1,25（OH）_2D_3缺失对膜内和软骨内成骨影响的比较研究

目的：比较1,25（OH）2D3缺失对膜内成骨和软骨内成骨影响的不同。方法：用免疫组织化学染色、HE染色和Western-blot等方法检测6周龄的野生型（wild type,WT）和1琢（OH）ase-/-小鼠的颅骨和股骨干骺端骨组织中I型胶原和甲状旁腺素受体（parathyroid hormone receptor,PTHR）的表达水平。结果：和WT小鼠相比较,1 ase-/-小鼠颅骨的I型胶原阳性面积明显减少,但是干骺端I型胶原阳性面积明显增加,差异有显著性（P〈0.01）;1琢（OH）ase-/-小鼠颅骨成骨细胞计数明显减少,差异存在统计学意义（P〈0.05）;但是干骺端成骨细胞计数明显增加,差异有显著性（P〈0.01）;1琢（OH）ase-/-小鼠颅骨的PHTR表达水平明显减少,但是在干骺端PHTR表达水平明显增加,差异均有显著性（P〈0.01）。结论：1,25（OH）2D3缺乏导致小鼠膜内成骨方式骨形成减少,而软骨内成骨骨形成增加。     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

细小炭角菌深层发酵产物的抗氧化活性研究

The antioxidant potency of components from Xylaria gracillima in submerged culture was investigated, employing various established in vitro systems, such as superoxide (O2?-) and hydroxyl (?OH) radical scavenging, reducing power, and ferrous ion chelating ability. Tocopherol (Ve), butylated hydroxytoluene (BHT) and ethylenediaminetetraacetic acid (EDTA) were used as positive controls. According to the results, components from X. gracillima in submerged culture showed significant effect on ferrous ion chelating ability, O2?- and ?OH radical scavenging ability at the range of concentration tested, and their highest antioxidant activities reached 89.72%, 70.90% and 77.46% respectively. The components also showed positive results of reducing power. These in vitro results suggested the possibility that components from X. gracillima in submerged culture could be effectively employed as an ingredient in healthy or functional food.     

羟自由基对心肌线粒体膜的影响及硒的效应

由Ｈ２Ｏ２和ＦｅＳＯ４体系产生的羟自由基（ＯＨ·）作用于大鼠心肌线粒体后,其膜脂双层内产生了脂类自由基。在观测时间内,其脂类自由基与自旋捕捉剂形成的自旋加合物的电子自旋共振（ＥＳＲ）信号强度随着孵育时间的延长而加强。一定浓度的硒代蛋氨酸（Ｓｅ—Ｍｅｔ）或Ｎａ２ＳｅＯ３可明显清除ＯＨ·并抑制脂类自由基的产生。在ＯＨ·的影响下,荧光探针ＤＰＨ在心肌线体膜脂中的荧光寿命和膜脂流动性的测试结果发生了明显变化。与此同时,心肌线粒体膜的能量转换过程（氧化磷酸化效率和呼吸控制率）也发生了显著的改变。１．０μｍｏｌ／Ｌ的Ｓｅ—Ｍｅｔ或２．３μｍｏｌ／Ｌ的Ｎａ２ＳｅＯ３可明显拮抗ＯＨ·的上述影响。前者的作用更为显著。     

肥胖儿童血清25- 羟维生素D 水平的研究

目的:探讨肥胖儿童血清25-(OH)D的水平,为临床评估维生素D营养状况与儿童肥胖症关系的研究提供参考。方法:将2009年5月至10月收治的儿童依据体重指数分成3组,抽取空腹静脉血检测血清中25-(OH)D水平,并进行组间比较。结果:通过分析25-(OH)D水平,肥胖组婴儿指标显著低于正常儿和瘦婴儿,统计学分析差异有显著性意义。结论:肥胖婴儿较正常儿和瘦婴儿25-(OH)D水平低,更易患佝偻病。提示防治佝偻病,对肥胖婴儿尤应重视,更要补充维生素D。     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

A multinuclear nmr relaxation study of the interaction of divalent metal ions with l-aspartic acid

Carbon-13 spin-lattice relaxation times, T₁, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu²⁺ and Mn²⁺, and Mg²⁺ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagaetically-induced relaxation. These results indicate that the Cu²⁺ interaction with L-aspartic acid is through α-amino and β-carboxyl groups while Mn²⁺ coordinates most strongly through α-and β-carboxyl groups, with the possibility of a weak interaction through the amino group.An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the β-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu²⁺ coordination is now probably through the α-amino and phosphate groups, while this analog is a monodentate ligand for Mn²⁺ coordinating through the phosphate group. Removal of the β-carboxyl group (L-alanine) also results in Cu²⁺ coordination through the α-carboxyl and α-amino groups, and the same ligand interactions are observed with Mn²⁺. Replacement of the α-amino group of L-aspartic acid with an - SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu²⁺ or Mn²⁺.     

Ethylenediaminetetraacetic Acid in Ammonium Hydroxide for Reducing Decalcification Time

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.