MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

蜂花粉抗脑衰老的实验动物研究

蜂花粉抗脑衰老的实验动物研究蒋滢,杨炳华,黄美英苏州医学院生化教研室苏州２１５００７２探索衰老机制,寻求延缓衰老的有效途径是生命科学中的重大问题,也是亟待解决的实际问题。脑是指挥全身一切活动的中枢,脑组织特别容易遭受自由基及活性氧的损伤,因此防治脑衰...     

GST-His-Heparinase Ⅰ双标签融合蛋白的原核表达与纯化

以pET15b-HepⅠ为模板,通过PCR技术扩增出上游合有6×His标签的HepⅠ基因序列,克隆至表达载体pGEX-4T-1。测序鉴定后,将重组表达质粒pGEX-His-HepⅠ转入E.coli BL21(DE3)感受态细菌,经IPTG诱导表达。表达产物可溶部分用GSTrap FF和HisTrap HP柱两步亲和纯化,所得产物经SDS-PAGE检测,在66 kDa和43 kDa处显示特异条带,分别与GST-His-HepⅠ和His-HepⅠ融合蛋白预期分子量相符;最终His-HepⅠ融合蛋白的比酶活为86.45 IU/mg,纯度高达99%,与仅一步亲和纯化得到的GST-His-HepⅠ融合蛋白相比,进一步提高了纯化后重组肝素酶的纯度。本研究为制备高纯度的HepⅠ提供了一种方法,对制备高安全性的LMwH和解析HepⅠ晶体结构具有重要意义。     

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.     

猫初级视皮层内GIu/GABA表达比率的衰老性变化

最近的一些研究结果显示,视皮层内抑制性递质系统作用减弱可能是导致老年性视觉功能衰退的重要因素.是否皮层内兴奋性递质系统亦伴随衰老而发牛改变并影响皮层内神经兴奋与抑制的平衡尚不清楚.为此,利用Nissl染色和免疫组织化学染色方法以及Image-Pro Express图像分析软件对青、老年猫初级视皮层(17区)内各层神经元密度、兴奋性递质谷氨酸免疫反应阳性(Glu-immunoreactive,Glu-IR)神经元密度以及抑制性递质γ-氨基丁酸免疫反应阳性(γ-aminobutyric acid.immunoreactive,GABA-IR)神经元密度进行了统汁分析.结果显示,青、老年猫初级视皮层各层神经元密度均没有明显的年龄性差异(P>0.05);与青年猫相比,老年猫初级视皮层Glu-IR、GABA-IR神经元密度均显著减少(P<0.01),而Glu.IR/GABA.IR神经元密度比率去却显著增大(P<0.01).结果提示,老年猫初级视皮层内兴奋性递质系统作用相对增强,而抑制性递质系统的作用相对减弱,导致皮层内兴奋-抑制平衡关系失调,这可能是引起老年个体视觉功能衰退的重要原因之一.