益生菌及益生元调节骨代谢的研究进展

骨质疏松症已成为威胁中老年人健康的主要疾病之一,越来越多的人受到该病症的危害.肠道菌群是定殖在机体肠道内,与宿主形成共生关系的微生物,对宿主的免疫及代谢等产生重要影响,研究发现,肠道菌群与骨代谢之间存在密切关系,本文从肠道菌群与免疫、骨代谢与免疫、肠道菌群与骨代谢、益生菌及益生元调节骨代谢等几个方面阐述,肠道菌群有望成为骨质疏松症治疗的一个新靶点,通过益生菌或益生元来干预肠道菌群组成,进而调节免疫系统状态,抑制促炎因子的生成,从而降低骨吸收作用,达到预防和治疗骨质疏松症的目的.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism

The adipose tissue is the site of expression and secretion of a range of biologically active proteins, called adipokines, for example, leptin, adiponectin, and resistin. Leptin has previously been shown to be expressed in osteoblasts and to promote bone mineralization, whereas adiponectin expression is enhanced during osteoblast differentiation. In the present study we explored the possible role of resistin in bone metabolism. We found that resistin is expressed in murine preosteoclasts and preosteoblasts (RAW 264.7, MC3T3-E1), in primary human bone marrow stem cells and in mature human osteoblasts. The expression of resistin mRNA in RAW 264.7 was increased during differentiation and seemed to be regulated through PKC- and PKA-dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NFkappaB promoter activity, indicating a role in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3-E1 cells in a PKA and PKC-dependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3-E1 into osteoblasts. All together, our results indicate that resistin may play a role in bone remodeling.     

Mineralocorticoid receptor function in bone metabolism and its role in glucocorticoid-induced osteopenia

Although the mineralocorticoid receptor (MR) is expressed in osteoblasts and osteocytes and frequently co-localizes with the glucocorticoid receptors (GR), its pathophysiological functions in bone remain elusive. We report here that pharmacologic inhibition of MR function with eplerenone resulted in increased bone mass, with stimulation of bone formation and suppression of resorption, while specific genetic deletion of MR in osteoblast lineage cells had no effect. Further, treatment with eplerenone as well as specific deletion of MR in osteocytes ameliorated the cortical bone thinning caused by slow-release prednisolone pellets. Thus, MR may be involved in the deleterious effects of glucocorticoid excess on cortical bone.     

The relationship between bone marrow adipose tissue and bone metabolism in postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a prevalent skeletal disorder associated with menopause-related estrogen withdrawal. PMOP is characterized by low bone mass, deterioration of the skeletal microarchitecture, and subsequent increased susceptibility to fragility fractures, thus contributing to disability and mortality. Accumulating evidence indicates that abnormal expansion of marrow adipose tissue (MAT) plays a crucial role in the onset and progression of PMOP, in part because both bone marrow adipocytes and osteoblasts share a common ancestor lineage. The cohabitation of MAT adipocytes, mesenchymal stromal cells, hematopoietic cells, osteoblasts and osteoclasts in the bone marrow creates a microenvironment that permits adipocytes to act directly on other cell types in the marrow. Furthermore, MAT, which is recognized as an endocrine organ, regulates bone remodeling through the secretion of adipokines and cytokines. Although an enhanced MAT volume is linked to low bone mass and fractures in PMOP, the detailed interactions between MAT and bone metabolism remain largely unknown. In this review, we examine the possible mechanisms of MAT expansion under estrogen withdrawal and further summarize emerging findings regarding the pathological roles of MAT in bone remodeling. We also discuss the current therapies targeting MAT in osteoporosis. A comprehensive understanding of the relationship between MAT expansion and bone metabolism in estrogen deficiency conditions will provide new insights into potential therapeutic targets for PMOP.     

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways.     

A study of hydrophobins-modified menaquinone-7 on osteoblastic cells differentiation

Molecular and Cellular Biochemistry - Menaquinone-7 is involved in bone metabolism and can be used to prevent and treat osteoporosis. However, as a fat-soluble vitamin, menaquinone-7 has poor water...     

Intracellular response of Bacillus natto in response to different oxygen supply and its influence on menaquinone-7 biosynthesis

Bioprocess and Biosystems Engineering - Menaquinone-7 (MK-7) plays an important role in blood clotting, cardiovascular disease and anti-osteoporosis, and has been wildly used in the food additives...     

Pre-receptorial regulation of steroid hormones in bone cells: insights on glucocorticoid-induced osteoporosis

In the past decades, concern on glucocorticoid-induced osteoporosis has increased with the widespread use of exogenous glucocorticoids (GC). Mature bone-forming cells (osteoblasts) are considered to be the principal site of action of GC in the skeleton. More likely, it is the entire cellular and molecular network surrounding these cells that is targeted by pharmacological doses of GC. Not only osteoblast and osteocyte metabolism, but the whole differentiation of mesenchymal stem cell toward the osteoblast lineage has been proven to be sensitive to GC. The effects of GC on this process are different according to the stage of differentiation of bone cell precursors. The presence of intact GC signalling is crucial for normal bone development and physiology, as opposed to the detrimental effect of high dose exposure. Both the physiological and pharmacological effects of GC are locally modulated by the activity of the 11β-hydroxysteroid dehydrogenase 1 (HSD1) that acts primarily as a glucocorticoid activator converting the inactive glucocorticoid (cortisone) into the active hormone (cortisol). We reviewed the metabolic and differentiation pathways controlled by GC signalling. These data have been merged with the recent evidences that 11β-HSD1 exert an important role by regulating the vulnerability of bone cells to GC. The different kinetics of 11β-HSD1 at various stage of differentiation and the GC-dependency of enzymatic activity have been presented.     

