The effect of maltobionic acid on bone metabolism markers in healthy Japanese postmenopausal women: A randomized double-blind placebo-controlled crossover study.

Osteoporosis is characterized by compromised bone strengthpredisposing to an increased risk of fracture and is a disease with a high incidence in postmenopausal women. Frequent estrogen deficiency, particularly in postmenopausal women, induces osteoclast activation and is a major contributor to reduced bone mineral density. Maltobionic acid (MB) reportedly promotes mineral resorption and maintains bone mineral density in human clinical trials, although no studies have confirmed that MB improves bone metabolism in humans. Therefore, this study aimed to investigate the effects of MB administration on bone-resorption markers in healthy Japanese postmenopausal women. This was a randomized, double-blind, placebo-controlled, crossover trial. Twenty-six healthy adult Japanese women who realized that they had passed through more than 1 year of natural menopause and were aged 40-69 years were categorized into three groups. The experimental groups were allowed to consume maltobionic acid syrup 4 g (MB syrup 4 g group), maltobionic acid syrup 2 g plus maltose syrup 2 g (MB syrup 2 g group), and maltose syrup 4 g (placebo group) for 4 weeks. All 26 participants completed the intervention. Continuous ingestion of MB syrup 2 g or 4 g for 4 weeks significantly reduced the levels of bone-resorption markers deoxypyridinoline (DPD) and urinary N-telopeptide (u-NTx), and significantly increased the bone formation marker osteocalcin (OC) compared with the placebo group. Maltobionic acid (MB) intake may improve bone metabolism and reduce bone health problems, including osteoporosis, in postmenopausal, adult Japanese women. (UMIN-CTR ID: UMIN000038627).

Effects of corn syrup solids containing maltobionic acid (maltobionic acid calcium salt) on bone resorption in healthy Japanese adult women: A randomized double-blind placebo-controlled crossover study.

Maltobionic acid is known to have an inhibitory effect on the differentiation of osteoclasts, and it has also been reported in an intervention trial that ingestion of corn syrup solids containing maltobionic acid maintained and increased the bone density of postmenopausal women. However, there is no information on whether maltobionic acid improves bone metabolism in humans. Therefore, we evaluated the influence of corn syrup solids containing maltobionic acid (maltobionic acid calcium salt) on bone resorption markers in healthy Japanese women. Forty-one individuals were selected from 68 participants and assigned to two groups: 21 individuals in the test food antecedent group and 20 individuals in the placebo food antecedent group; individuals in the first group ingested 4 g of corn syrup solids containing maltobionic acid, and subjects in the second group ingested 4 g of placebo (hydrous crystalline maltose and calcium carbonate) for 4 weeks. Bone resorption marker levels (DPD and u-NTx) were evaluated by urinalysis. Forty subjects completed the study, and no adverse events related to the test food were observed. Fourteen subjects were excluded prior to the efficacy analysis because of conflict with the control criteria; the remaining 33 subjects were analyzed. Consumption of corn syrup solids containing maltobionic acid was maintained; DPD and u-NTx values were improved (p < .05). These results indicate that corn syrup solids containing maltobionic acid might contribute to suppress bone resorption and improve bone metabolism in postmenstrual women. (UMIN-CTR ID: UMIN000034257; Foundation: San-ei Sucrochemical Co., Ltd.).

In Vitro Utilization Characteristics of Maltobionic Acid and Its Effects on Bowel Movements in Healthy Subjects.

We examined the in vitro digestibility of maltobionic acid, obtained from enzymatic oxidation of maltose, its utilization by intestinal bacteria, and its biological effects on the bowel movements in healthy subjects. We found that maltobionic acid is not digested in vitro by saliva, gastric juice, or pancreatic juice. Moreover, it is digested only to a small extent by small intestinal enzymes. Among the 24 strains of intestinal bacteria, maltobionic acid was selectively utilized by Bifidobacterium dentium and Bi. adolescentis. We also evaluated the influence of long-term ingestion of maltobionic acid calcium salt on bowel movements in healthy Japanese women by a randomized, double-blind, placebo-controlled, crossover trial. Thirty-four subjects completed the study, and no adverse events related to the test food were observed. Ten subjects were excluded prior to the efficacy analysis because of conflict with the control criteria; the remaining 24 subjects were analyzed. Intake of test food containing 4 g maltobionic acid for 4 weeks caused a significant increase in the stool frequency, significant improvement in stool form scale and CAS-MT total scores as compared with the placebo group. These results suggest that maltobionic acid is an indigestible carbohydrate and is a promising therapeutic agent for improving the intestinal environment.

