Human induced pluripotent stem cell-derived fiber-shaped cardiac tissue on a chip.

We propose a method for the production of a fiber-shaped three-dimensional (3D) cellular construct of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for the quantification of the contractile force. By culturing the cardiomyocytes in a patterned hydrogel structure with fixed edges, we succeeded in fabricating hiPS-CM fibers with aligned cardiomyocytes. The fiber generated contractile force along the fiber direction due to the hiPS-CM alignment, and we were able to measure its contractile force accurately. Furthermore, to demonstrate the drug reactivity of hiPS-CM fibers, the changes in the contractile frequency and force following treatment with isoproterenol and propranolol were observed. We believe that hiPS-CM fibers will be a useful tool for pharmacokinetic analyses during drug development.

Anomalous magnetization behaviour in a single crystal of vanadium spinel FeV2O4.

Spinel oxide FeV2O4, having the orbital degrees of freedom at Fe(2+) and V(3+) ions, exhibits multi-step magnetic phase transitions and successive structural phase transitions at low temperatures. In order to clarify the magnetic properties of FeV2O4, we have measured the temperature dependence of magnetization, isothermal magnetization curves and specific heat using a single crystal of FeV2O4. Temperature-induced magnetization jumps below the 110 K were observed in the zero-field-cooled magnetization curves. Furthermore, we found that the behaviours of the isothermal magnetization curves were quite different between the zero-field-cooled and field-cooled conditions. We suggest that the change of the magnetic domain structure under the magnetic field associated with the orbital states of Fe(2+) ions is the possible origin of these intriguing and anomalous magnetic properties in a single crystal of FeV2O4.

White matter impairment in Rett syndrome: diffusion tensor imaging study with clinical correlations.

BACKGROUND AND PURPOSE: RTT, caused by mutations in the methyl CPG binding protein 2 (MeCP2) gene, is a disorder of neuronal maturation and connections. Our aim was to prospectively examine FA by DTI and correlate this with certain clinical features in patients with RTT. MATERIALS AND METHODS: Thirty-two patients with RTT underwent neurologic assessments and DTI. Thirty-seven age-matched healthy female control subjects were studied for comparison. With use of a 1.5T MR imaging unit, DTI data were acquired, and FA was evaluated to investigate multiple regional tract-specific abnormalities in patients with RTT. RESULTS: In RTT, significant reductions in FA were noted in the genu and splenium of the corpus callosum and external capsule, with regions of significant reductions in the cingulate, internal capsule, posterior thalamic radiation, and frontal white matter. In contrast, FA of visual pathways was similar to control subjects. FA in the superior longitudinal fasciculus, which is associated with speech, was equal to control subjects in patients with preserved speech (phrases and sentences) (P = .542), whereas FA was reduced in those patients who were nonverbal or speaking only single words (P < .001). No correlations between FA values for tracts and clinical features such as seizures, gross or fine motor skills, and head circumference were identified. CONCLUSIONS: DTI, a noninvasive technique to assess white matter tract pathologic features, may add specificity to the assessment of RTT clinical severity that is presently based on the classification of MeCP2 gene mutation and X-inactivation.

Collagen maturity, glycation induced-pentosidine, and mineralization are increased following 3-year treatment with incadronate in dogs.

UNLABELLED: Collagen cross-linking is a determinant of bone quality. A three-year treatment of bisphosphonate-incadronate disodium-in beagles increased degree of mineralization, collagen maturity, and pentosidine, a compound with advanced glycation end products. The treatment had no effect on the total amount of enzymatic cross-link formation. INTRODUCTION: Collagen cross-linking is a determinant of bone quality. Recently, we reported that long-term treatment with bisphosphonate increased microdamage accumulation. The aim of this study was to clarify the effect of a three-year treatment with bisphosphonate on degree of mineralization and immature and mature enzymatic cross-links and non-enzymatic collagen cross-link, pentosidine, in cortical bone in the same dogs. METHODS: Twenty-nine 1-year-old beagles (15 males, 14 females) were divided into three groups that daily were given vehicle or incadronate at doses of 0.3 or 0.6 mg/kg/day orally for three years. A cortex of a rib was fractionated into low- and high-density portions. The contents of calcium, phosphorus, enzymatic immature and mature cross-links, and the non-enzymatic glycation product pentosidine were determined in each fraction. RESULTS: Calcium, phosphorus, and pentosidine contents and the ratio of mature to immature cross-links increased significantly with incadronate in a dose-dependent manner, but the total amount of enzymatic cross-links was unchanged. The pentosidine content correlated inversely with cortical activation frequency (p < 0.01). CONCLUSION: Long-term suppression of bone remodeling by bisphosphonate increases degree of mineralization, collagen maturity, and non-enzymatic cross-linking.

