尕海湿地草甸土退化过程土壤氮矿化演变特征

氮矿化是生态系统循环的重要环节之一,影响着生态系统功能和氮素生物地球化学循环,因此研究高寒湿地退化过程中土壤氮矿化演变特征,对揭示气候变化和人为活动干扰背景下的湿地土壤氮素循环过程具有重要意义。以尕海湿地4种不同退化梯度(未退化、轻度退化、中度退化、重度退化)土壤为研究对象,采用野外树脂芯原位培养方法,通过对植物生长季不同生长阶段(生长初期、生长盛期、枯萎期)土壤氮素矿化作用研究,分析湿地退化演替过程中土壤氮矿化时空变化特征及其与土壤环境因子和酶活性之间的关系。结果表明：尕海湿地退化对土壤氮矿化过程有显著抑制作用,与未退化(0.143 mg kg^-1 d^-1)相比,轻度退化、中度退化、重度退化的土壤净氮矿化速率分别减小了0.018 mg kg^-1 d^-1、0.025 mg kg^-1 d^-1、0.020 mg kg^-1 d^-1;随着退化程度加剧,土壤净氨化速率逐渐减小或者不变,而净硝化速率却增大。随时间推移,各退化...     

ICAM-1-mediated osteoblast-T lymphocyte direct interaction increases mineralization through TGF-β1 suppression

Modulation of osteoblast functions by T lymphocytes is important in inflammation-associated mineralized tissue diseases. The study aimed to determine whether direct interaction between these two cell types affects osteoblast functions and mineralization. The results showed that direct contact between the two cell types was evident by scanning electron microscopy and transmission electron microscopy. Under osteogenic induction, higher hydroxyapatite precipitation was observed in cocultures with direct contact with T lymphocytes compared with that by osteoblasts cultured alone. Cocultures without direct cell contact caused a decrease in mineralization. Direct cell contact also upregulated intercellular adhesion molecule (ICAM)-1 and simultaneously downregulated transforming growth factor (TGF)-β1 in osteoblasts. However, the downregulation of TGF-β1 was reversed by ICAM-1 blocking. Exogenously added TGF-β1 in cocultures with direct cell contact suppressed mineralization. In conclusion, studies are consistent with ICAM-1-mediated direct contact between osteoblasts and T lymphocytes increasing mineralization via downregulation of TGF-β1 in osteoblasts in vitro. This suggests a possible unexpected, but crucial, role of T lymphocytes in enhancing matrix mineralization during the repair process in vivo. The study identifies ICAM-1/TGF-β1 as possible novel therapeutic targets for the treatment and prevention of inflammation-associated mineralized tissue diseases.     

Phosphodiesterase activity of alkaline phosphatase in ATP-initiated Ca(2+) and phosphate deposition in isolated chicken matrix vesicles

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.     

腾格里沙漠东南缘不同类型生物土壤结皮对土壤有机碳矿化的影响

生物土壤结皮(BSCs)是荒漠生态系统的重要组成部分,是该区土壤碳循环及碳平衡的关键影响因素。研究了腾格里沙漠东南缘不同类型生物土壤结皮覆盖下土壤碳矿化过程及其对温度(10℃、25℃和35℃)和水分(土壤含水量10%和25%)变化响应特征,分析了土壤碳矿化过程与土壤理化性质的关系。结果表明:(1)结皮的形成和发育显著影响土壤有机碳矿化过程,藻类、地衣和藓类结皮覆盖的土壤碳矿化速率和CO_2-C累积释放量均显著高于去除结皮的土壤,不同类型BSCs覆盖土壤和去除结皮土壤之间均表现为藓类结皮土壤地衣结皮土壤藻类结皮。(2)含结皮层土壤的平均和最大矿化速率均随温度升高和水分增加而逐渐增大,有结皮覆盖的土壤和去除结皮的土壤对温度和水分变化的响应规律相同。(3)有结皮土壤和去除结皮土壤碳矿化速率的温度敏感性(Q_(10))与结皮类型密切相关,均表现为藓类结皮地衣结皮藻类结皮。结果表明生物土壤结皮由以藻类为主向以藓类为主的演变进一步促进了土壤碳矿化过程,结皮对土壤碳循环的调控作用受水热等环境因子的共同影响。     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.     

Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.     