A heterozygous deficiency in protein phosphatase Ppm1b results in an altered ovulation number in mice.

Ppm1b, a metal­dependent serine/threonine protein phosphatase, catalyzes the dephosphorylation of a variety of phosphorylated proteins. Ppm1b­/­ mouse embryos die at the fertilized oocyte stage, whereas Ppm1b+/­ mice with a C57BL/6 background exhibit no phenotypic abnormalities. Because the C57BL/6 strain produces a limited number of pups, in an attempt to produce Ppm1b­/­ mice, congenic Ppm1b+/­ mice with an ICR background were established, which are more fertile and gave birth to more pups. As a result, however, no Ppm1b­/­ offspring were obtained when pairs of Ppm1b+/­ ICR mice were bred again. Ppm1b+/­ male and female ICR mice were analyzed from the viewpoint of fecundity. The Ppm1b haploinsufficiency had no effect on testicular weight or the number of sperm in male mice. Despite the fact that the levels of Ppm1b protein in the ovaries of sexually mature Ppm1b+/­ mice were decreased compared with those of Ppm1b+/+ mice, there appeared to be no significant difference in the histological appearance of the ovaries, litter sizes or plasma progesterone levels at the estrous stage. When superovulation was induced by stimulation using a hormone treatment, the number of ovulated oocytes were the same for Ppm1b+/­ and Ppm1b+/+ mice at 4 weeks of age when the estrous cycle did not proceed, however, the number of ovulated oocytes was lower in sexually mature Ppm1b+/­ mice at 11 weeks of age compared with Ppm1b+/+ mice in the first and the second superovulation cycles. These collective results suggest that follicle development is excessive in Ppm1b+/­ mice, and that this leads to a partial depletion of matured follicles and a corresponding decrease in the number of ovulated oocytes.

Isolation of a spirolactone norditerpenoid as a yeast Ca2+ signal transduction inhibitor from Kuji amber and evaluation of its effects on PPM1A activity.

A different type of biologically active compound from Kuji amber (Late Cretaceous, Japan) before the K-Pg boundary [65 million years ago (Ma)] was isolated based on the growth-restoring activity of a mutant yeast involving Ca2+ signal transduction. It was identified as a spirolactone norditerpenoid, (4R*, 5S*, 8R*, 9R*, 10S*)-14,15,16,19-tetranor-labdan-13,9-olide (1) from spectral analyses with high-resolution electron ionization mass spectrometry (HREIMS), 1D and 2D nuclear magnetic resonance (NMR). Although the planar structure of 1 is known as an artificial derivative from marrubiin, it was isolated as a natural product from Kuji amber and its structure was elucidated for the first time. It had a growth-restoring activity against the mutant yeast through the direct or indirect inhibition of calcineurin activity [protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) activation]. Furthermore, the compound had potent inhibitory effect against the degranulation of rat basophilic leukemia 2H3 (RBL-2H3) cells.

Targeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain.

PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1lΔ/Δ mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.

Calcineurin inhibitors suppress the high-temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: a new method of screening for calcineurin inhibitors.

The ubiquitin/proteasome system plays significant and important roles in the regulation of metabolism of various proteins. The dysfunction of this system is involved in several diseases, for example, cancer, neurogenic diseases and chronic inflammation. Therefore, the compounds, which regulate the ubiquitin/proteasome system, might be candidates for the development use as clinical drugs. The Saccharomyces cerevisiae mutant (rsp5(A401E)) has a single amino acid change, Ala401Glu, in the RSP5 gene, which encodes an essential E3 ubiquitin ligase, is hypersensitive to high-temperature stress. Here, we found that the immunosuppressants FK506 and cyclosporin A, both known as calcineurin inhibitors, complemented the high-temperature stress-induced growth defect of rsp5(A401E) strain. The defect of calcineurin pathway by disrupting the CNB1 and CRZ1 gene also partially complemented the high-temperature stress sensitivity of rsp5(A401E) cells. Thus, these results suggest that inhibition of the calcineurin pathway confers the tolerance to high-temperature stress on rsp5(A401E) cells. Furthermore, some diterpenoid compounds, which restore the growth of rsp5(A401E) cells, showed the activities of calcineurin inhibition and protein phosphatase 2C activation. These results indicate that calcineurin inhibitors suppress the high-temperature stress sensitivity of rsp5(A401E) cells and that analysis of their physiological function is effective for the screening of calcineurin inhibitors in yeast cells.

