Biological activity of gamma-irradiated serum albumin

Gamma-irradiated serum albumin activated L-tyrosine oxidation to 3,4-dioxyphenyl alanine (DOPA) and forms adducts with DOPA oxidation products. These adducts are more resistant to proteolysis and have bactericidal and mutagenic capacity. A possible role of such adducts in a radiation damage to the organism is discussed.     

Refolding human serum albumin at relatively high protein concentration

The conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2-mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4-8 M urea and 14 mM 2-mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 microM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg.ml-1 whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding.     

Fatty acids bound to serum albumin potentiate platelet prostaglandin biosynthesis.

Utilization of arachidonic acid by human platelets is increased when the fatty acid content of serum albumin is increased as well. Platelet aggregation induced by arachidonic acid and by low concentrations of thrombin is thus potentiated, suggesting that platelet responsiveness to aggregating agents is influenced by the plasma content in free fatty acids.     

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.     

Molten globule intermediates of human serum albumin in low concentration of urea

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.     

In vivo and in vitro effects of sizofiran on the human neutrophils and the serum opsonic activity

In this paper, we examine the effects of SPG, which is a well known BRM, both in vivo and in vitro on the neutrophilic ROS production and the serum opsonic activity by the chemiluminescence technique using luminol as a probe. To investigate the in vivo effects, SPG was administered to 12 healthy male volunteers and two phases of enhancement of the neutrophilic ROS production and the serum opsonic activity were observed. In vitro, the addition of SPG showed a dose-dependent suppression. To investigate the mechanisms in these contradictory effects of SPG, supernatants of a lymphocytes culture medium in the presence of SPG with or without mitogen were added to the neutrophils. The addition of supernatants at a lower concentration of SPG (0.01 mg/ml) with mitogens showed significant preventive effects on the neutrophilic ROS production for the duration of incubation. This suggests that cytokines derived from lymphocytes may contribute to the in vivo effects of SPG. SPG can play an important role in the host’s defense against microbe infections by enhancing it’s effect on non-specific immunity when administered in vivo     

RNA-hydrolyzing activity of human serum albumin and its recombinant analogue

The comparative analysis of RNA-hydrolyzing activity of albumin from human serum and albumin expressed in methylotrophic yeast Pichia pastoris has been carried out. The rate of polyribonucleotide phosphodiester bond cleavage in the presence of recombinant albumin has been found to be similar to that of the reaction mediated by the native protein. According to ³¹P NMR data, RNA hydrolysis follows the mechanism of intermolecular trans-esterification to yield 2′,3′-cyclophosphodiester reaction products that are further slowly hydrolyzed to form nucleoside-3′- and nucleoside-2′-phosphates. Analysis of pH dependence suggests an acid–base mechanism of catalysis. The catalytic activity and substrate specificity of albumin in RNA hydrolysis distinguish it from human ribonucleases.     

Amine oxidase activity in commercial preparations of bovine serum albumin

Amine oxidase activity has been identified in commercial samples of bovine serum albumin (BSA). Benzylamine, phenylethylamines and to a lesser extent, indoleamines, were found to be substrates. The amine oxidase activity was inhibited by semicarbazide and was virtually absent in electrophoretically purified samples. Kinetic analysis of benzylamine deamination and experiments utilizing mixed substrates indicate that more than one catalytic activity may be involved. The results show that amine deamination should be considered as a potential source of error in experiments employing high concentrations of commercially available BSA preparations. This would be of particular importance for $\text{in vitro}$ studies with dopamine since this amine was found to be deaminated at a rapid rate.     

In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters

The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.     

In vivo activity of nuclease-resistant siRNAs

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.