The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Simplified Aeroelastic Model for Fluid Structure Interaction between Microcantilever Sensors and Fluid Surroundings

Fluid-structural coupling occurs when microcantilever sensors vibrate in a fluid. Due to the complexity of the mechanical characteristics of microcantilevers and lack of high-precision microscopic mechanical testing instruments, effective methods for studying the fluid-structural coupling of microcantilevers are lacking, especially for non-rectangular microcantilevers. Here, we report fluid-structure interactions (FSI) of the cable-membrane structure via a macroscopic study. The simplified aeroelastic model was introduced into the microscopic field to establish a fluid-structure coupling vibration model for microcantilever sensors. We used the finite element method to solve the coupled FSI system. Based on the simplified aeroelastic model, simulation analysis of the effects of the air environment on the vibration of the commonly used rectangular microcantilever was also performed. The obtained results are consistent with the literature. The proposed model can also be applied to the auxiliary design of rectangular and non-rectangular sensors used in fluid environments.     

eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.     

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

The influence of stomach distention on feeding in the nudibranch mollusk Melibe leonina

Although research on satiation has revealed much about the effect of sensory inputs on motivational state, we have yet to fully understand exactly how satiating signals influence the neural circuits underlying specific behaviors. One organism that is well suited for addressing this question is the nudibranch Melibe leonina, because its feeding activity is easily quantified, it has translucent skin that makes the stomach easy to observe, and it has large, identifiable neurons that are very suitable for subsequent analysis of the neural correlates of satiation. In this study our goal was to document the time course of satiation in Melibe, and determine if stomach distention contributes to satiation. When exposed to brine shrimp (Artemia), Melibe immediately commenced stereotypic oral hood movements to capture prey, and continued to do so for approximately five hours. Individuals eventually stopped, despite the continued presence of food, and the slowing and eventual termination of oral hood closures was correlated with distension of the stomach caused by the ingested Artemia. We obtained further evidence that stomach distension is one of the underlying causes of satiation by injecting artificial non-nutritive food into the stomach, and by cutting open part of the stomach wall to prevent it from filling and distending. The first treatment caused satiation to occur more rapidly, while the second treatment delayed satiation. Both results demonstrate that in Melibe stomach distention has a major impact on the motivation to feed. These findings provide the framework for subsequent studies designed to determine precisely how stomach distention influences feeding circuits.