Rietveld refinement on x-ray diffraction patterns of bioapatite in human fetal bones

Bioapatite, the main constituent of mineralized tissue in mammalian bones, is a calcium-phosphate-based mineral that is similar in structure and composition to hydroxyapatite. In this work, the crystallographic structure of bioapatite in human fetuses was investigated by synchrotron radiation x-ray diffraction (XRD) and microdiffraction ( micro -XRD) techniques. Rietveld refinement analyses of XRD and micro -XRD data allow for quantitative probing of the structural modifications of bioapatite as functions of the mineralization process and gestational age.     

Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system

Chick limb‐bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non‐mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K‐cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT‐PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co‐transporter, NaPi IIb, matrix metalloprotease 13. MMP‐13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. J. Cell. Biochem. 112: 607–613, 2011. © 2010 Wiley‐Liss, Inc.     

Limitations of a calculated N mineralization potential in studies of the N mineralization process

Soil is often incubated under controlled conditions to assess its capacity to mineralize nitrogen and to define the N mineralization potential (N_o) by fitting a negative exponential curve to N mineralization data. The specificity of N_o for a given soil and its relevance in N mineralization studies was examined as part of an overall study of the N mineralization process. Soil mixed with an equal amount of sand was aerobically incubated at 35 °C and leached at specific time intervals. Upon leaching, ammonium and nitrate were measured in the extract.It was found that N mineralization data did not always follow first-order kinetics making it difficult to calculate N_o. The computed N_o value was influenced by the shape of the curve, the duration of the incubation experiment and was reciprocally related to the N mineralization constant (k_exp). N_o did not always give an adequate indication of the amount of N mineralized and was not soil specific as the time of sampling largely affected its size. The usefulness of N_o in the simulation of the N mineralization process with a k_exp value valid for all soils was limited and a k_exp value specific for each soil was required. A value combining the soil specific N_o and k_exp values and reflecting the amount of N mineralized over one year was proposed as a suitable alternative to the use of N_o in comparative studies of the N mineralization process. It was concluded that a calculated N_o could not be used in studies comparing the N mineralization of different soils. In addition, the simulation of the N mineralization required the use of the soil specific k_exp and could not be carried out with a k_exp valid for all soils.     

Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.     

Pre-adsorbed type-I collagen structure-dependent changes in osteoblastic phenotype

Type-I collagen is the most abundant extracellular matrix in bones and modulates various functions of osteoblasts. We prepared two different structures of type-I collagen on tissue culture grade polystylene (TCPS) surfaces, one is feltwork structure of filamentous molecules from acid solutions (ACs) and the other is network structure of fibrils from neutral solutions (NCs), to examine effects of the structures on the maturation process of osteoblast-like cells. No significant differences of cell proliferation were observed between TCPS and ACs, but NCs delayed the proliferation. In initial cell attachment, the cells on ACs had tense lamellipodia with sharp tips, while those on NCs had loose lamellipodia. No detectable differences in levels of expressed integrin alpha2- and alpha5-subunits were observed between the structures. Although the matrix mineralization in NCs was also delayed in comparison with TCPS and ACs, fully mineralized levels in NCs were the same as those of TCPS and ACs. In addition, although we examined the effects of densities of pre-adsorbed collagen molecules on osteoblast maturation, the effects were less serious than those of the structures. This study suggests that the structures of collagen affect proliferation and mineralization of osteoblast-like cells.     

Chondrocyte-derived transglutaminase promotes maturation of preosteoblasts in periosteal bone

