N-acetyl-L-methionine is a superior protectant of human serum albumin against post-translational oxidation as compared to N-acetyl-L-tryptophan

Sodium octanoate and N-acetyl-L-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, as compared with N-AcTrp, N-acetyl-L-methionine (N-AcMet) is superior in protecting albumin exposed to light during storage. Here, we examine, whether N-AcMet also is better than N-AcTrp to protect albumin against oxidation. Recombinant human serum albumin (rHSA) without and with N-AcMet or N-AcTrp was oxidized by using chloramine-T (CT) as a model compound for mimicking oxidative stress. Oxidation of rHSA was examined by determining carbonyl groups and advanced oxidation protein products. Structural changes were studied by native-PAGE, circular dichroism, intrinsic fluorescence and differential scanning calorimetry. The anti-oxidant capacity of CT-treated rHSA was quantified by its ability to scavenge peroxynitrite and the hydroxyl radical. The pharmacokinetics of indocyanine green-labeled albumin preparations was studied in male mice. We found that the number of chemical modifications and the structural changes of rHSA were significantly smaller in the presence of N-AcMet than in the presence of N-AcTrp. The anti-oxidant properties of CT-exposed rHSA were best protected by adding N-AcMet. Finally, N-AcMet is superior in preserving the normal pharmacokinetics of rHSA. Thus, N-AcMet is superior to N-AcTrp in protecting albumin preparations against oxidation. In addition, N-AcMet is probable also useful for protecting other proteins. Therefore, N-AcMet should be useful as a new and effective stabilizer and antioxidant for albumin isolated from blood, rHSA, albumin-fusion proteins and for preparations of rHSA-therapeutic complexes.     

3ircular dichroism simulation shows a site-II-to-site-I displacement of human serum albumin-bound diclofenac by ibuprofen

Purpose: The purpose of this study was to confirm the hypothesis that a site-II-to-site-I displacement takes place when some nonsteroidal anti-inflammatory drugs are displaced by another drug from their high-affinity binding site to a site of lower affinity on human serum albumin (HSA).Methods: Diclofenac, sodium salt, was used as a representative example because of its prominent reversal of the Cotton effect. Effects of site-specific drugs on the free fraction of diclofenac were determined by equilibrium dialysis, and effects on induced circular dichroism (CD) of diclifenac bound to HSA were studied by CD and CD simulation techniques.Results: Ibuprofen, a site-II-specific drug, altered the CD spectrum of the diclofenac-HSA complex at a molar ratio of 0.5∶1 to that obtained at a higher ratio (5∶1) without ibuprofen. The induced CD spectrum obtained in the presence of ibuprofen was very similar to one that assumed that all diclofenac displaced from its high-affinity binding site (site II) became rebound to a lower-affinity site (site I). The rebinding could be influenced by a free energy linkage between the two sites which would make site I (or parts thereof) more suitable for diclofenac binding.Conclusion: We have confirmed the existence of a site II-to-site displacement, which is very striking and pharmacologically important, because the concentration of unbound drug being displaced is much lower than expected for a competitive mechanism.     

A comparison of avoidance to acoustic stimuli in fish with different auditory capabilities: juvenile chum salmon (Oncorhynchus keta) and common carp (Cyprinus carpio)

This study compared the repulsive effects of sound playbacks of intermittent 30, 150, 300, 600 and 900 Hz tones on two fish with different auditory capabilities: juvenile chum salmon (Oncorhynchus keta) and common carp (Cyprinus carpio). When 150 and 300 Hz tones were emitted from an underwater speaker, O. keta exhibited a moderate repulse reaction. Conversely, C. carpio exhibited a moderate repulse reaction to a tone with a frequency of 30 Hz, which indicates that a low-frequency component in complex broadband sound may be important for inducing a repulse reaction in cyprinids.     

Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.     

Differences in dynamics between indigenous and inoculated Sphingomonas paucimobilis strain SS86 in soils

Abstract Survival of γ-HCH-degrading Sphingomonas paucimobilis strain SS86 indigenous or inoculated to soil was examined under laboratory conditions. Strain SS86 inoculated to soil declined to undectectable levels though amendment of soil with γ-HCH or starvation-treatment of the cells enhanced its survivability. About 10³–10⁴ cells/g soil of strain SS86 indigenous to soil survived stably for long periods and was more tolerant to soil treatment by desiccation, percolation, and chloroform-fumigation than inoculated SS86. The haritable micro-pores in soil and/or physiological properties peculiar to the indigenous SS86 were supposed to ensure its good ability to survive by protecting it against protozoan grazing.     

Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.