RNAi Methodologies for the Functional Study of Signaling Molecules

RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules.     

植物多肽信号分子CLE家族

动物中存在众多多肽信号分子,它们在信号转导方面发挥重要作用。近几年,对植物中多肽信号分子的研究取得了重大突破,它们积极参与调控植物生长发育的众多过程,同时也表明多肽信号分子在细胞之间的"交流"过程中发挥作用在进化上是保守的。CLE(CLAVATA3/EMBRYO SURROUNDING REGION)家族是目前植物领域研究较热的多肽信号分子家族,通过对拟南芥CLV3和百日草TDIF等CLE多肽信号分子的研究发现,CLE蛋白在成为有功能活性的信号分子之前,存在翻译后蛋白剪切和修饰的过程,这方面与动物中多肽信使的成熟过程相似。对CLE家族成员的分子特征、生物学功能、翻译后的加工修饰和研究中出现的问题进行综述,并对本领域未来的发展方向作出展望。     

Wnt Signaling is Essential for Bone Regeneration

This article has no abstract.     

高效的骨诱导生长因子——成骨蛋白-1

成骨蛋白-1(OP-1)又称骨形态发生蛋白-7(BMP-7),属转化生长因子β(TGF-β)超家族成员.重组人OP-1(rhOP-1)在体内和体外都显示了高效的骨诱导活性,可使多种实验动物的骨缺损满意愈合,有良好的临床应用前景.     

NO-cGMP Signaling and Regenerative Medicine Involving Stem Cells

Nitric oxide (NO) is a short lived diatomic free radical species synthesized by nitric oxide synthases (NOS). The physiological roles of NO depend on its local concentrations as well as availability and the nature of downstream target molecules. At low nanomolar concentrations, activation of soluble guanylyl cyclase (sGC) is the major event initiated by NO. The resulting elevation in the intracellular cyclic GMP (cGMP) levels serves as signals for regulating diverse cellular and physiological processes. The participation of NO and cGMP in diverse physiological processes is made possible through cell type specific spatio-temporal regulation of NO and cGMP synthesis and signal diversity downstream of cGMP achieved through specific target selection. Thus cyclic GMP directly regulates the activities of its downstream effectors such as Protein Kinase G (PKG), Cyclic Nucleotide Gated channels (CNG) and Cyclic nucleotide phosphodiesterases, which in turn regulate the activities of a number of proteins that are involved in regulating diverse cellular and physiological processes. Localization and activity of the NO-cGMP signaling pathway components are regulated by G-protein coupled receptors, receptor and non receptor tyrosine kinases, phosphatases and other signaling molecules. NO also serves as a powerful paracrine factor. At micromolar concentrations, NO reacts with superoxide anion to form reactive peroxinitrite, thereby leading to the oxidation of important cellular proteins. Extensive research efforts over the past two decades have shown that NO is an important modulator of axon outgrowth and guidance, synaptic plasticity, neural precursor proliferation as well as neuronal survival. Excessive NO production as that evoked by inflammatory signals has been identified as one of the major causative reasons for the pathogenesis of a number of neurodegenerative diseases such as ALS, Alzheimers and Parkinson diseases. Regenerative therapies involving transplantation of embryonic stem cells (ES cells) and ES cell derived lineage committed neural precursor cells have recently shown promising results in animal models of Parkinson disease (PD). Recent studies from our laboratory have shown that a functional NO-cGMP signaling system is operative early during the differentiation of embryonic stem cells. The cell type specific, spatio-temporally regulated NO-cGMP signaling pathways are well suited for inductive signals to use them for important cell fate decision making and lineage commitment processes. We believe that manipulating the NO-cGMP signaling system will be an important tool for large scale generation of lineage committed precursor cells to be used for regenerative therapies. Special issue dedicated to John P. Blass.     