Analysis of thirteen populations of black cohosh for formononetin.

Black cohosh (Actaea racemosa L. syn. Cimicifuga racemosa (L.) Nutt.), a North American perennial plant, is a promising natural alternative to hormone replacement therapy for treating menopausal symptoms, but the mechanism of action is not understood. The clinical actions of this plant have been attributed to the isoflavonone formononetin since 1985, when its presence was reported in a black cohosh extract. Others have since looked for formononetin, but have not detected it. We looked for formononetin in extracts of black cohosh roots and rhizomes collected in thirteen locations in the eastern United States, including Maryland, New Jersey, New York, North Carolina, Pennsylvania, Virginia, and Tennessee. The rhizome samples were extracted using 80% methanol, and the extracts were partially purified using solid-phase extraction to concentrate any isoflavonoids that might be present. We tested for formononetin in these partially purified samples using thin-layer chromatography and high-performance liquid chromatography with a photodiode array detector and a mass spectrometer. Formononetin was not detected in any of the thirteen plant populations examined. Remifemin, a German product now on the United States market, and CimiPure, a commercially available black cohosh rhizome extract, were also analyzed. We did not detect formononetin, or ononin (formononetin-7-glucoside), in any sample tested by the above-mentioned chemical analyses. Therefore, the clinically observed estrogen-like actions of black cohosh, such as reduction of hot flashes, are likely due to a compound, or combination of compounds, other than formononetin.

Effect of calcium supplementation on bone dynamics of the maxilla, mandible and proximal tibia in experimental osteoporosis.

The effect of calcium supplementation on the bone dynamics in the hard palate of the molar region (maxilla), mandible and proximal tibia in experimental osteoporotic rats was examined. Ninety ovariectomized (OVX) and 45 sham-OVX Wistar female rats were used in this study. All the rats received surgical operation at 6 weeks of age. Ovariectomized rats were fed on a low calcium diet (0.02%) for 12 weeks post-operation, and then randomly divided into the two following groups. One group was fed on high calcium diet (2.30%) (OVX-HCa) and the other group was remained on the low calcium diet (OVX-LCa). Sham-OVX rats were fed on regular calcium diet (1.15%) during the experimental period (Sham-OVX). Histomorphological analysis was carried out from 12 to 32 weeks post-operation. On undecalcified thin section, bone volume, eroded surface, osteoid surface and bone formation rate were calculated for cortical bone of the maxilla, and for cancellous bone of the mandible and proximal tibia. In the OVX-LCa group, compared with the Sham-OVX group, decrease of the bone volume and increase of the bone resorption and formation parameters were detected throughout the observation periods. In the OVX-HCa group, compared with the OVX-LCa group, increase of the bone volume and temporarily increased parameters of bone formation at 1 week after feeding on high calcium diet were observed in the maxilla, but these changes were not observed in the mandible and proximal tibia. Moreover, the bone resorption and formation parameters in the maxilla, mandible and proximal tibia in the OVX-HCa group became equivalent to the Sham-OVX levels with the passage of time.

Octreotide-induced suppression of the hyperglycemic response to neostigmine or bombesin: relationship to hypothalamic noradrenergic drive.

Neostigmine (cholinesterase inhibitor) or bombesin, when injected into the third cerebral ventricle of awake rat, dose-dependently increased serum glucose with the simultaneous rise in hypothalamic noradrenergic neuronal activity (NAA). Co-administration of octreotide with neostigmine or bombesin suppressed the hypothalamic NNA response with the simultaneous inhibition of the hyperglycemic response. There was a close relationship between hypothalamic NNA and serum glucose in these studies. On the basis of the concept that hypothalamic noradrenergic drive plays an important role in mediating the hyperglycemic response to stressful stimuli, the present findings suggest that the hyperglycemic response to neostigmine or bombesin is mediated via the interaction with hypothalamic noradrenergic neurons.