Pisiferdiol restores the growth of a mutant yeast suffering from hyperactivated Ca2+ signalling through calcineurin inhibition.

In the course of our screening program for a new inhibitor of the Ca(2+) signalling pathway using mutant yeast [Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)], a mouse PP2Cα activator, pisiferdiol, isolated from Chamaecyparis pisifera, was found to alleviate the Ca(2+) signal-mediated growth inhibition. Pisiferdiol showed growth inhibition activity against the mpk1Δ strain compared with the cnb1Δ strain and induced Li(+) sensitivity to the wild-type strain, indicating that it suppresses the calcineurin pathway in the yeast. However, the Li(+) sensitivity to ptc1Δ strain by pisiferdiol was diminished. Pisiferdiol showed growth restored activity in the zds1Δ strain without immunophilins Fkb1p or Cph1p, and in the pmc1Δ strain. It inhibited calcineurin-induced expression in the reporter gene assay and decreased the protein expression (Western blots) of calcineurin (Cnb1p) in addition to a decrease of Swe1p and phosphorylation of Cdc28p in the mutant yeast. These results showed that pisiferdiol could suppress indirectly the action of calcineurin and restored the growth inhibition of the mutant yeast through Ptc1p activation.

Inhibitory effects of wasabi isothiocyanates on chemical mediator release in RBL-2H3 rat basophilic leukemia cells.

Wasabi is a plant of Japanese origin. It belongs to the family Brassicaceae and produces various isothiocyanates (ITCs). To clarify the type I allergies inhibited by wasabi ITCs, we investigated the inhibitory effect on chemical mediator release from dinitrophenylated bovine serum albumin (DNP-BSA)-stimulated RBL-2H3 rat basophilic leukemia cells. Allyl ITC (AITC), sec-butyl ITC (s-BuITC), and 3-butenyl ITC (3-BuITC), which have 3 or 4 carbon chains, inhibited histamine release but did not inhibit the release of leukotriene B4 (LTB4) or cysteinyl LTs (CysLTs). 4-Pentenyl ITC (4-PeITC) and 5-hexenyl ITC (5-HeITC), which have 5 or 6 carbon chains and an unsaturated bond at the end, inhibited LTB4 release but did not inhibit the release of histamine or CysLTs. 6-Methylthiohexyl ITC (6-MTITC), 6-methylsulfinylhexyl ITC (6-MSITC), and 6-methylsulfonylhexyl ITC (6-MSFITC), which have a sulfur atom inserted at the end of a 6-carbon chain, inhibited the release of histamine, LTB4, and CysLTs and the elevation in intracellular Ca(2+). These results suggest that wasabi ITCs inhibited type I allergies by inhibiting chemical mediator release and that the inhibitory effects on each chemical mediator were due to differences in the side chain structure of the wasabi ITCs.

Effects of veraguensin and galgravin on osteoclast differentiation and function.

The dried flower buds of Magnolia sp. are widely used as herbal medicines because of their anti-inflammatory, anti-malarial and anti-platelet activities. Here, we found that veraguensin and galgravin, lignan compounds derived from Magnolia sp., dose-dependently inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblastic cells. These compounds also inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells and bone marrow macrophages. In the RANKL-induced signaling pathway, veraguensin and galgravin reduced p38 phosphorylation and suppressed the expression of c-Fos, a key transcription factor for osteoclastogenesis. Veraguensin and galgravin also inhibited osteoclastic pit formation, which was accompanied by decreased mature osteoclast viability. In conclusion, these results indicate that veraguensin and galgravin can inhibit bone resorption and may offer novel compounds for the development of drugs to treat bone-destructive diseases such as osteoporosis.

Sanguinarine as a potent and specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via phosphorylation of p38 in HL60 cells.

