Anthocyanin glucosylation mediated by a glycoside hydrolase family 3 protein in purple carrot.

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.

Lack of Musashi-2 induces type IIa fiber-dominated muscle atrophy.

Skeletal muscle is a highly plastic tissue, adapting its structure and metabolism in response to diverse conditions such as contractile activity, nutrients, and diseases. Finding a novel master regulator of muscle mass and quality will provide new therapeutic targets for the prevention and treatment of muscle weakness. Musashi is an RNA-binding protein that dynamically regulates protein expression; it was originally discovered as a cell fate determination factor in neural cells. Here, we report that Musashi-2 (Msi2) is dominantly expressed in slow-type muscle fibers, fibers characterized by high metabolism and endurance. Msi2 knockout (KO) mice exhibited a decrease in both soleus myofiber size and number compared to control mice. Biochemical and histological analyses revealed that type IIa fibers, which are of the fast type but have high metabolic capacity, were decreased in Msi2 KO mice. The contraction force of isolated soleus muscle was lower in KO mice, and the expression of the metabolic proteins, cytochrome c oxidase and myoglobin, was also decreased in KO muscle. Our data demonstrate the critical role of Msi2 in the maintenance of normal fiber-type composition and metabolism.

Isoproterenol, an adrenergic ß receptor agonist, induces metallothionein synthesis followed by canceling amyloid ß1-42-induced neurodegeneration.

Adrenergic ß receptor activation may ameliorate amyloid ß toxicity. We examined whether isoproterenol, an adrenergic ß receptor agonist reduces neurodegeneration caused by Aß1-42, for which intracellular Zn2+ dysregulation is a trigger. Neurodegeneration was assessed in the dentate granule cell layer 14 days after intracerebroventricular injection of human Aß1-42 into the mouse brain. Neurodegeneration was canceled after co-injection of isoproterenol. Isoproterenol did not affect Aß staining (uptake) in the dentate granule cell layer 1 h after Aß injection. In contrast, isoproterenol reduced intracellular Zn2+ level increased by Aß. The synthesis of intracellular metallothioneins (MTs), Zn2+-binding proteins was not enhanced in the dentate granule cell layer 24 h after Aß1-42 injection, but significantly enhanced after co-injection of isoproterenol. These data indicate that isoproterenol enhances MT synthesis and cancels neurodegeneration via intracellular Zn2+ toxicity after Aß1-42 injection. It is likely that MT synthesis enhanced by adrenergic ß receptor-mediated signaling contributes to ameliorating Aß1-42 toxicity in the brain.

A fragmented form of annexin A1 is secreted from C2C12 myotubes by electric pulse-induced contraction.

The main function of annexin A1 (ANXA1), a member of the annexin superfamily, is to bind to cellular membranes in a Ca(2+)-dependent manner. In skeletal muscle, ANXA1 is thought to be involved in the repair of damaged membrane tissue and in the migration of muscle cells. We hypothesized that ANXA1 is one of the myokines secreted during muscle contractions to accelerate the repair of cell damage after contraction. Here we performed cell contractions by electric pulse stimulation; the results revealed that a fragmented form of ANXA1 was cleaved by calpain and selectively secreted from skeletal muscle cells by contraction. We therefore realized that muscle-contraction-induced calpain-dependent ANXA1 fragmentation has a wound-healing effect on damaged cells. This suggested that not the intact form but rather fragmented ANXA1 is a contraction-induced myokine.

An improved glucose transport assay system for isolated mouse skeletal muscle tissues.

There is a growing demand for a system in the field of sarcopenia and diabetes research that could be used to evaluate the effects of functional food ingredients that enhance muscle mass/contractile force or muscle glucose uptake. In this study, we developed a new type of in vitro muscle incubation system that systemizes an apparatus for muscle incubation, using an electrode, a transducer, an incubator, and a pulse generator in a compact design. The new system enables us to analyze the muscle force stimulated by the electric pulses and glucose uptake during contraction and it may thus be a useful tool for analyzing the metabolic changes that occur during muscle contraction. The system may also contribute to the assessments of new food ingredients that act directly on skeletal muscle in the treatment of sarcopenia and diabetes.

