)-signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.

A single set of low intensity resistance exercise immediately following high intensity resistance exercise stimulates growth hormone secretion in men.

AIM: The purpose of the present study was to examine the effects of an additional set immediately following high intensity resistance exercise on growth hormone (GH) response. METHODS: Subjects (n=8) performed 4 resistance exercise protocols (bilateral knee extension exercise) on separate days. The protocols were categorized into 2 types of protocol, namely "Strength-up type (S-type)" and "Combination type (Combi-type)". The S-type was resistance exercise which consisted of 5 sets at 90% of 1 repetition maximum (RM) with 3-min rest periods between sets, whereas the Combi-type is a training protocol which adds an additional set (either 50% of 1 RM [C50-type], 70% of 1 RM [C70-type] or 90% of 1 RM [C90-type]) to the S-type. Serum GH concentration and blood lactate concentration were determined pre-exercise and at 0-60 min postexercise. Relative changes in thigh girth and maximal unilateral isometric strength were determined pre-exercise and immediately postexercise. RESULTS: The increasing values of GH concentration (DGH) in the S-type was the lowest of all protocols. On the other hand, DGH in the C50-type showed a significantly (p<0.05) higher increase than in the S-type and C90-type, and a relatively higher increase than in the C70-type. CONCLUSION: These results suggests that a high intensity, low volume training protocol to induce neural adaptation resulted in little GH response, but GH secretion was increased by performing a single set of low intensity resistance exercise at the end of a series of high intensity resistance sets.

Equation-of-state measurements of polyimide at pressures up to 5.8 TPa using low-density foam with laser-driven shock waves.

The laser-driven equation-of-state (EOS) experiments for polyimide are presented. The experiments were performed with emission measurements from the rear sides of shocked targets at up to a laser intensity of 10(14) W/cm(2) or higher with 351 nm wavelength and 2.5 ns duration. Polyimide Hugoniot data were obtained up to 0.6 TPa with good accuracy. Applying low-density foam ablator to the EOS unknown material, we also obtained the data at a highest pressure of 5.8 TPa in the nonmetal materials. Those data were in agreement with the theoretical curves.

Effects of soy protein diet on the expression of adipose genes and plasma adiponectin.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.

Effects of counseling on climacteric symptoms in Japanese postmenopausal women.

OBJECTIVES: We objectively assessed the effects of counseling on climacteric symptoms in Japanese postmenopausal women. METHODS: Symptoms in 44 women (age, 51.4 +/- 3.4 years; period after menopause, 3.6 +/- 3.4 years) treated with counseling were evaluated according to the Keio modified menopause index. The response to counseling was compared with that to hormone replacement therapy (HRT). RESULTS: Forty cases (90.9%) showed an improvement in index score. There were no significant relationships between improvement and age, the period after menopause, or the severity or type of symptoms before counseling. The most improved symptom was headache, followed by palpitation and insomnia. Physical symptoms accounted for most of the common symptoms. The pattern of improvement with counseling was markedly different from that with HRT. CONCLUSIONS: We suggest that counseling is effective for treating climacteric symptoms, since it improves not only psychological symptoms, but also physical ones. Counseling may deserve evaluation as a complementary treatment to HRT.

Reduction by phytate-reduced soybean beta-conglycinin of plasma triglyceride level of young and adult rats.

This study examined the effects of soybean beta-conglycinin, from which phytate was mostly removed, on the plasma lipids in young and adult rats. Male Wistar young (6 week-old) and adult (21 week-old) rats were fed high cholesterol diets containing 20% casein, soy protein isolate (SPI), or soybean beta-conglycinin for 10 days. In young rats, although the food intake of the beta-conglycinin group was higher than those of the casein and SPI groups, the weight gain was significantly lower than those of the other groups. However, in adult rats, the weight gain was not different among the groups. In young and adult rats, relative liver weights of SPI and beta-conglycinin groups were significantly lower than that of the casein group, and the degree of the reduction was more marked in the beta-conglycinin group than in the SPI group. In young rats, the plasma triglyceride level was significantly lower in the SPI and beta-conglycinin groups than that in the casein group. In addition, the plasma triglyceride level of the beta-conglycinin group was significantly lower than that of the SPI group. Plasma total cholesterol levels of the SPI and beta-conglycinin groups were significantly lower than that of the casein group. However, there was little difference in the lowering effect between SPI and beta-conglycinin. These results indicate that soybean beta-conglycinin may have lowering functions not only on plasma total cholesterol level, but also on plasma triglyceride level.

Immunochemical assessment of neural visinin-like calcium-binding protein 3 expression in rat brain.

Expression of neural visinin-like calcium-binding protein 3 (NVP3) was assessed by immunoblot and immunohistochemical analyses in rat brain. NVP3 was markedly expressed in the cerebellum, at a concentration of 9.5microM. On SDS-PAGE, native NVP3 migrated at 23kDa, identical to the recombinant myristoyl-form, but somewhat faster than the non-myristoyl-form. Both forms bound 3 moles of calcium. The myristoyl-form exhibited a cooperativity in binding calcium and calcium-dependent membrane-binding, but the non-myristoyl-form did not. At 3 months, NVP3 was primarily localized in the Purkinje cells, with intense staining in the cell bodies, dendrites and axons. The cerebellar granule cells and basal nuclear neurons were faintly stained. During development of the cerebellum, NVP3-positive Purkinje cells first appeared on post-natal day 14 (P14). The staining intensity then increased and plateaued on P28. Labeling showed a tendency to accumulate in the dendrites and nerve terminals in a fine granular pattern. During aging process, NVP3 levels decreased by 43% at 12 months and 68% at 24 months, while the levels of NVP1, synaptophysin and drebrin were preferentially preserved. These results suggest that NVP3 is involved in dendritic arborization and postsynaptic function in cerebellar Purkinje cells and that presynaptic nerve terminals are another functional site of the protein.

Vaginal symptoms in Japanese postmenopausal women: comparison with other climacteric symptoms.

OBJECTIVES: This study was designed to investigate the current status of vaginal symptoms in Japanese postmenopausal women, and to clarify the significance of vaginal symptoms by analyzing their relationship to other climacteric symptoms. METHODS: The complaints of 613 women attending a menopause clinic (mean age +/- standard deviation 53.5 +/- 7.2 years; time since menopause or bilateral oophorectomy 6.4 +/- 6.5 years) were evaluated according to the Keio modified menopause index. RESULTS: In total, 56.4% of subjects had at least one vaginal symptom. The most frequent vaginal symptom was dyspareunia, followed by vaginal dryness, discharge and itching, and the mean prevalence of the four symptoms was 31.7%. The peak intensity of vaginal symptoms occurred in the sixth decade, or 3-5 years after menopause or oophorectomy. Vaginal symptoms were usually associated with other climacteric symptoms, but some occurred independently. CONCLUSIONS: A moderate proportion of Japanese postmenopausal women experience vaginal symptoms, which can occur independently of other climacteric symptoms. Vaginal symptoms therefore warrant careful attention during the treatment of postmenopausal women.