重组人骨形态发生蛋白2在CHO细胞中的表达、鉴定及活性分析

构建真核表达载体pCDNA3.1( )-hBMP-2,与质粒pSV2-dhfr共转染CHO-dhfr-细胞,以含有700μg/mLG418的IMDM进行选择性培养,筛选抗性克隆,并用MTX扩增,提高rhBMP-2的表达量。收集的rhBMP-2蛋白进行Westernblot检测,还原蛋白样品电泳产生一条大小约为18kD的特异性条带,非还原蛋白样品电泳产生一条大小约为30kD的特异性条带,提示表达的rhBMP-2是经过糖基化修饰的,且以同源二聚体形式分泌表达。单细胞分离培养得到14株rCHO(hBMP-2)单克隆细胞株,ELISA法检测rhBMP-2表达水平,最高可达7.83μg/24h/106cells。活性分析结果表明,表达的rhBMP-2具有很强的生物学活性。     

Osteoclast migration on phosphorylated osteopontin is regulated by endogenous tartrate-resistant acid phosphatase

Osteopontin (OPN) is a multifunctional protein implicated in cellular adhesion and migration. Phosphorylation has emerged as a post-translational modification important for certain biological activities of OPN. This study demonstrates that adhesion of isolated neonatal rat osteoclasts in vitro was augmented on bovine milk osteopontin (bmOPN) with post-translational modifications (PTMs) compared to human Escherichia-coli-derived recombinant OPN (hrOPN) without PTMs. The difference in adhesiveness between these OPN variants was more pronounced at low coating concentrations (≤ 10 μg/ml). Both OPN forms adhered exclusively using a β₃-integrin. Partial (≤50%) dephosphorylation by tartrate-resistant acid phosphatase (TRAP) in vitro reduced osteoclast attachment to bmOPN to the same level as to hrOPN, demonstrating the importance of specific phosphorylations in OPN-dependent osteoclast adhesion.The involvement of PTMs of OPN in migration of primary rat and mouse osteoclasts was assessed on culture dishes coated with the different OPN forms and then overlaid with gold particles. Here, osteoclasts exhibited haptotactic migration on bmOPN but did not migrate on hrOPN. The presence of neutralizing antibodies to TRAP inhibited migration on bmOPN. Moreover, migration of osteoclasts isolated from TRAP-overexpressing transgenic mice was augmented on bmOPN, but not on hrOPN or type I collagen.These data collectively provide evidence in favor of a role for endogenous TRAP in regulating osteoclast migration on post-translationally modified OPN. In a tissue context, modulation of the phosphorylation level of OPN by extracellular phosphatases, e.g., TRAP, could regulate the extent of degradation such as depth and area at each bone resorption site by triggering osteoclast detachment and facilitate subsequent migration on the bone surface.     

Poly(vinyl chloride) tubing with covalently bound alkaline phosphatase and alternative approach for investigations of open-tubular bioreactors

A one-step carbodiimide method was found to allow covalent binding of enzymes to the inner wall of poly(vinyl chloride) (PVC) tubing. The immobilization is performed under mild conditions without laborious pretreatment or activation of reactor surface. In these preliminary studies, alkaline phosphatase (ALP, EC 3.1.3.1) and p-nitrophenyl phosphate (NPP) were applied as a model enzyme and substrate, respectively. The resulting open-tubular bioreactor exhibits satisfactory operational and storage stability. In addition, a novel and very simple instrumental concept for optical monitoring of the biocatalytic process directly inside the microbioreactor using a system of paired emitter-detector diodes is presented.     

