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101.
102.
Basu A Yang K Wang M Liu S Chintala R Palm T Zhao H Peng P Wu D Zhang Z Hua J Hsieh MC Zhou J Petti G Li X Janjua A Mendez M Liu J Longley C Zhang Z Mehlig M Borowski V Viswanathan M Filpula D 《Bioconjugate chemistry》2006,17(3):618-630
Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies. 相似文献
103.
Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1 000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis continued with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal. 相似文献
104.
Li Y Wo JM Cai L Zhou Z Rosenbaum D Mendez C Ray MB Jones WF Kang YJ 《Experimental biology and medicine (Maywood, N.J.)》2003,228(3):286-292
Barrett's esophagus is the transformation of normal esophageal squamous epithelium to specialized intestinal metaplasia (SIM). Among the Barrett's specialized cells, those that can develop protective mechanisms against apoptosis may have potential to become malignant. Studies have shown that overexpression of metallothionein (MT), low molecular protein that protects cells from apoptotic stimuli, appears to be associated with more advanced, highly malignant tumors. We thus investigated the relationship between MT expression and apoptosis in different stages of Barrett's carcinogenesis. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling and immunohistochemical dual-staining assay were performed in human biopsy samples of normal, SIM, dysplasia, and adenocarcinoma. Apoptotic index and MT expression were quantified by using an image system to analyze the converted digital data. A negative correlation between MT expression and apoptotic index was found. MT expression was significantly increased along with the histologic progression towards adenocarcinoma. This study thus suggests that MT may contribute to cytoprotection, thereby inhibiting apoptosis and leading to carcinogenesis of Barrett's esophageal cells. 相似文献
105.
Cardona-Gómez GP Mendez P DonCarlos LL Azcoitia I Garcia-Segura LM 《The Journal of steroid biochemistry and molecular biology》2002,83(1-5):211-217
In the brain, as in other tissues, estradiol interacts with growth factors. One of the growth factors that is involved in the neural actions of estradiol is insulin-like growth factor-I (IGF-I). Estradiol and IGF-I cooperate in the central nervous system to regulate neuronal development, neural plasticity, neuroendocrine events and the response of neural tissue to injury. The precise molecular mechanisms involved in these interactions are still not well understood. In the central nervous system there is abundant co-expression of estrogen receptors (ERs) and IGF-I receptors (IGF-IRs) in the same cells. Furthermore, the expression of estrogen receptors and IGF-I receptors in the brain is cross-regulated. In addition, using specific antibodies for the phosphorylated forms of extracellular-signal regulated kinase (ERK) 1 and ERK2 and Akt/protein kinase B (Akt/PKB) it has been shown that estradiol affects IGF-I signaling pathways in the brain. Estradiol treatment results in a dose-dependent increase in the phosphorylation of ERK and Akt/PKB in the brain of adult ovariectomized rats. In addition, estradiol and IGF-I have a synergistic effects on the activation of Akt/PKB in the adult rat brain. These findings suggest that estrogen effects in the brain may be mediated in part by the activation of the signaling pathways of the IGF-I receptor. 相似文献
106.
Translational activation of several dormant mRNAs in vertebrate oocytes is mediated by cytoplasmic polyadenylation, a process controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein CPEB. The translation of CPE-containing mRNAs does not occur en masse at any one time, but instead is temporally regulated. We show here that in Xenopus, partial destruction of CPEB controls the temporal translation of CPE-containing mRNAs. While some mRNAs, such as the one encoding Mos, are polyadenylated at prophase I, the polyadenylation of cyclin B1 mRNA requires the partial destruction of CPEB that occurs at metaphase I. CPEB destruction is mediated by a PEST box and Cdc2-catalyzed phosphorylation, and is essential for meiotic progression to metaphase II. CPEB destruction is also necessary for mitosis in the early embryo. These data indicate that a change in the CPEB:CPE ratio is necessary to activate mRNAs at metaphase I and drive the cells' entry into metaphase II. 相似文献
107.
108.
A. Gálvez E. Valdivia Hikmate Abriouel Emilio Camafeita Enrique Mendez Manuel Martínez-Bueno Mercedes Maqueda 《Archives of microbiology》1998,171(1):59-65
The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass
(5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence
of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin
EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic
effect on E. faecalis S-47.
Received: 15 July 1998 / Accepted 28 October 1998 相似文献
109.
Review: Anaerobic treatment of municipal sanitary landfill leachates: the problem of refractory and toxic components 总被引:5,自引:0,他引:5
Alkalay D. Guerrero L. Lema J.M. Mendez R. Chamy R. 《World journal of microbiology & biotechnology》1998,14(3):309-320
The wide use of municipal sanitary landfills has drawn attention to the leaching effluent generated, this may be problematic to the site's environment, whether by infiltration or other contaminating modes. Anaerobic digestion has been shown to be one of the most efficient systems with which to treat this type of effluent. This article reviews the techniques used by different authors for leachate characterization, specifically related to refractory and toxic components and their effect on anaerobic treatability. In addition, it covers the treatment of refractory organics, organic and inorganic toxic materials and the nutrient balance for adequate system operation. The main conclusions are that there is ample availability of methods by which to identify the different components present in leachates as well as for their toxicity assessment and that nutrients are in general available in sufficient amounts. Treatability studies are presented which are shown to be of general value and can be used in a straightforward manner. 相似文献
110.
Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane. 相似文献