Ficus carica L. (Fig) promotes nerve regeneration in a mouse model of sciatic nerve crush.

Peripheral nerve injuries result in significant loss of motor and sensory function, and the slow rate of nerve regeneration can prolong recovery time. Thus, approaches that promote axonal regeneration are critical to improve the outcomes for patients with peripheral nerve injuries. In this study, we investigated the effects of Ficus carica L. (fig) and Vaccinium macrocarpon Ait. (cranberry), which are rich in phytochemicals with demonstrable and diverse medicinal properties, on nerve regeneration in a mouse model of sciatic nerve crush. Our investigation revealed that fig extract, but not cranberry extract, prevented the decline in muscle weight and nerve conduction velocity induced by nerve crush. The fig extract also mitigated motor function impairment, myelin thinning, and axon diameter reduction, indicating its potential to promote nerve regeneration. Furthermore, the fig extract enhanced macrophage infiltration into the nerve tissue, suggesting that it could ameliorate nerve injury by promoting tissue repair via increased macrophage infiltration. The study provides valuable insights into the potential of the fig extract as a novel agent promoting nerve regeneration. Further investigation into the mechanisms underlying the action of fig extracts is needed to translate these findings into clinical applications for patients with peripheral nerve injuries.

Tenc1-deficient mice develop glomerular disease in a strain-specific manner.

BACKGROUND/AIMS: Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome. METHODS: To elucidate the function of Tenc1 in the kidney, Tenc1(ICGN) was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease. RESULTS: Biochemical and histological analysis revealed that homozygous Tenc1(ICGN) mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway. CONCLUSION: This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Gene expression analysis detected a low expression level of C1s gene in ICR-derived glomerulonephritis (ICGN) mice.

BACKGROUND: ICR-derived glomerulonephritis (ICGN) strain is a novel inbred strain of mice with a hereditary nephrotic syndrome. Deletion mutation of tensin 2 (Tns2), a focal adhesion molecule, has been suggested to be responsible for nephrotic syndrome in ICGN mice; however, the existence of other associative factors has been suggested. METHODS AND RESULTS: To identify additional associative factors and to better understand the onset mechanism of nephrotic syndrome in ICGN mice, we conducted a comprehensive gene expression analysis using DNA microarray. Immune-related pathways were markedly altered in ICGN mice kidney as compared with ICR mice. Furthermore, the gene expression level of complement component 1, s subcomponent (C1s), whose human homologue has been reported to associate with lupus nephritis, was markedly low in ICGN mouse kidney. Real-time quantitative reverse transcription-polymerase chain reaction confirmed a low expression level of C1s in ICGN mouse liver where the C1s protein is mainly synthesized. A high serum level of anti-dsDNA antibody and deposits of immune complexes were also detected in ICGN mice by enzyme-linked immunosorbent assay and immunohistochemical analyses, respectively. CONCLUSION: Our results suggest that the immune system, especially the complement system, is associated with nephrotic syndrome in ICGN mice. We identified a low expression level of C1s gene as an additional associative factor for nephrotic syndrome in ICGN mice. Further studies are needed to elucidate the role of the complement system in the onset of nephrotic syndrome in ICGN mice.

Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.

Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

The Impact of Milk Fat Globule Membrane with Exercise on Age-Related Degeneration of Neuromuscular Junctions.

