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81.
Genetically engineered strains of Escherichia coli and Pseudomonas aeruginosa were prepared harboring the gene cluster nirFDLGH from Pseudomonas stutzeri substrain ZoBell on a high copy plasmid. These genes have been previously implicated as being essential for the biosynthesis of heme d(1), the prosthetic group of dissimilatory nitrite reductases in anaerobic, denitryfying bacteria. Tetrapyrroles detectable at steady-state levels were identified from both organisms, and cell-free extracts from each were also used to transform uroporphyrinogen in vitro. E. coli does not naturally produce d(1), and the engineered strain failed to produce d(1) or any tetrapyrrole foreign to E. coli. Therefore, while nirFDLGHmay be necessary for d(1) biosynthesis, it is not sufficient. In the denitrifier P. aeruginosa, the results were more positive. The presence of the plasmid led to increased levels of d(1). In addition, a previously unidentified tetrapyrrole was detected. This compound was characterized by visible absorption spectroscopy, infrared spectroscopy, X-ray photoelectron spectroscopy, mass spectrometry, and NMR, and a tentative structure was proposed for this compound. The tetrapyrrole has structural features similar to sirohydrochlorin (as precorrin-2 or sirotetrahydrochlorin, a known intermediate of d(1)) and d(1) itself. The most unusual substituents are epoxide and sulfoxide moieties. When this tetrapyrrole was treated with strong mineral acid and heat, it was converted into natural d(1). 相似文献
82.
Prajapati S Verma U Yamamoto Y Kwak YT Gaynor RB 《The Journal of biological chemistry》2004,279(3):1739-1746
The NF-kappaB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IkappaB kinases (IKKs) by cytokines such as tumor necrosis factor-alpha and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cbeta (PP2Cbeta) can associate with the IKK complex. PP2Cbeta association with the IKK complex led to the dephosphorylation of IKKbeta and decreased its kinase activity. The binding of PP2Cbeta to IKKbeta was decreased at early times post-tumor necrosis factor-alpha treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cbeta demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cbeta to down-regulate cytokine-induced NF-kappaB activation by altering IKK activity. 相似文献
83.
Choi SO Um SY Jung SH Jung SJ Kim JI Lee HJ Chung SY 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,830(2):301-305
A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen. 相似文献
84.
We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner. 相似文献
85.
Jin Young Chung Jung Hoon Choi Choong Hyun Lee Ki-Yeon Yoo Moo-Ho Won Dae Young Yoo Dae Won Kim Soo Young Choi Hwa Young Youn Seung Myung Moon In Koo Hwang 《Neurochemical research》2010,35(4):620-627
Microglia are main form of active immune defense, and they are constantly moving and analyzing the CNS for damaged neurons and infectious agents. In this study, we compared microglia in the spinal cord of the young adult (1–2 years old) and aged (10–12 years old) German Shepherd dogs via immunohistochemistry and western blot analysis for ionized calcium-binding adapter molecule 1 (Iba-1), a microglial marker. In addition, we also observed the interferon-γ (IFN-γ), a pro-inflammatory cytokine, and interleukin-1β (IL-1β), produced by activated microglia/macrophage, protein levels in these groups. At first, we found that neuronal nuclei (NeuN, a neuronal marker)-immunoreactive neurons were distributed throughout the grey mate of the spinal cord, and there were no significant differences between the adult and aged groups. Most of Iba-1-immunoreactive microglia were morphologically ramified microglia (resting form) in the adult group, while some Iba-1-immunoreactive microglia were morphologically activated microglia in the aged group. In western blot analysis, Iba-1, IFN-γ and IL-1β expression were increased in the aged group. This result may be associated with age-dependent changes in the spinal cord. 相似文献
86.
Jung Ha Kim Kabsun Kim Hye Mi Jin Insun Song Bang Ung Youn Seoung-Hoon Lee Yongwon Choi Nacksung Kim 《The Journal of biological chemistry》2010,285(8):5224-5231
The regulation of NFATc1 expression is important for osteoclast differentiation and function. Herein, we demonstrate that macrophage-colony-stimulating factor induces NFATc1 degradation via Cbl proteins in a Src kinase-dependent manner. NFATc1 proteins are ubiquitinated and rapidly degraded during late stage osteoclastogenesis, and this degradation is mediated by Cbl-b and c-Cbl ubiquitin ligases in a Src-dependent manner. In addition, NFATc1 interacts endogenously with c-Src, c-Cbl, and Cbl-b in osteoclasts. Overexpression of c-Src induces down-regulation of NFATc1, and depletion of Cbl proteins blocks NFATc1 degradation during late stage osteoclastogenesis. Taken together, our data provide a negative regulatory mechanism by which macrophage-colony-stimulating factor activates Src family kinases and Cbl proteins, and subsequently, induces NFATc1 degradation during osteoclast differentiation. 相似文献
87.
Bcl‐xL, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐xL overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐xL expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐xL overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐xL overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐xL not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc. 相似文献
88.
Merryman WD Youn I Lukoff HD Krueger PM Guilak F Hopkins RA Sacks MS 《American journal of physiology. Heart and circulatory physiology》2006,290(1):H224-H231
It has been speculated that heart valve interstitial cells (VICs) maintain valvular tissue homeostasis through regulated extracellular matrix (primarily collagen) biosynthesis. VICs appear to be phenotypically plastic, inasmuch as they transdifferentiate into myofibroblasts during valve development, disease, and remodeling. Under normal physiological conditions, transvalvular pressures (TVPs) on the right and left side of the heart are vastly different. Hence, we hypothesize that higher left-side TVPs impose larger local tissue stress on VICs, which increases their stiffness through cytoskeletal composition, and that this relation affects collagen biosynthesis. To evaluate this hypothesis, isolated ovine VICs from the four heart valves were subjected to micropipette aspiration to assess cellular stiffness, and cytoskeletal composition and collagen biosynthesis were quantified by using the surrogates smooth muscle alpha-actin (SMA) and heat shock protein 47 (HSP47), respectively. VICs from the aortic and mitral valves were significantly stiffer (P < 0.001) than those from the pulmonary and tricuspid valves. Left-side isolated VICs contained significantly more (P < 0.001) SMA and HSP47 than right-side VICs. Mean VIC stiffness correlated well (r = 0.973) with TVP; SMA and HSP47 also correlated well (r = 0.996) with one another. Assays were repeated for VICs in situ, and, as with in vitro results, left-side VIC protein levels were significantly greater (P < 0.05). These findings suggest that VICs respond to local tissue stress by altering cellular stiffness (through SMA content) and collagen biosynthesis. This functional VIC stress-dependent biosynthetic relation may be crucial in maintaining valvular tissue homeostasis and also prove useful in understanding valvular pathologies. 相似文献
89.
90.