Association of Grb7 with phosphoinositides and its role in the regulation of cell migration

Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.     

Secondary metabolism: regulation and role in fungal biology

Filamentous fungi produce a diverse array of secondary metabolites--small molecules that are not necessary for normal growth or development. Secondary metabolites have a tremendous impact on society; some are exploited for their antibiotic and pharmaceutical activities, others are involved in disease interactions with plants or animals. The availability of fungal genome sequences has led to an enhanced effort at identifying biosynthetic genes for these molecules. Genes that regulate production of secondary metabolites have been identified and a link between secondary metabolism, light and sexual/asexual reproduction established. However, the role of secondary metabolites in the fungi that produce them remains a mystery. Many of these fungi live saprophytically in the soil and such molecules may provide protection against other inhabitants in this ecological niche.     

Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis

The kinetics of yeast phosphofructo-1-kinase has been studied in vitro. Effector concentrations (Fru-6-P, ATP, ADP, AMP, Pi, Fru-1,6-P2, and Fru-2,6-P2) and pH were adjusted so as to mimic intracellular concentrations in yeast. Under these conditions we were able to reproduce the measured in vivo rate of PFK. In addition, by reconstituting the intracellular conditions existing during aerobic and anaerobic glycolysis, we were able to reproduce in vitro the changes in the rate of PFK observed under these conditions. Without the addition of the newly discovered effector Fru-2,6-P2, in vitro rates of PFK are much lower than its in vivo rate. Changes in Fru-2,6-P2, Fru-1,6-P2, ATP, AMP, Pi, and pH in going from aerobic to anaerobic conditions all contributed somewhat to the change in the rate of PFK observed during the Pasteur effect, with no contribution coming from ADP. These studies show that the control of PFK under the condition of the Pasteur effect cannot be ascribed to changes in any one particular effector but rather to contributions from a variety of effectors. Also, the net change in the rate of PFK in the switch from anaerobic to aerobic glycolysis is small compared with the change in its dependence upon its substrate Fru-6-P, indicating a compensation mechanism.     

Anaerobic citrate metabolism and its regulation in enterobacteria

Several species of enterobacteria are able to utilize citrate as carbon and energy source. Under oxic conditions in the presence of a functional tricarboxylic acid cycle, growth on this compound solely depends on an appropriate transport system. During anaerobiosis, when 2-oxoglutarate dehydrogenase is repressed, some species such as Klebsiella pneumoniae and Salmonella typhimurium, but not Escherichia coli, are capable of growth on citrate by a Na⁺-dependent pathway forming acetate, formate, and CO₂ as products. During the last decade, several novel features associated with this type of fermentation have been discovered in K. pneumoniae. The biotin protein oxaloacetate decarboxylase, one of the key enzymes of the pathway besides citrate lyase, is a Na⁺ pump. Recently it has been shown that the proton required for the decarboxylation of carboxybiotin is taken up from the side to which Na⁺ ions are pumped, and a membrane-embedded aspartate residue that is probably involved both in Na⁺ and in H⁺ transport was identified. The Na⁺ gradient established by oxaloacetate decarboxylase drives citrate uptake via CitS, a homodimeric carrier protein with a simultaneous-type reaction mechanism, and NADH formation by reversed electron transfer involving formate dehydrogenase, quinone, and a Na⁺-dependent NADH:quinone oxidoreductase. All enzymes specifically required for citrate fermentation are induced under anoxic conditions in the presence of citrate and Na⁺ ions. The corresponding genes form a cluster on the chromosome and are organized as two divergently transcribed operons. Their co-ordinate expression is dependent on a two-component system consisting of the sensor kinase CitA and the response regulator CitB. The citAB genes are part of the cluster and are positively autoregulated. In addition to CitA/CitB, the cAMP receptor protein (Crp) is involved in the regulation of the citrate fermentation enzymes, subjecting them to catabolite repression. Received: 25 September 1996 / Accepted: 18 November 1996     

一株产甲萘醌-7(MK-7)菌的分离鉴定及产物合成条件的初步研究

本文从豆豉中分离出了一株高产MK-7的菌株并对其进行了鉴定,同时对该菌合成MK-7的条件进行了初步研究.在参照《伯杰氏细菌鉴定手册》,并根据形态学特征、生理生化特性、并结合16S rDNA序列分析结果,该菌属于解淀粉芽孢杆菌.该菌合成MK-7的最佳条件为:发酵温度37℃、装液量为30 mL/250 mL、接种量为2％和摇瓶培养时间为3h.研究发现,菌株Y-2合成的MK-7主要存在于细胞内.以上结果可为基于菌株Y-2选育高产MK-7菌株和实现MK-7的产业化提供理论依据.