Long-term effect of incadronate disodium (YM-175) on fracture healing of femoral shaft in growing rats.

The aim of this study was to investigate the long-term effect of incadronate on fracture healing of the femoral shaft in rats. Female Sprague-Dawley 8-week-old rats were injected subcutaneously (sc) with either vehicle (V group) or two doses of incadronate (10 microg/kg and 100 microg/kg) three times a week for 2 weeks. Right femoral diaphysis was then fractured and fixed with intramedullary stainless wire. Just after fracture, incadronate treatment was stopped in pretreatment groups (P groups: P-10 and P-100) or continued in continuous treatment groups (C groups: C-10 and C-100). All rats were killed at 25 weeks or 49 weeks after surgery. Fractured femur was evaluated radiologically and mechanically and then stained in Villanueva bone stain and embedded in methyl methacrylate. Undecalcified cross-sections from the fracture area were evaluated microradiologically and histomorphometrically. Radiographic observation showed that the fracture line disappeared in all groups. Cross-sectional area in the C-100 group was the biggest among all groups and in the C-10 group was larger than that in the V group at 25 weeks. Histological and histomorphometric observations showed that the process of fracture healing was delayed under continuous treatment with incadronate as evidenced by the delay of both lamellar cortical shell formation and resolution of original cortex in C groups. Percent linear labeling perimeter, mineral apposition rate (MAR), and bone formation rate (BFR) in C groups significantly decreased compared with the other groups, indicating that the callus remodeling was suppressed under continuous treatment, especially with a high dose. Mechanical study showed that the stiffness and ultimate load of the fractured femur in the C 100 group were the highest among all groups at both 25 weeks and 49 weeks. In conclusion, this study showed that long-term continuous treatment with incadronate delayed the process of fracture healing of femur in rats, especially under high dose but it did not impair the recovery of mechanical integrity of the fracture.

Analysis of anti-rotavirus activity of extract from Stevia rebaudiana.

Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions.

Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants. In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis. Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants. Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA. Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts. In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots. We also isolated a genomic fragment containing both OsNAS1 and OsNAS2. In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves. The amounts of endogenous NA in both the roots and leaves were higher than in barley. We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation. Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes. Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.

Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores.

A cDNA clone, Ids3 (iron deficiency-specific clone 3), was isolated from an Fe-deficient-root cDNA library of Hordeum vulgare. Ids3 encodes a protein of 339 amino acids with a calculated molecular mass of 37.7 kDa, and its amino acid sequence shows a high degree of similarity with those of plant and fungal 2-oxoglutarate-dependent dioxygenases. One aspartate and two histidine residues for ferrous Fe binding (Asp-211, His-209, His-265) and arginine and serine residues for 2-oxoglutarate binding (Arg-275, Ser-277) are conserved in the predicted amino acid sequence of Ids3. Ids3 expression was rapidly induced by Fe deficiency, and was suppressed by re-supply of Fe. Among eight graminaceous species tested, Ids3 expression was observed only in Fe-deficient roots of H. vulgare and Secale cereale. which not only secrete 2'-deoxymugineic acid (DMA), but also mugineic acid (MA) and 3-epihydroxymugineic acid (epiHMA, H. vulgare), and 3-hydroxymugineic acid (HMA, S. cereale). The Ids3 gene is encoded on the long arm of chromosome 4H of H. vulgare, which also carries the hydroxylase gene that converts DMA to MA. Moreover, the Ids2 gene, which is the plant dioxygenase with the highest homology to Ids3, is encoded on the long arm of chromosome 7H of H. vulgate, which carries the hydroxylase gene that converts MA to epiHMA. The observed expression patterns of the Ids3 and Ids2 genes strongly suggest that IDS3 is an enzyme that hydroxylates the C-2' positions of DMA and epiHDMA, while IDS2 hydroxylates the C-3 positions of MA and DMA.

Isolation and characterization of IDI2, a new Fe-deficiency-induced cDNA from barley roots, which encodes a protein related to the alpha subunit of eukaryotic initiation factor 2B (eIF2Balpha).

