Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.     

The properties of bioengineered chondrocyte sheets for cartilage regeneration

Background  

Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage.     

Tungstate-induced color-pattern modifications of butterfly wings are independent of stress response and ecdysteroid effect

Systemic injections of sodium tungstate, a protein-tyrosine phosphatase (PTPase) inhibitor, to pupae immediately after pupation have been shown to efficiently produce characteristic color-pattern modifications on the wings of many species of butterflies. Here we demonstrated that the tungstate-induced modification pattern was entirely different from other chemically-induced ones in a species of nymphalid butterfly Junonia (Precis) orithya. In this species, the systemic injections of tungstate produced characteristic expansion of black area and shrinkage of white area together with the move of parafocal elements toward the wing base. Overall, pattern boundaries became obscure. In contrast, an entirely different modification pattern, overall darkening of wings, was observed by the injections of stress-inducing chemicals, thapsigargin, ionomycin, or geldanamycin, to pupae under the rearing conditions for the adult summer form. On the ventral wings, this darkening was due to an increase of the proportion of peppered dark scales, which was reminiscent of the natural fall form of this species. Under the same rearing conditions, the injections of ecdysteroid, which is a well-known hormone being responsible for the seasonal polyphenism of nymphalid butterflies, yielded overall expansion of orange area especially around eyespots. Taken together, we conclude that the tungstate-induced modifications are clearly distinguishable from those of stress response and ecdysteroid effect. This conclusion then suggests that the putative PTPase signaling pathway that is sensitive to tungstate uniquely contributes to the wing-wide color-pattern development in butterflies.     

Induction of an endogenous tumor necrosis factor in mice by murine recombinant interferon-γ combined with a lipid A subunit analog (GLA-60) of low toxicity

Summary We have investigated the endogenous production of a serum cytotoxic factor when recombinant interferon- (rIFN-) is combined with synthetic lipid A subunit analogs of low toxicity (GLA compounds). The cytotoxic activity of the serum was measured by the crystal violet staining method with L929 cells as a target. Intravenous administration of rIFN- followed by intravenous administration of lipopolysaccharide induced the endogenous production of a cytotoxic factor in the serum. The priming effect of rIFN- appeared immediately and persisted for approximately 20 h after the injection. Administration of lipopolysaccharide as a trigger enhanced the production of the cytotoxic factor in the serum maximally 2 h after the injection. The cytotoxic activity in the serum was completely inhibited by anti-(mouse tumor necrosis factor) (TNF) antibody. A synthetic lipid A subunit analog (GLA-60), which is much less toxic in its endotoxin activities than lipopolysaccharide or synthetic lipid A (compound 506), induced the endogenous production of serum TNF in rIFN--primed mice. GLA-60 entrapped within liposomes induced the production of serum TNF in rIFN--primed mice more effectively than GLA-60 solubilized in phosphate-buffered saline. Intravenous or intranasal administrations of rIFN- followed by intranasal administration of GLA-60 produced TNF in the lung washing fluid but not in the serum, indicating that TNF production can be induced locally rather than systemically by the alteration of the administration route of the primer and trigger. These results indicate that GLA-60, a lipid A subunit analog of low toxicity, is a beneficial triggering agent in the production of endogenous TNF, as well as having other immunopharmacological properties, and may provide a basis for cancer (metastases) treatment as a result of its ability to induce endogenous TNF.     

[11C]BCTC: Radiosynthesis and in vivo binding to transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in the mouse trigeminal nerve

The purpose of this study was to synthesize a new positron emission tomography radiotracer, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-[¹¹C]carboxamide ([¹¹C]BCTC, [¹¹C]1), and assess its in vivo binding to the transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in mice. [¹¹C]BCTC was synthesized by reacting the hydrochloride of 4-tertiarybutylaniline (2·HCl) with [¹¹C]phosgene ([¹¹C]COCl₂) to give isocyanate [¹¹C]4, followed by reaction with 4-(3-chloropyridin-2-yl)tetrahydropyrazine (3). [¹¹C]BCTC was obtained at a 16–20% radiochemical yield, based on the cyclotron-produced [¹¹C]CO₂ (decay-corrected to the end of bombardment), with >98% radiochemical purity and 65–110 GBq/μmol specific activity at the end of synthesis. An ex vivo biodistribution study in mice confirmed the specific binding of [¹¹C]BCTC to TRPV1 in the trigeminal nerve, which is a tissue with high TRPV1 expression.     

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic,but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.     

Two‐module organization of the mandible in the yellow‐necked mouse: a comparison between two different morphometric approaches

Mandibles of yellow‐necked mouse (Apodemus flavicollis) were used to explore modularity. We tested a biological hypothesis that two separate modules (alveolar region and ascending ramus) can be recognized within the mandible. As a second research goal, we compared two different morphometric procedures under the assumption that methodological approaches that use either geometric or traditional morphometric techniques should give similar results. Besides confirmation of the predicted hypothesis of modularity, the application of both approaches revealed that: (i) modularity was somewhat more evident when it was analysed on the asymmetric (fluctuating asymmetry, FA) than on the symmetric (individual variation) component of variation; (ii) there is correspondence in the patterns of individual variation and FA, which indicates that integration of mandibular traits among individuals is primarily due to direct developmental interactions; and (iii) allometry does not obscure the hypothesized modularity for individual variation or for FA. In addition, traditional morphometric method allowed us to check whether allometry influenced each module to the same extent and to conclude that the ascending ramus is more heavily influenced by general size than the alveolar region. In studies of modularity, differences in methods can lead to discrepancies in the results, and therefore, some caution is required when comparing findings from different investigations. The substantial agreement between our results provides evidence that, when considering two‐module organization of the mouse mandible, direct comparison among studies that use the methods applied herein is, in great part, reliable.     

Characterization of a novel acetamidobenzoxazolone‐based PET ligand for translocator protein (18 kDa) imaging of neuroinflammation in the brain

We developed the novel positron emission tomography (PET) ligand 2‐[5‐(4‐[¹¹C]methoxyphenyl)‐2‐oxo‐1,3‐benzoxazol‐3(2H)‐yl]‐N‐methyl‐N‐phenylacetamide ([¹¹C]MBMP) for translocator protein (18 kDa, TSPO) imaging and evaluated its efficacy in ischemic rat brains. [¹¹C]MBMP was synthesized by reacting desmethyl precursor ( 1 ) with [¹¹C]CH₃I in radiochemical purity of ≥ 98% and specific activity of 85 ± 30 GBq/μmol (n = 18) at the end of synthesis. Biodistribution study on mice showed high accumulation of radioactivity in the TSPO‐rich organs, e.g., the lungs, heart, kidneys, and adrenal glands. The metabolite analysis in mice brain homogenate showed 80.1 ± 2.7% intact [¹¹C]MBMP at 60 min after injection. To determine the specific binding of [¹¹C]MBMP with TSPO in the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity between the ipsilateral and contralateral sides in PET images. Displacement experiments with unlabelled MBMP or PK11195 minimized the difference in uptake between the two sides. In summary, [¹¹C]MBMP is a potential PET imaging agent for TSPO and, consequently, for the up‐regulation of microglia during neuroinflammation.

     

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis

Background

Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.

Results

Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.

Conclusion

Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.