Morphological changes in neuromuscular junctions (NMJs), which are synapses formed between α-motor neurons and skeletal muscle fibers, are considered to be important in age-related motor dysfunction. We have previously shown that the intake of dietary milk fat globule membrane (MFGM) combined with exercise attenuates age-related NMJ alterations in the early phase of aging. However, it is unclear whether the effect of MFGM with exercise on age-related NMJ alterations persists into old age, and whether intervention from old age is still effective when age-related changes in NMJs have already occurred. In this study, 6- or 18-month-old mice were treated with a 1% MFGM diet and daily running wheel exercise until 23 or 24 months of age, respectively. MFGM treatment with exercise was effective in suppressing the progression of age-related NMJ alterations in old age, and even after age-related changes in NMJs had already occurred. Moreover, the effect of MFGM intake with exercise was not restricted to NMJs but extended to the structure and function of peripheral nerves. This study demonstrates that MFGM intake with exercise may be a novel approach for improving motor function in the elderly by suppressing age-related NMJ alterations.

Effect of treatment with conditioned media derived from C2C12 myotube on adipogenesis and lipolysis in 3T3-L1 adipocytes.

Regular exercise is an effective strategy that is used to prevent and treat obesity as well as type 2 diabetes. Exercise-induced myokine secretion is considered a mechanism that coordinates communication between muscles and other organs. In order to examine the possibility of novel communications from muscle to adipose tissue mediated by myokines, we treated 3T3-L1 adipocytes with C2C12 myotube electrical pulse stimulation-conditioned media (EPS-CM), using a C2C12 myotube contraction system stimulated by an electrical pulse. Continuous treatment with myotube EPS-CM promoted adipogenesis of 3T3-L1 pre-adipocytes via the upregulation of the peroxisome proliferator-activated receptor-gamma (PPARγ) 2 and PPARγ-regulated gene expression. Furthermore, our results revealed that myotube EPS-CM induces lipolysis and secretion of adiponectin in mature adipocytes. EPS-CM obtained from a C2C12 myoblast culture did not induce such changes in these genes, suggesting that contraction-induced myokine(s) secretion occurs particularly in differentiated myotubes. Thus, contraction-induced secretion of myokine(s) promotes adipogenesis and lipid metabolism in 3T3-L1 adipocytes. These findings suggest the possibility that skeletal muscle communicates to adipose tissues during exercise, probably by the intermediary of unidentified myokines.

TRPM8-mediated cutaneous stimulation modulates motor neuron activity during treadmill stepping in mice.

Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos+ MNs, but increased the average soma size of the c-fos+ MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos+ cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.

Rapid Intracellular Zn2+ Dysregulation via Membrane Corticosteroid Receptor Activation Affects In Vivo CA1 LTP.

Involvement of membrane mineralocorticoid (MC) and glucocorticoid (GC) receptors in synaptic Zn2+ dynamics remains unclear. Here, we tested whether synaptic plasticity is affected by rapid intracellular Zn2+ dysregulation via membrane MC and GC receptor activation, in comparison with intracellular Ca2+ dysregulation. In anesthetized rats, extracellular Zn2+ level was increased under local perfusion of the hippocampal CA1 with 500 ng/ml corticosterone. In vivo CA1 long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal cell synapses was attenuated by the pre-perfusion with corticosterone prior to tetanic stimulation, and the attenuation was canceled by co-perfusion with CaEDTA, an extracellular Zn2+ chelator, suggesting that corticosterone-induced increase in extracellular Zn2+ is involved in the subsequent attenuation of LTP. In rat brain slices, corticosterone-induced increases in extracellular and intracellular Zn2+ were blocked in the presence of spironolactone, a MC receptor antagonist that canceled corticosterone-induced attenuation of LTP. Mifepristone, a GC receptor antagonist, which canceled corticosterone-induced attenuation of LTP, also blocked corticosterone-induced increase in intracellular Zn2+, but not extracellular Zn2+. Moreover, corticosterone-induced decrease in phosphorylated CaMKII was restored in the presence of CaEDTA or spironolactone. These results indicate that glucocorticoid rapidly induces the increase in intracellular Zn2+, which occurs via membrane MC and GC receptor activations, and decreases phosphorylated CaMKII level, resulting in attenuating LTP. Membrane MC and GC receptors induce intracellular Zn2+ dysregulation via differential mechanisms. In contrast, glucocorticoid-induced intracellular Ca2+ dysregulation is not crucial for affecting LTP.

