Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function

Osteogenesis requires close co-operation with angiogenesis to create vascularized bone tissue. In this study, an indirect co-culture model using osteoblasts (OBs), primary endothelial cells (ECs) and Matrigel interlayer was established to understand the impact of each cell type on the other. ECs synergistically enhanced osteoblastic gene expression by OBs, while OBs were capable of supporting tubule-like structures formed by ECs on Matrigel, enhancing mean tubule length from 146.5 ± 23.5 μm in ECs alone to 192 ± 28.6 μm in co-culture (p < 0.05). Similar improvements were noted in terms of tubule number. An applicability study of the co-culture model to bone tissue engineering, performed on a biopolymer fibrous membrane, showed substantially enhanced deposition of calcified nodules. These results demonstrate the efficacy of co-culture with ECs to improve osteogenesis for bone tissue engineering.     

Molecular network and functional implications of macromolecular tRNA synthetase complex

Understanding the complex network and multi-functionality of proteins is one of the main objectives of post-genome research. Aminoacyl-tRNA synthetases (ARSs) are the family of enzymes that are essential for cellular protein synthesis and viability that catalyze the attachment of specific amino acids to their cognate tRNAs. However, a lot of evidence has shown that these enzymes are multi-functional proteins that are involved in diverse cellular processes, such as tRNA processing, RNA splicing and trafficking, rRNA synthesis, apoptosis, angiogenesis, and inflammation. In addition, mammalian ARSs form a macromolecular complex with three auxiliary factors or with the elongation factor complex. Although the functional meaning and physiological significance of these complexes are poorly understood, recent data on the molecular interactions among the components for the multi-ARS complex are beginning to provide insights into the structural organization and cellular functions. In this review, the molecular mechanism for the assembly and functional implications of the multi-ARS complex will be discussed.     

减毒活疫苗中逆转录酶活性检测

逆转录酶 (RT)是目前发现的所有逆转录病毒的一个重要标志 ,凡是逆转录病毒均携带逆转录酶 ,逆转录酶活性的测定可以反映出制品中是否有逆转录病毒的污染 ,不受病毒基因序列的影响。采用PCR法进行逆转录酶活性检测方法 ,以整个生活周期中无DNA过程的MS2RNA为模板 ,设计一对引物 ,在逆转录系统中加入待检样品 ,经过RT PCR扩增后 ,放大检测对象物进行观察 ,从而了解制品中是否存在逆转录酶的污染 ,继而间接推测其是否污染逆转录病毒。对不同生物制品厂家生产的减毒活疫苗的逆转录酶活性进行检测 ,发现在一些疫苗里有逆转录酶活性阳性。     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

A neutralizing monoclonal antibody specific for the dimer interface region of IL-8

We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.     

Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca^2 + concentration. The oligomerization of STIM1, which triggers extracellular Ca^2 + influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca^2 + concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca^2 + concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca^2 + concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca^2 + influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca^2 + loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca^2 +-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.