Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti‐proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α‐2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN‐α‐MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN‐α‐MIP cryogel was found as 254.8 × 10⁴ IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption–desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α‐2b on HeLa cell line was found between the values 3.45 × 10⁸ IU/mg and 3.75 × 10⁸ IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α‐2b. Copyright © 2013 John Wiley & Sons, Ltd.     

Pyrolytic Yields of 2-Amino-9H-pyrido[2,3-b]indole and 3-Amino-1-methyl-5H-pyrido[4,3-b]indole as Matagens from Proteins

An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] from a Klebsiella pneumoniae (Aerobacter aerogenes) mutant produced about 40% maltohexaose (G₆) from short-chain amylose ( =23). Mostly G₆ was produced from maltooligosaccharides larger than G₆ by an exo-mechanism action. It also hydrolyzed G₆ and shorter maltooligosaccharides to give smaller maltooligosaccharides. Its position specificity of action on G₃ through G₈ was studied with maltodextrins specifically labeled at the reducing-end glucose unit with ¹⁴C. The highest frequency of cleavage was at the second bond from the reducing end in G₃ through G₆. For G₇ and G₈, the sixth bond from the nonreducing end of the substrate was cleaved with absolute specificity by the exo-mechanism action.

Kinetic parameters of the exo-maltohexaohydrolase on various substrates were also studied. The Michaelis constant (Km) for short-chain amylose was the smallest among the various substrates examined.

G₆ was also formed from G₄ by a transfer action of the enzyme, with an action pattern dependent on the substrate concentration.     

Design of a Calorimeter for the Continuous Study of Heat Production during Anaerobic Microbial Growth

A conduction type calorimeter has been designed to chase microbial growth in batch system. The calorimeter is of a twin structure having thermopile plates as a temperature sensor, The heat evolution during the microbial growth at a required temperature can be observed as an output-voltage generated at thermopile terminals with a sensitivity of 58.5 mV K^?1

A stainless steel cell with a volume of 300 cm³ serves as a culture cell which is capable of being autoclaved prior to the initiation of calorimetric run, taking out from the calorimeter body.

Because of the twin structure, the apparatus works with sufficient stability in detecting small heat evolution for long duration. Its operation has been demonstrated with the growth of Sacch. cerevisiae grown on liquid synthetic medium under anaerobic condition.     

Lowering Effect of Phenolic Glycosides on the Rise in Postprandial Glucose in Mice

Glycosides were screened for their lowering effect on the postprandial blood glucose rise in vivo. The effect of phlorizin and other phenolic glycosides on the postprandial blood glucose response to glucose ingestion was evaluated in Std ddY mice. When phlorizin was simultaneously added, the peak blood glucose level was significantly decreased by 51% (p < 0.01) compared to vehicles following glucose ingestion by mice, while the blood insulin responses were generally similar. Screening experiments were conducted with different classes of phenolic glycosides added to a glucose solution. Reductions of 40–52% (p < 0.05) were observed in vehicles containing arbutin, 4-hydroxyphenyl-α-d-glucopyranoside (hydroquinone-α-glucoside) or glycyrrhizin, and of only 15–31% (not significant) in vehicles containing neohesperidin dihydrochalcone, glycyrrhetinic acid monoglucuronide, or 3,4-dimethoxyphenyl-β-d-glucopyranoside. No lowering effect was observed in vehicles containing salicin. Since glycyrrhizin, arbutin, and hydroquinone-α-glucoside blunted to varying degrees the postprandial blood glucose rise following glucose ingestion, they may be useful adjuvants for the treatment of diabetic subjects.     

A Novel Synthesis of 3-Substituted Furans: Synthesis of Perillen and Dendrolasin

A novel synthetic method of 3-substituted furans was developed and syntheses of perillen and dendrolasin are described.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Subunits and Sulfhydryl Groups of Beef Liver d-Glycerate Dehydrogenase

The amino acid composition of beef liver d-glycerate dehydrogenase (EC 1.1.1.29) was determined. Results of sodium dodecyl sulfate gel electrophoresis and measurements of the number of NADH bound by the enzyme and the number of the essential sulfhydryl groups suggested that the enzyme was composed of two identical subunits with the molecular weight of 36,000. Close relation between the essential sulfhydryl groups and the coenzyme binding site was also suggested. Effect of an alkylating agent (bromopyruvate) with the structure similar to the substrate was studied. Effects of iodoacetate and iodoacetamide were also studied. It was suggested that these reagents behaved as active-site-directed irreversible inhibitors of the enzyme. Bromopyruvate exhibited a high affinity to the enzyme. Iodoacetate (anionic reagent) had a higher affinity than iodoacetamide (neutral reagent).     

Structural Conversion from Non-native to Native form of Recombinant Human Epidermal Growth Factor by Brevibacillus choshinensis

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40°C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.     

Inhibitory Potency of Erythrina variegata Proteinase Inhibitors toward Serine Proteinases in the Blood Coagulation and Fibrinolytic Systems

The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman–Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIa inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward β-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.