缺氧信号在骨中的核心作用

缺氧信号在维持氧稳态和细胞生存中起着至关重要的作用。在胚胎发育时期处于快速增殖的细胞和肿瘤组织中快速生长的细胞中都能观察到缺氧现象的存在。为了应对缺氧胁迫,生物有机体形成了一系列的调节机制。在多种调节途径中,缺氧诱导因子HIF-1和HIF-2是最主要的能够应答细胞内氧气浓度的降低而对多种基因进行调控的转录因子,与生物体的生长发育及一些疾病的发病机理都存在着密切关系。最近的研究发现在骨骼发育,骨骼的形成和再生,以及关节的形成和动态平衡的调节中HIF-1和HIF-2的具有重要作用。此外,HIF-1和HIF-2的过度表达在临床上与骨肉瘤和骨关节炎明显相关。总之,这些发现预示着缺氧的信号在骨骼的生物学及其疾病中起到中心调节的作用。     

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.     

Cell Interaction Molecules Utilized in Bone Marrow

Many aspects of blood cell formation can now be modeled in culture and rapid progress is being made in understanding how blood cell precursors interact with unique components of their environment. This brief review considers some cell interaction molecules that may be important for controlling the position of cells within, as well as their egress from, bone marrow.     

高效的骨诱导生长因子——成骨蛋白-1

成骨蛋白１（ＯＰ１）又称骨形态发生蛋白７（ＢＭＰ７）,属转化生长因子β（ＴＧＦβ）超家族成员．重组人ＯＰ１（ｒｈＯＰ１）在体内和体外都显示了高效的骨诱导活性,可使多种实验动物的骨缺损满意愈合,有良好的临床应用前景     

Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin''s ability to arrest protein synthesis in cells or on ricin''s enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins.     

Design and Evaluation of a Simple Signaling Device for Live Traps

Abstract: Frequent checks of live traps require enormous amounts of labor and add human scents associated with repeated monitoring, which may reduce capture efficiency. To reduce efforts and increase efficiency, we developed a trap-signaling device with long-distance reception, durability in adverse weather, and ease of transport, deployment, and use. Modifications from previous designs include a normally open magnetic switch and a mounting configuration to maximize reception. The system weighed <225 g, was effective ≤17.1 km, and failed in <1% of trap-nights. Employing this system, researchers and wildlife managers may reduce the amount of effort checking traps while improving the welfare of trapped animals.     

DNA 分子简谐特性的研究

考虑周期驱动力下的ＤＮＡ分子系统的福克普朗克方程。通过数值计算，研究期望值〈ｘ（ｔ）〉的简谐性。根据随机力强度对简谐特性的研究，我们得到温度有益于癌症治疗的结论。     

Intracellular Signaling Molecules Activated by Epstein-Barr Virus for Induction of Interferon Regulatory Factor 7

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is the principal oncogenic protein in the EBV transformation process. LMP-1 induces the expression of interferon regulatory factor 7 (IRF-7) and activates IRF-7 protein by phosphorylation and nuclear translocation. LMP-1 is an integral membrane protein with two regions in its C terminus that initiate signaling processes, the C-terminal activator regions 1 (CTAR-1) and CTAR-2. Here, genetic analysis of LMP-1 has determined that the PXQXT motif that governs the interaction between LMP-1 CTAR-1 and tumor necrosis factor receptor-associated factors (TRAFs) is needed to induce the expression of IRF-7. Mutations in the PXQXT motif in CTAR-1 that disrupt the interaction between LMP-1 and TRAFs abolished the induction of IRF-7. Also, dominant-negative mutants of TRAFs inhibited the induction of IRF-7 by CTAR-1. The last three amino acids (YYD) of CTAR-2 are also important for the induction of IRF-7. When both PXQXT and YYD were mutated (LMP-DM), the LMP-1 mutant failed to induce IRF-7. Also, LMP-DM blocked the induction of IRF-7 by wild-type LMP-1. These data strongly suggest that both CTAR-1 and CTAR-2 of LMP-1 independently induce the expression of IRF-7. In addition, NF-kappaB is involved in the induction of IRF-7. A superrepressor of IkappaB (sr-IkappaB) could block the induction of IRF-7 by LMP-1, and overexpression of NF-kappaB (p65 plus p50) could induce the expression of IRF-7. In addition, we have found that human IRF-7 is a stable protein, and sodium butyrate, a modifier of chromatin structure, induces IRF-7.     