A new Fe-deficiency-inducible cDNA, IDI2, was isolated from Fe-deficient barley roots using the cDNA MACRO Array Technique. Accumulation of IDI2 transcripts in barley roots was strongly correlated with iron nutritional status. IDI2 encoded a protein with a low similarity to the alpha subunit of eukaryotic initiation factor 2B (eIF2Balpha). In addition, many hypothetical proteins homologous to IDI2 were also found in a database search. These proteins had limited similarity to eIF2Balpha as well as IDI2. It has been reported that these eIF2Balpha-like proteins (eIF2Balpha-LPs) are a family that is distinct from the eIF2Balpha/beta/delta family and widely distributed in the archaea, bacteria, and eukarya. A phylogenic analysis revealed that IDI2 is the first member of the eIF2Balpha-LP family to be found in higher plants. A possible role of IDI2 protein in regulating protein synthesis in Fe-deficient barley roots is proposed.

[Evaluation of antiviral agents for adenovirus using the MTT method in vitro].

PURPOSE: Currently, there is no effective treatment for adenoviral conjunctivitis. We evaluated the antiviral inhibitory effect of four antiviral agents against adenovirus (ADV) in vitro. METHODS: Viruses used for the experiment were ADV type 4 (ADV 4), type 8 (ADV 8) and type 37 (ADV 37). We examined four antiviral agents, i.e., cidofovir (HPMPC), zalcitabine (ddC), foscarnet (PFA), and acyclovir (ACV). 50% effective concentration (EC50), 50% cytotoxic concentration (CC50) and selectivity index (SI) of compounds were determined for ADV infection in HEp-2 cells using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. RESULTS: HPMPC and ddC showed an inhibitory effect against all three ADVs. In particular, ddC showed more potent and selective inhibition against ADV than HPMPC. PFA and ACV were ineffective against ADV. CONCLUSIONS: HPMPC and ddC were inhibitory against ADV 4, ADV 8 and ADV 37 replication in vitro. The MTT method is rapid and simple for the screening of antiviral agents. We think this method is also very useful for the screening of anti-ADV agents.

Effect of bisphosphonate (incadronate) on fracture healing of long bones in rats.

This study was designed to test whether bisphosphonates disturb the process of fracture healing. Female Sprague-Dawley rats were injected with either two doses of bisphosphonate (incadronate) (10 microg/kg and 100 microg/kg) or vehicle three times a week for 2 weeks. Right femora were then fractured and fixed with intramedullary wires. Incadronate treatment was stopped in pretreatment groups (P-10 and P-100 groups), while the treatment was continued in continuous treatment groups (C-10 and C-100 groups). Animals were sacrificed at 6 and 16 weeks after surgery. Soft X-ray of all fractured femora was taken. After mechanical testing, fractured femora were stained in Villanueva bone stain and embedded in methyl methacrylate. Cross-sections near fracture line were analyzed by microradiography and histomorphometry. Radiographic study showed that bony callus was present in all the fractures and incadronate treatment led to a larger callus, especially in C-100 group at both 6 and 16 weeks. Histologic study showed that the process of fracture healing in pretreatment groups was delayed at 6 weeks, but reached control level thereafter and showed same characteristics as in control at 16 weeks. Woven bony callus could still be seen in continuous treatment groups at 16 weeks. Mechanical study indicated that the ultimate load of C-100 group was slightly higher than the other treatment groups and control. The results suggest that pretreatment with incadronate did not affect fracture healing at 16 weeks after fracture. However, continuous incadronate treatment could lead to larger callus, but it delayed remodeling process during fracture healing, especially with high-dose treatment.

Cloning of nicotianamine synthase genes, novel genes involved in the biosynthesis of phytosiderophores.

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

Modifications in brain cAMP- and calcium/calmodulin-dependent protein kinases induced by treatment with S-adenosylmethionine.

