Physiological ER Stress Mediates the Differentiation of Fibroblasts

Recently, accumulating reports have suggested the importance of endoplasmic reticulum (ER) stress signaling in the differentiation of several tissues and cells, including myoblasts and osteoblasts. Secretory cells are easily subjected to ER stress during maturation of their secreted proteins. Skin fibroblasts produce and release several proteins, such as collagens, matrix metalloproteinases (MMPs), the tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAGs), and the production of these proteins is increased at wound sites. Differentiation of fibroblasts into myofibroblasts is one of the key factors for wound healing and that TGF-β can induce fibroblast differentiation into myofibroblasts, which express α-smooth muscle actin. Well-differentiated myofibroblasts show increased production of collagen and TGF-β, and bring about wound healing. In this study, we examined the effects of ER stress signaling on the differentiation of fibroblasts, which is required for wound healing, using constitutively ER stress-activated primary cultured fibroblasts. The cells expressed positive α-smooth muscle actin signals without TGF-β stimulation compared with control fibroblasts. Gel-contraction assays suggested that ER stress-treated primary fibroblasts caused stronger shrinkage of collagen gels than control cells. These results suggest that ER stress signaling could accelerate the differentiation of fibroblasts to myofibroblasts at injured sites. The present findings may provide important insights for developing therapies to improve wound healing.     

PET Imaging of Lung Inflammation with [18F]FEDAC, a Radioligand for Translocator Protein (18 kDa)

Purpose

The translocator protein (18 kDa) (TSPO) is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lung. This study aimed to perform positron emission tomography (PET) imaging of lung inflammation with [¹⁸F]FEDAC, a specific TSPO radioligand, and to determine cellular sources enriching TSPO expression in the lung.

Methods

An acute lung injury model was prepared by intratracheal administration of lipopolysaccharide (LPS) to rat. Uptake of radioactivity in the rat lungs was measured with small-animal PET after injection of [¹⁸F]FEDAC. Presence of TSPO was examined in the lung tissue using Western blot and immunohistochemical assays.

Results

The uptake of [¹⁸F]FEDAC increased in the lung with the progress of inflammation by treatment with LPS. Pretreatment with a TSPO-selective ligand PK11195 showed a significant decrease in the lung uptake of [¹⁸F]FEDAC due to competitive binding to TSPO. TSPO expression was elevated in the inflamed lung section and its level responded to the [¹⁸F]FEDAC uptake and severity of inflammation. Increase of TSPO expression was mainly found in the neutrophils and macrophages of inflamed lungs.

Conclusion

From this study we conclude that PET with [¹⁸F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation. Study on human lung using [¹⁸F]FEDAC-PET is promising.     

Radiosynthesis of three [11C]ureido-substituted benzenesulfonamides as PET probes for carbonic anhydrase IX in tumors

Three ureido-substituted benzenesulfonamides 1a-c have been developed as potent inhibitors for carbonic anhydrase IX, which is overexpressed in hypoxic tumors. In this study, we labeled these unsymmetrical ureas 1a-c using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]1a-c could become promising positron tomography probes for imaging carbonic anhydrase IX in tumors. The strategy for radiosynthesis of [(11)C]1a-c was to react hydrochloride of anilines 2a-c with [(11)C]COCl(2) to give isocyanate [(11)C]4a-c, followed by a reaction with 4-aminobenzenesulfonamide (3).     

Synthesis and evaluation of 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/??mol specific activity using 3300-4000 MBq of [¹⁸F]F⁻. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.     

Improved Visualization and Specific Binding for Metabotropic Glutamate Receptor Subtype 1 (mGluR1) Using [11C]ITMM with Ultra-High Specific Activity in Small-Animal PET

Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial target in the development of new medications to treat central nervous system (CNS) disorders. Recently, we developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[¹¹C]methoxy-N-methyl-benzamide ([¹¹C]ITMM) as a useful positron emission tomography (PET) probe for mGluR1 in clinical studies. Here, we aimed to improve visualization and threshold of specific binding for mGluR1 using [¹¹C]ITMM with ultra-high specific activity (SA) of > 3,500 GBq/μmol in rat brains. A two-tissue compartment model indicated large differences between the two SAs in the constants k₃ and k₄, representing binding ability for mGluR1, while constants K ₁ and k₂ showed no differences. The total distribution volume (V_T) values of conventional and ultra-high SA were 9.1 and 11.2 in the thalamus, 7.7 and 9.7 in the striatum, and 6.4 and 8.5 mL/cm³ in the substantia nigra, respectively. The specific binding of [¹¹C]ITMM with ultra-high SA was significantly higher than the conventional SA, especially in the basal ganglia. Parametric PET images scaled with V_T of the ultra-high SA clearly identified regional differences in the rat brain. In conclusion, PET studies using [¹¹C]ITMM with ultra-high SA could sufficiently improve visualization and specific binding for mGluR1, which could help further understanding for mGluR1 functions in CNS disorders.     

When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation

Amplified genes are frequently localized on extrachromosomal double minutes (DMs) or in chromosomal homogeneously staining regions (HSRs). We previously showed that a plasmid bearing a mammalian replication initiation region could efficiently generate DMs and HSRs after transfection into human tumor cell lines. The Breakage-Fusion-Bridge (BFB) cycle model, a classical model that explains how HSRs form, could also be used to explain how the transfected plasmids generate HSRs. The BFB cycle model involves anaphase bridge formation due to the presence of dicentric chromosomes, followed by the breakage of the bridge. In this study, we used our plasmid-based model system to analyze how anaphase bridges break during mitosis. Dual-color fluorescence in situ hybridization analyses revealed that anaphase bridges were most frequently severed in their middle irrespective of their lengths, which suggests that a structurally fragile site exists in the middle of the anaphase bridge. Breakage of the chromosomal bridges occurred prior to nuclear membrane reformation and the completion of cytokinesis, which indicates that mechanical tension rather than cytokinesis is primarily responsible for severing anaphase bridges. Time-lapse observation of living cells revealed that the bridges rapidly shrink after being severed. If HSR length was extended too far, the bridge could no longer be resolved and became tangled depending on the tension. The unbroken bridge appeared to inhibit the completion of cytokinesis. These observations strongly suggest that anaphase bridges are highly elastic and that the length of the spindle axis determines the maximal HSR length.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.     

Synthesis and in vivo evaluation of ¹⁸F-fluoroethyl GF120918 and XR9576 as positron emission tomography probes for assessing the function of drug efflux transporters

The purpose of this study was to synthesize two new positron emission tomography (PET) probes, N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[1?F]fluoroethoxy-9-oxo-4-acridine carboxamide ([1?F]3) and quinoline-3-carboxylic acid [2-(4-{2-[7-(2-[1?F]fluoroethoxy)-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxyphenyl]amide ([1?F]4), and to evaluate the potential of these PET probes for assessing the function of two major drug efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). [1?F]3 and [1?F]4 were synthesized by 1?F-alkylation of each O-desmethyl precursor with [1?F]2-fluoroethyl bromide for injection as PET probes. In vitro accumulation assay showed that treatment with P-gp/BCRP inhibitors (1 and 2) enhanced the intracellular accumulation capacity of P-gp- and BCRP-overexpressing MES-SA/Dx5 cells. In PET studies, the uptake (AUC(brain[0-)?? (min])) of [1?F]3 and [1?F]4 in wild-type mice co-injected with 1 were approximately sevenfold higher than that in wild-type mice, and the uptake of [1?F]3 and [1?F]4 in P-gp/Bcrp knockout mice were eight- to ninefold higher than that in wild-type mice. The increased uptake of [1?F]3 and [1?F]4 was similar to that of parent compounds ([11C]1 and [11C]2) previously described, indicating that radioactivity levels in the brain after injection of [1?F]3 and [1?F]4 are related to the function of drug efflux transporters. Also, these results suggest that the structural difference between parent compounds ([11C]1 and [11C]2) and fluoroethyl analogs ([1?F]3 and [1?F]4) do not obviously affect the potency against drug efflux transporters. In metabolite analysis of mice, the unchanged form in the brain and plasma at 60 min after co-injection of [1?F]4 plus 1 were higher (95% for brain; 81% for plasma) than that after co-injection of [1?F]3 plus 1. [1?F]4 is a promising PET probe to assess the function of drug efflux transporters.