Octadecanoid-derived Signaling Molecules Involved in Touch Perception in a Higher Plant

Recent evidence suggests that octadecanoid derived metabolites, notably jasmonic acid and/or methyl jasmonate, are involved in the reaction of touch-sensitive tendrils to mechanical stimulation.     

Wnt Signaling in Bone Development and Disease: Making Stronger Bone with Wnts

The skeleton as an organ is widely distributed throughout the entire vertebrate body. Wnt signaling has emerged to play major roles in almost all aspects of skeletal development and homeostasis. Because abnormal Wnt signaling causes various human skeletal diseases, Wnt signaling has become a focal point of intensive studies in skeletal development and disease. As a result, promising effective therapeutic agents for bone diseases are being developed by targeting the Wnt signaling pathway. Understanding the functional mechanisms of Wnt signaling in skeletal biology and diseases highlights how basic and clinical studies can stimulate each other to push a quick and productive advancement of the entire field. Here we review the current understanding of Wnt signaling in critical aspects of skeletal biology such as bone development, remodeling, mechanotransduction, and fracture healing. We took special efforts to place fundamentally important discoveries in the context of human skeletal diseases.The skeleton has many important functions related to human health. Aside from the classical functions of the skeleton in structural support and movement, the bone matrix forms a major reservoir of calcium and other inorganic ions, and bone cells are active regulators of calcium homeostasis. Recent data suggest that bone cells can secrete hormones (e.g., FGF23 and osteocalcin) and likely play a physiologically significant role in regulating phosphate and energy homeostasis. It has emerged that Wnt signaling plays a major role controlling multiple aspects of skeletal development and maintenance. Thus, understanding how the Wnt pathway controls skeletal growth and homeostasis has broad implications for human health and disease.Cartilage and bone define the skeleton and are produced by chondrocytes and osteoblasts, respectively. During embryonic development, bones are formed by two distinct processes: intramembranous and endochondral ossification (Fig. 1A). A number of cranial bones and the lateral portion of the clavicles are formed by intramembranous ossification. In this process, mesenchymal progenitor cells condense and differentiate directly into bone-forming osteoblasts. The majority of bones in our body are formed by endochondral ossification, during which mesenchymal progenitor cells condense and differentiate first into cartilage-forming chondrocytes to generate an avascular template of the future bone. Chondrocytes in these templates undergo a program of proliferation and progressive cellular maturation. Eventually, they exit the cell cycle and become pre-hypertrophic, then terminally differentiating into hypertrophic chondrocytes, which are eliminated ultimately by apoptosis. Hypertrophic chondrocytes produce a matrix that is calcified and functions as a scaffold for new bone formation. Concomitant with chondrocyte hypertrophy, osteoclasts, osteoblasts, and blood vessels migrate in from perichondral regions and remodel this template into bone.Open in a separate windowFigure 1.Mechanisms of skeleton formation. (A) Bones can form by either intramembranous or endochondral ossification. Both processes are initiated by the condensation of mesenchymal cells. During intramembranous ossification, mesenchymal cells differentiate directly into osteoblasts and deposit bone. During endochondral ossification, mesenchymal cells differentiate into chondrocytes and first make a cartilage intermediate. Chondrocytes in the center of the bone initiate a growth plate, stop proliferating, and undergo hypertrophy. Hypertrophic chondrocytes mineralize their matrix and undergo apoptosis, attracting blood vessels and osteoblasts that remodel the intermediate into bone. (B) The first histologic sign of synovial joint formation is the gathering and flattening of cells, forming the interzone. Cavitation occurs within the presumptive joint separating the two cartilaginous structures. Remodeling and maturation proceed to give