Several lines of evidence suggest that the mechanism of action of antidepressant drugs (AD) involves adaptive changes occurring in intraneuronal post-receptor signal transduction cascades. Protein phosphorylation has a key role in signal transduction and was previously found to be a target in the action of AD (5-HT and/or NA reuptake blockers). Several studies showed that cAMP- and type II Ca2+/calmodulin-dependent protein kinases (PKA and CaMKII) are markedly affected by typical AD in two different and complementary cellular districts, respectively microtubules (a somatodendritic compartment) and synaptic vesicles (a presynaptic terminal compartment). In order to investigate whether the effect on protein kinases may be involved in the therapeutic action of drugs it is interesting to compare the effect of atypical AD with that of typical drugs. In this study the effect of the atypical AD S-adenosylmethionine (SAMe) was tested. Repeated (12 days) SAMe treatment induced in cerebrocortical microtubules an increase in the binding of cAMP to the RII PKA regulatory subunit and an increase in the endogenous phosphorylation of microtubule-associated protein 2, an effect resembling that of typical AD. In synaptic terminals the treatment induced an increase in the activity of CaMKII and in the endogenous phosphorylation of vesicular substrates. However, this modification was found in the cerebral cortex rather than in the hippocampus, where typical AD affect CaMKII. In addition the synapsin I level was decreased in the hippocampus and increased in the cerebral cortex, an effect not detected with typical AD.

F344/Shi rats bearing type b catalase-1 locus are sensitive to sodium L-ascorbate promotion of two-stage urinary bladder carcinogenesis.

Our previous data showed that F344/DuCrj and LEW/Crj rat strains bearing the type a catalase-1 locus (CS1a) are sensitive to the promoting activity of sodium L-ascorbate (Na-AsA) in 2-stage urinary bladder carcinogenesis, whereas ODS/Shi and WS/ Shi rat strains bearing the type b catalase-1 locus (CS1b) are resistant. In present study, we investigated the susceptibility of F344/Shi rats also bearing the CS1 to the Na-AsA-promoting effects on bladder tumor development. Male rats, 6 weeks old, were given 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 4 weeks, then fed either basal diet supplemented with 5% Na-AsA or no chemicals for 32 weeks. The rats given BBN alone had a few small carcinomas in the urinary bladder. In contrast, animals administered BBN-Na-AsA had many large carcinomas. Administration of Na-AsA was associated with significant elevation of urinary pH and L-ascorbic acid. The results indicate that F344/Shi rats are sensitive to the promoting effects of Na-AsA on 2-stage urinary bladder carcinogenesis, and thus that the CS1 locus may not influence susceptibility to promotion.

Evaluation of altered myocardial high energy phosphate metabolism in patients on maintenance dialysis using phosphorus-31 magnetic resonance spectroscopy.

RATIONALE AND OBJECTIVES: Assessment of left ventricular metabolism and function is important in patients on maintenance dialysis because congestive heart failure occurs quite frequently and has a poor prognosis. The purpose of this study was to evaluate the changes of myocardial high energy metabolism in dialysis patients by using phosphorus-31 (31P) magnetic resonance (MR) spectroscopy. METHODS: Phosphorus-31 spectra were obtained from anteroseptal wall of the heart in six normal subjects (mean age, 24 +/- 1 years) and 14 dialysis patients (mean age, 52 +/- 11 years), using a 1.5-tesla clinical MR system. Four patients had previous history of heart failure. Echocardiography was performed in all patients to evaluate left ventricular (LV) hypertrophy and LV function. RESULTS: The averaged ratio of phosphocreatine (PCr)/beta-adenosine triphosphate (beta-ATP) in dialysis patients (1.15 +/- 0.25 mean +/- standard deviation), was significantly lower than that in healthy subjects (1.63 +/- 0.21; P < 0.01). There was no significant difference in PCr/beta-ATP ratios between the non-LV hypertrophy group (1.21 +/- 0.24; n = 7) and the LV hypertrophy group (1.09 +/- 0.24; n = 7). The averaged PCr/beta-ATP ratio in four patients with history of heart failure (0.96 +/- 0.18) was significantly lower than that of the 10 patients without history of heart failure (1.22 +/- 0.23; P < 0.05). CONCLUSIONS: These results indicate that patients on maintenance dialysis have decreased PCr/beta-ATP ratio and 31P MR spectroscopy can provide noninvasive assessment of altered high energy phosphate metabolism.

Formate dehydrogenase, an enzyme of anaerobic metabolism, is induced by iron deficiency in barley roots.

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the lds3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.