Nitrite toxicity in Indian major carps: sublethal effect on selected enzymes in fingerlings of Catla catla, Labeo rohita and Cirrhinus mrigala

The effects of 96-h sublethal exposure of nitrite (1, 2, 4, 8 and 10.4 mg l(-1)) on selected enzymatic activities in serum and tissues of fingerlings of catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) were studied for the first time in these species. All three species responded almost identically to nitrite exposure. With increasing nitrite concentration, reduction in activities was observed in acetylcholinesterase (AChE) in brain and liver; alkaline phosphatase (ALP) in serum, brain and gill; and acid phosphatase (ACP) in gill, while progressive increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities in brain, gill and serum, and ACP activity in serum and brain was observed. Lactate dehydrogenase (LDH) activity increased in gill, liver, kidney, brain and serum of all three species with increasing nitrite concentration up to 8 mg l(-1) followed by reduction at 10.4 mg l(-1). The study revealed nitrite stress causing alteration in activities of all measured tissue and serum enzymes in the fingerlings, and so stresses the need for proper management of this particular nutrient in water during carp culture.     

植酸钠对菌根真菌根内菌丝碱性磷酸酶活性及根外菌丝生长的影响

采用三室隔网培养装置,以玉米为宿主植物,接种丛枝菌根真菌(AM)(Glomus intraradices),研究了不同用量的植酸钠对AM真菌生长和代谢活性的影响．研究发现,接种AM真菌的植株地上部和根系的P浓度和吸P量,比非菌根植物的提高了1～2倍．外源植酸钠的存在,显著降低了AM真菌根内菌丝的碱性磷酸酶活性,增加了AM真菌在土壤中的菌丝密度．结果表明,外源植酸钠对根内AM真菌碱性磷酸酶活性和真菌根外菌丝的生长具有调控(增减)作用,并且AM真菌提高了植物对土壤固有养分和外源植酸钠中P的吸收和利用．     

Nephronectin promotes osteoblast differentiation via the epidermal growth factor-like repeats

We found that nephronectin was significantly down-regulated by TGF-β1. To determine the function of nephronectin in osteogenesis, we generated various constructs to produce stable MC3T3-E1 cell lines, expressing and secreting nephronectin protein, including full-length (Npnt), lacking EGF-like repeats (Np-MAM), and lacking RGD and MAM domains (Np-EGF). We demonstrated that nephronectin promotes differentiation during osteoblast differentiation and the EGF-like repeats were essential. Lack of these repeats resulted in inhibiting the change in morphology. Over-expression of nephronectin results in earlier formation of bone nodules than the vector control. ERK activation is essential for nephronectin-induced osteoblast differentiation.     

Understanding the local actions of lipids in bone physiology

The adult skeleton is a metabolically active organ system that undergoes continuous remodeling to remove old and/or stressed bone (resorption) and replace it with new bone (formation) in order to maintain a constant bone mass and preserve bone strength from micro-damage accumulation. In that remodeling process, cellular balances – adipocytogenesis/osteoblastogenesis and osteoblastogenesis/osteoclastogenesis – are critical and tightly controlled by many factors, including lipids as discussed in the present review.Interest in the bone lipid area has increased as a result of in vivo evidences indicating a reciprocal relationship between bone mass and marrow adiposity. Lipids in bones are usually assumed to be present only in the bone marrow. However, the mineralized bone tissue itself also contains small amounts of lipids which might play an important role in bone physiology. Fatty acids, cholesterol, phospholipids and several endogenous metabolites (i.e., prostaglandins, oxysterols) have been purported to act on bone cell survival and functions, the bone mineralization process, and critical signaling pathways. Thus, they can be regarded as regulatory molecules important in bone health. Recently, several specific lipids derived from membrane phospholipids (i.e., sphingosine-1-phosphate, lysophosphatidic acid and different fatty acid amides) have emerged as important mediators in bone physiology and the number of such molecules will probably increase in the near future. The present paper reviews the current knowledge about: (1°) bone lipid composition in both bone marrow and mineralized tissue compartments, and (2°) local actions of lipids on bone physiology in relation to their metabolism. Understanding the roles of lipids in bone is essential to knowing how an imbalance in their signaling pathways might contribute to bone pathologies, such as osteoporosis.     

Implication of intestinal VDR deficiency in inflammatory bowel disease

Background

To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice.

Methods

Vdr^ΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics.

Results

Vdr^ΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr^ΔIEpC mice.

Conclusions

Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD.General significanceVDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.