During endochondral development, elongation of the bone collar occurs coordinately with growth of the underlying cartilaginous growth plate. Transglutaminases (TGases) are upregulated in hypertrophic chondrocytes, and correlative evidence suggests a relationship between these enzymes and mineralization. To examine whether TGases are involved in regulating mineralization/osteogenesis during bone development, we devised a coculture system in which one cellular component (characterized as preosteoblastic) is derived from the nonmineralized region of the bone, and the other cellular component is hypertrophic chondrocytes. In these cocultures, mineralization is extensive, with the preosteoblasts producing the mineralized matrix, and the chondrocytes regulating this process. Secreted regulators are involved, as conditioned medium from chondrocytes induces mineralization in preosteoblasts, but not vice versa. One factor is TGase. In the cocultures, inhibition of TGase reduces mineralization, and addition of the enzyme enhances it. Exogenous TGase also induces markers of osteoblastic differentiation (i.e., bone sialoprotein and osteocalcin) in the preosteoblasts, suggesting their differentiation into osteoblasts. Two possible signaling pathways may be affected by TGase and result in increased mineralization (i.e., TGF-beta and protein kinase A pathways). Addition of exogenous TGF-beta2 to the cocultures increases mineralization; though, when mineralization is induced by TGase, there is no detectible elevation of TGF-beta, suggesting that these two factors stimulate osteogenesis by different pathways. However, an interrelationship seems to exist between TGase and PKA-dependent signaling. When mineralization of the cocultures is stimulated through the addition of TGase, a concomitant reduction (50%) in PKA activity occurs. Consistent with this observation, addition of an activator of PKA (cyclic AMP) to the cultures inhibits matrix mineralization, while known inhibitors of PKA (H-89 and a peptide inhibitor) cause an increase in mineralization. Thus, at least one mechanism of TGase stimulation probably involves inhibition of the PKA-mediated signaling.     

Rates of net nitrogen mineralization in disturbed and undisturbed soils

Quantification of net nitrogen mineralization (NNM) in soils is indispensable in order to optimize N fertilization of crops. Two long-term laboratory incubation methods were applied to determine rates of net nitrogen mineralization (r_NNM) of soils from two sites of arable land (sandy loam soil, silty loam soil) at four temperature levels (2°C, 8°C, 14°C, 21°C). Since variability within replicates was small, the modified 12-week incubation method of Stanford and Smith (1972) using disturbed soils allowed to establish reliable Arrhenius functions with reasonable expenditure. The fit of the functions derived from the 5-month incubation of 23 undisturbed soil columns (4420 cm³) was worse. This was caused by greater variability and less differentiation between temperature levels. Results of both experiments could be described best by zero-order kinetics. Mean mineralization rates of disturbed samples were approximately twice as high than those of undisturbed samples. The suitability of both methods for the prediction of NNM at site conditions is discussed. Actual respiration (AR) at incubation temperatures and substrate induced respiration (SIR) were measured at the end of the incubation of undisturbed soil columns. The results presented reveal that soil microbial communities develop in a different manner during long-term incubation at different temperatures. This behavior offends the underlying assumption that soil microbes remain in steady-state during incubation and that rising rates are physiological reactions to temperature enhancement. Therefore soil microbial biomass (SMB) dynamics during the experiment has to be accounted for when rates of NNM and Arrhenius functions are established. R Merck Section editor     

Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice

X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice.     

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.     

Long-term effects of tripeptide Ile-Pro-Pro on osteoblast differentiation in vitro

Bone mineralization is a result of the function of bone-forming osteoblasts. Osteoblast differentiation from their precursors is a carefully controlled process that is affected by many signaling molecules. Protein-rich food-derived bioactive peptides are reported to express a variety of functions in vivo. We studied the long-term in vitro effect of bioactive tripeptide Ile-Pro-Pro (IPP) on osteoblasts differentiated from human mesenchymal stem cells. Osteoblast bone alkaline phosphatase activity (bALP), bone-forming capacity and gene expression were investigated. Treatment with 50 microM IPP had no effect on bALP activity, but osteoblast mineralization was increased. Gene expression of beta-catenin, Cbfa1/Runx2, PTHrP, CREB-5, osteoglycin, osteocalcin, caspase-8, osteoprotegerin (OPG) and RANKL was analyzed by quantitative real-time PCR on Days 13, 17 and 20 of culture. The results indicate that IPP increased mineral formation due to enhanced cell survival and matrix formation. In addition, IPP reduced the RANKL/OPG ratio. Bioactive peptides, such as IPP, could be one method by which a protein-rich diet promotes bone integrity.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

土壤氮素矿化的生态模型研究

土壤氮素矿化是反映土壤供氮能力的重要因素之一,也是目前国内外土壤生态学研究热点之一。土壤氮素矿化可表示为土壤有机氮的含量、矿化过程中土壤环境因素和矿化持续时间的复合函数;土壤氮素矿化的基本模型有3类,即动力学模型、热力学模型及经验模型;影响矿化过程并可定量表示的土壤环境条件如土壤温度、湿度、pH及有机质形态。     

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.