rise to the mature synovial joint. Wnt signaling plays a significant role in controlling almost all aspects of skeleton formation. Osteoblasts (purple); chondrocytes (blue); osteochondroprogenitor cells (brown).The developing skeletal elements are often segmented to form joints, which are required to support mobility. Synovial joints, which allow movement via smooth articulation between bones, form when chondrogenic cells in a newly formed cartilage undergo a program of dedifferentiation and flattening to form an interzone (Fig. 1B). Cavitation occurs within the flattened cells, allowing physical separation of the skeletal elements, and the formation of the synovial cavity. Cells and tissues in and around the interzone are remodeled at the same time to form the articular cartilage and other joint structures. Failure to form or maintain joints leads to joint fusion or osteoarthritis, a major skeletal disease.Following its formation, bone remains a regenerative tissue and is maintained during postnatal life by continuous remodeling. This highly active, homeostatic process is required for its functions and is controlled by three cell types: osteoblasts on the bone surface that deposit new bone matrix; osteocytes embedded in bone that are terminally differentiated from osteoblasts and function as mechanical and metabolic sensors; and the matrix-resorbing osteoclasts (Fig. 2). Osteoblasts are derived from mesenchymal stem cells (MSCs), whereas osteoclasts differentiate from hematopoietic progenitors. Decreased bone mass may be due to reduced osteoblast function or elevated osteoclast activity, and, conversely, increased bone mass may result from increased osteoblast function or decreased osteoclast activity. The precise balance of formation and resorption is critical for maintaining normal bone mass, and alterations in this balance lead to common bone diseases such as osteoporosis and osteopetrosis.Open in a separate windowFigure 2.Anatomy of bone. Cortical and trabecular bone represent the two major forms of bone. Osteoblasts (dark purple) are present on the surface and form new bone. Osteocytes (brown) are terminally differentiated osteoblasts that have become embedded in bone and communicate information to one another and to cells on the surface to regulate bone homeostasis. Osteoclasts (blue) are of hematopoietic origin and catabolize bone. A major function of Wnt/β-catenin signaling in osteoblasts is to suppress RANKL and to promote OPG production, thereby inhibiting osteoclast formation.There are two major bone types, cortical and trabecular, which show different anatomical properties (Fig. 2). Cortical (or compact) bone is the solid, densely packed bone that forms the outer layer of most bones and gives strength and rigidity. Trabecular (or cancellous) bone is present mostly in the marrow cavities of long bones and is the dominant bone type in vertebral bodies. Trabecular bone forms a porous, cobweb-like network of trabeculae whose large surface area is thought to facilitate the metabolic activity of bones mediated by osteoblasts and osteoclasts. Trabeculae are sites of active remodeling and will often orient in the direction of mechanical loading, dissipating the energy of loading and adding to bone strength. It is trabecular bone, rather than cortical bone, which is most severely affected in osteoporosis.The Wnt/β-catenin pathway plays a major role in controlling skeletal development and homeostasis, which are the focus of this work. We focus not only on differentiation of skeletal cells and formation of skeletal tissues, but also on the role of the Wnt/β-catenin signaling pathway on bone homeostasis, mechanotransduction, and wound healing, paying particular attention to human and mouse studies.     

Quorum Sensing Signaling Molecules Involved in the Production of Violacein by Pseudoalteromonas

The production of violacein by Pseudoalteromonas sp. 520P1 has many features of quorum sensing. Signaling molecules were extracted from bacterial culture and subsequently identified as N-(3-oxooctanoyl)-homoserine lactone and N-tetradecanoyl-homoserine lactone. The former but not the latter induced the production of violacein in strain 520P1. We conclude that N-(3-oxooctanoyl)-homoserine lactone is a signaling molecule involved in the production of violacein.