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181.
Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.  相似文献   
182.
Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZEZZZ) Pfr to Pr back-conversion.  相似文献   
183.
184.
The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg2+, cdG·Ca2+ complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.  相似文献   
185.
Inhibition of CYP 17 is a promising strategy for the treatment of prostate cancer. Recently two non-steroidal compounds with high in vitro activity were synthesized in our group (BW19 and BW95). However, after a few hours they showed in vivo a strong decrease in their activity. This might be due to a fast biodegradation. Potential hydroxy and epoxy metabolites were synthesized and their inhibitory activities were tested by a new non-cellular assay using recombinant enzyme. As source, membrane fractions of E. coli pJL17/OR coexpressing human CYP 17 and rat NADPH-P450-reductase were, used. Showing a high and constant CYP 17 activity and a fast and easy isolation procedure the new method was advantageous compared with the microsomal assay. Interestingly, all the new synthesized hydroxy and epoxy compounds except one showed a lower inhibition of CYP 17 than the parent compounds. Thus, the loss of in vivo activity may be partly explained.  相似文献   
186.
A new aminopeptidase — aminopeptidase Co — has been detected in the yeast Saccharomycescerevisiae. The enzyme is only active in the presence of Co2+ions. Zn2+- and Mn2+ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.  相似文献   
187.
Leaching bacteria such as Thiobacillus ferrooxidans attach to pyrite or sulfur by means of extracellular polymeric substances (EPS) (lipopolysaccharides). The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrochemical interaction with the negatively charged pyrite surface. EPS from sulfur cells possess increased hydrophobic properties and do not attach to pyrite, indicating adaptability to the substrate or substratum.  相似文献   
188.
Summary We tested whether grasshoppers in experimental field environments, i.e. cages (40×40 cm) placed on existing old field vegetation, (1) were limited in density by plant abundance and/or nitrogen content, (2) exhibited interspecific competition, and (3) altered the relative abundance of different plant species. We examined interactions among a pair of early season grasshopper species (May–June; Arphia conspersa and Pardalophora apiculata) and a late season pair (July–August; Melanoplus femur-rubrum and Melanoplus bivittatus). Each grasshopper species was placed in cages by itself and with another grasshopper species. Grasshoppers generally survived at higher density in fertilized cages and they reduced plant abundance relative to empty cages, suggesting that grasshoppers may be food limited at these densities. In unfertilized plots, early season grasshoppers preferred grasses (Schizachyrium scoparium and Poa pratensis) and favored the growth of forbs (especially Solidago spp.). However, late in summer, Melanoplus spp. preferred Solidago spp. and favored the growth of grasses.The pattern of grasshopper survivorship and plant reduction within these experimental environments provide preliminary support for some of the predictions of resource competition theory. Grasshoppers exhibited interspecific competition only if they significantly reduced plant biomass. If two species competed, a grasshopper species was eliminated only if the superior competitor, when living by itself, could reduce plant biomass to a significantly lower level than the inferior competitor. Competitors persisted only if they did not differ in their ability to reduce plant biomass or reduced the abundance of different plant species.  相似文献   
189.
Over the last few years, a relative decline of the morbidity and mortality of human immunodeficiency virus (HIV) infection in industrialised countries has been observed due to the use of a potent combined therapy known as high active antiretroviral therapies (HAARTs). It has led to a decrease of viral load and a quantitative and qualitative improvement of immune function in patients, especially CD4+ T-lymphocyte count, having as a consequence a decrease of infectious complications and a global clinical improvement. Besides the positive effects of HAARTs on immune and metabolic alterations during HIV infection, it has been reported that the commonly used drugs AZT, ddI, and ddC are toxic to hepatocytes. Recent reports continue to point to the mitochondria as targets for toxicity. The prevalence of these symptoms is continued during acquired immunodeficiency syndrome (AIDS). The effects of oxidative stress occurring as a consequence of mitochondrial toxicity may amplify some of the pathophysiological and phenotypic events during infection. Mitochondrial stabilisation and antioxidative strategies are possible new therapeutic aims since the antiretroviral treatment is prolonged with increased longevity from AIDS, which has become a more manageable chronic illness. The aim of the present review article is to summarize the current knowledge about mitochondrial dysfunction during HAART and its consequence for patients with chronic treatment. Oxidative stress may serve as one pathway for cellular damage in AIDS and its treatment. One important future goal is to prevent or attenuate the side effects of HAART so that improved disease management can be achieved.  相似文献   
190.
Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.  相似文献   
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