Distribution of Small Integrin-binding Ligand, N-linked Glycoproteins (SIBLING) in the Articular Cartilage of the Rat Femoral Head

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis. (J Histochem Cytochem 58:1033–1043, 2010)     

Synthesis, structure and property of manganese(II) complexes with mixed tetradentate imidazole-containing ligand and benzenedicarboxylate

Three new coordination complexes [Mn(L)(H₂O)₂](1,4-BDC)·2H₂O (1), [Mn(L)_0.5(1,4-BDC)]CH₃OH·H₂O (2) and [Mn(L)(H₂O)₂](1,2-HBDC)₂·2H₂O (3) were synthesized by solvothermal reactions of 1,2,4,5-tetrakis(imidazol-1-ylmethyl)benzene (L) and 1,4-benzenedicarboxylic acid (1,4-H₂BDC) or 1,2-benzenedicarboxylic acid (1,2-H₂BDC) with Mn(II) salt, and characterized by single crystal X-ray diffraction, IR, thermogravimetric and elemental analyses. In complexes 1 and 3, each ligand L links four Mn(II) atoms to form two-dimensional (2D) cationic network with non-coordinated 1,4-BDC²⁻ and 1,2-HBDC⁻ anions lying in the voids between the two adjacent layers, respectively. The 2D layers are further connected together by hydrogen bonds to give three-dimensional (3D) supramolecular structures. However, the 1,4-BDC²⁻ in 2 acts not only as counteranion, but also as bridging ligand leading to the formation of 2-fold interpenetrated 3D framework with pcu (primitive cubic unit) topology. The Mn(II) atoms bridged by carboxylate groups in 2 show antiferromagnetic interactions.     

Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis

Hyperlipidemia is a major cause of atherosclerosis and atherosclerosis-associated conditions in cardiovascular diseases. Oxidative stress, as a main risk factor causes vascular endothelial cell apoptosis, which is implicated in the pathogenesis of cardiovascular disorders. Diosgenin, an aglycone of steroidal saponins, has been reported to exert anti-proliferative and proapoptotic actions on cancer cells widely. In this study, we propose that diosgenin can protect the hyperlipidemic rats and prevent endothelial apoptosis under oxidative stress. We investigated the hypolipidemic and antioxidative effects of diosgenin on rats fed with high cholesterol and high fat diet for 6 weeks. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), glutathione peroxidase (GSH-PX), nitric oxide synthase (NOS), hepatic malondialdehyde (MDA), lipoprotein lipase (LPL), hepaticlipase (HL) and superoxide dismutase (SOD) activities were evaluated. Then we explored the effects and mechanism of diosgenin against hydrogen peroxide-induced apoptosis of human vein endothelium cells (HUVECs). Intracellular reactive oxygen species (ROS), glutathione (GSH), nitric oxide (NO), DNA fragment formation and mitochondrial membrane potentials (ΔΨm) were determined. Diosgenin treatment increased LPL, HL, SOD, GSH-PX and NOS activities, thus attenuated oxygen free radicals, decreased MDA, TC, TG and LDL-C levels in hyperlipidemic rats. Diosgenin pretreatment significantly attenuated H₂O₂-induced apoptosis in HUVECs, intracellular ROS, GSH depletion, DNA fragment formation, and restored NO, ΔΨm. These results suggested that diosgenin is a very useful compound to control hyperlipidemia by both improving the lipid profile and modulating oxidative stress and prevent H₂O₂-induced apoptosis of HUVECs, in partly through regulating mitochondrial dysfunction pathway.     

Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln¹² and Glu¹⁴ and ephrin-B2. Mutation of Gln¹² and Glu¹⁴ does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ∼10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln¹² and Glu¹⁴ with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.     

A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization

A gene encoding a Na(+)/H(+) antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8(T) by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43,481 Da, which was 54% identical and 76% similar to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with highest activities at pH 8.5-9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.     

巨胞饮的机制及功能的研究进展

巨胞饮（macropinocytosis）是内吞的一种形式,指在某些因素刺激下,细胞膜皱褶形成大且不规则的原始内吞小泡,它们被称为巨胞饮体。巨胞饮体的直径一般为0．5～2μm,有时可达5μm。与其它内吞形式的小泡相比,巨胞饮体直径较大,为非选择性地内吞细胞外营养物质和液相大分子提供了一条有效途径。最近的研究表明,巨胞饮具有清除凋亡细胞、参与免疫反应、介导某些病原菌侵袭细胞、更新细胞膜等功能。     

Early melatonin supplementation alleviates oxidative stress in a transgenic mouse model of Alzheimer's disease

Multiple lines of evidence demonstrated that increased brain oxidative stress is a key feature of Alzheimer's disease (AD). Melatonin is a potent endogenous antioxidant and free radical scavenger. A transgenic mouse model for AD mimics the accumulation of senile plaques, neuronal loss, and memory impairment. Four-month-old transgenic mice were administrated melatonin at 10 mg/kg for 4 months. We investigated the long-term influence of melatonin on these mice before amyloid plaques were deposited. We found an increase in the levels of brain thiobarbituric acid-reactive substances (TBARS) and a decrease in glutathione (GSH) content, as well as accelerated upregulation of the apoptotic-related factors, such as Bax, caspase-3, and prostate apoptosis response-4 (Par-4) in transgenic mice, but not in wild-type (WT) littermates. Significantly, the increase in TBARS levels, reduction in superoxide dismutase activity, and GSH content were reinstated by melatonin. In addition, transgenic mice administered melatonin (10 mg/kg) showed a significant reduction in upregulated expression of Bax, caspase-3 and Par-4, indicating inhibited triggering of neuronal apoptosis. These results supported the hypothesis that oxidative stress was an early event in AD pathogenesis and that antioxidant therapy may be beneficial only if given at this stage of the disease process. In sharp contrast to conventional antioxidants, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential therapeutic candidate in AD treatment.     

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.