Comparison of gene expression profiles among papilla, medulla and cortex in rat kidney.

The aim of this study was to compare gene expression profiles in the different kidney regions as the basis for toxicogenomics. Rat kidney was separated into papilla, medulla and cortex, and total RNA was isolated from these and from the whole slice. Gene expression profiling was performed using Affymetrix Rat Genome 230 2.0 Array. When global normalization was applied, the expression of beta-actin or GAPDH varied among the regions. It was considered that such a comparison could not be made, especially between papilla and other portions, since the production of total mRNA in the former was relatively low. In fact, ANOVA was performed on the gene expression values with global normalization in papilla, medulla, cortex, and whole slice, and the numbers of genes appeared to be the highest in papilla. It was also observed that many genes showed their maximum or minimum in the whole slice, which was theoretically impossible. To overcome the problems associated with global normalization, the "percellome" normalization (a way to obtain the values directly related to the copies of mRNA per cell) was employed to compare the regions. In applying this procedure, probe sets with regional difference in expression were efficiently extracted by ANOVA. When they were sorted by the fold difference to other regions, the higher rank was occupied by genes characteristic of the functions of kidney, i.e., channels, transporters and metabolic enzymes. Some of them were consistent with the literature and were related to pathophysiological phenomena. Comprehensive comparison of data of gene expression in the renal anatomical area will greatly enhance studies of the physiological function and mechanism of toxicity in kidney.

Profiling of gene expression in rat liver and rat primary cultured hepatocytes treated with peroxisome proliferators.

The Toxicogenomics project has been constructing a large-scale database of about 150 compounds exposed to rat (single dose, 3, 6, 9, 24 hrs and repeated dose for 3, 7, 14 28 days with 3 dose levels) and rat hepatocytes (2, 8, 24 hr with 3 concentrations) and data of transcriptome in liver using GeneChip, and the related toxicological measures are being accumulated. In the present study, the data of three ligands of peroxisome proliferator activated receptor alpha (PPARalpha), i.e., clofibrate, WY-14643 and gemfibrozil in our database were analyzed. Many of the beta-oxidation-related genes were commonly induced in vivo and in vitro, whereas expression changes in genes related to cell proliferation, apoptosis, were detected in vivo (single and repeated dose) but not in vitro. Changes in those related to the immune response, coagulation and the stress response were also detectable exclusively in vivo. Using the genes mobilized in two or three PPARalpha agonists, hierarchical clustering was performed on 32 compounds stored in our database. In the profiling of an in vivo single dose, benzbromarone and aspirin were located in the same cluster of the three PPARalpha agonists. The clustering of in vitro data revealed that benzbromarone, three NSAIDs (aspirin, indomethacin and diclofenac sodium) and valproic acid belonged to the same cluster of PPARalpha agonists, supporting the reports that benzbromarone,valproic acid and some NSAIDs were reported to be PPARalpha agonists. Using the genes commonly up-regulated both in vivo and in vitro, principal component analysis was performed in 32 compounds, and principal component 1 was found to be the convenient parameter to extract PPARalpha agonist-like compounds from the database.

Localization of endothelin receptor subtype-A in the testis of rats, dogs, and monkeys.

In order to evaluate the physiological roles of the testicular endothelin (Edn) signaling via Edn receptor subtype-A (Ednra) in mammals, the localization of Ednra was investigated by in situ hybridization and immunohistochemistry in the testis of rats, dogs, and monkeys. For in situ hybridization, a rat Ednra RNA probe which is highly homologous to the subcloned canine and monkey Ednra (88.7% and 87.9% identical, respectively) was used. Both Ednra mRNA and protein were detected in interstitial cells and cells in the basal compartment of the seminiferous tubules, mainly Sertoli cells, as well as spermatogonia and some early spermatocytes, but not spermatids. The localization pattern of Ednra was exhibited in a same manner among species, indicating that the physiological role of Edn signaling throughout Ednra was maintained in the mammalian testis.