Sanguinarine, a plant alkaloid, was identified as a potent and specific protein phosphatase (PP) 2C inhibitor. It inhibited PP2C competitively with respect to alpha-casein (Ki=0.68 microM) and showed selectivity for PP2C as compared with PP1, PP2A, and PP2B in vitro. In vivo, sanguinarine showed cytotoxicity toward human promyelocytic leukemia cell line HL60, with an IC(50) value of 0.37 microM, and induced apoptosis through a caspase-3/7-dependent mechanism involving the phosphorylation of p38, a PP2Calpha substrate. The apoptosis activity induced by sanguinarine was partially inhibited by a p38 inhibitor, SB203580, and was involved in the phospho-p38 protein in HL60 cells.

Disruption of the mouse protein Ser/Thr phosphatase 2Cbeta gene leads to early pre-implantation lethality.

Protein phosphatase 2Cbeta (PP2Cbeta) is a member of a family of protein Ser/Thr phosphatases (PP2C) that is composed of at least twelve different gene products. Recent studies have revealed that PP2Cbeta mRNA accumulates in mature sperm, unfertilized metaphase II-arrested oocytes and zygotes, but that the mRNA level then decreases sharply between the early two-cell and eight-cell stages, remaining at low levels during the 16-cell to blastocyst stages of mice. These observations raised the possibility that PP2Cbeta plays a crucial role during gametogenesis, fertilization, and/or early stages of embryonic development. In this study, we employed a gene knockout technique in mice to test this possibility. We found that PP2Cbeta(Delta/wt) mice generate normal mature gametes. However, PP2Cbeta(Delta/Delta) embryos die between the two-cell and eight-cell stages. To our interest, PP2Cbeta(Delta/Delta) ES cells which had been generated by transfecting PP2Cbeta(3lox/3lox) ES cells with Cre-expressing plasmid were viable. In addition, knockdown of PP2Cbeta using siRNA did not affect the proliferation of wild-type ES cells. These observations suggest that relatively high PP2Cbeta expression is specifically required during the early stages of pre-implantation development. The possible mechanisms for the early pre-implantation lethality of PP2Cbeta(Delta/Delta) mice are discussed.

Cadmium cation increases the production and mRNA levels of insulin-like growth factor-binding protein-1 in HepG2.

The production of insulin-like growth factor-binding protein-1 (IGFBP-1) in HepG2 was increased by cadmium cation (Cd2+) at 3 microM, but not by other divalent cations. The mRNA level of IGFBP-1 was also increased by the administration of 3 microM of Cd(2+). These results suggest that Cd(2+) impacts the gene expression of IGFBP-1, which leads to production of IGFBP-1.

Quercetin suppresses bone resorption by inhibiting the differentiation and activation of osteoclasts.

Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells [M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells]. Quercetin dose-dependently inhibited osteoclast-like (OCL) cell formation at 2-5 microM concentration in both the coculture and MD cell culture. Quercetin inhibited the increase of tartrate-resistant acid phosphatase (TRAP) activity of mononuclear preosteoclasts (pOCs) induced by receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) in both MD and RAW cell cultures. Quercetin reversely induced the disruption of actin rings in OCLs. Quercetin also suppressed both pit formation induced by osteoclasts on dentine slices and PTH-stimulated (45)Ca release in mouse long bone cultures. These results suggest that osteoclast progenitors as well as mature osteoclasts, are quercetin's target cells in relation to bone resorption, and that quercetin's suppressive effect on bone resorption results from both its inhibitory effect on the differentiation of osteoclast progenitor cells into pOCs and from its disruptive effect on actin rings in mature osteoclasts.

Molecular cloning of PP2Ceta, a novel member of the protein phosphatase 2C family.

We have cloned a novel member of the mouse protein phosphatase 2C family, PP2Ceta. Sequence analysis suggests that PP2Ceta, PP2Czeta and NERPP-2C constitute a unique subgroup of the PP2C family. PP2Ceta had extremely low activity against alpha-casein compared with PP2Calpha and was localized mainly in cell nuclei, suggesting that PP2Ceta dephosphorylates a unique nuclear protein(s) in the cells.

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9.

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.

Regulation of stress-activated protein kinase signaling pathways by protein phosphatases.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.