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.     

In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice.

The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development.     

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.     

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.     

Glutathione-dependent ascorbate recycling activity of rat serum albumin

An efficient regeneration of vitamin C (ascorbate) from its oxidized byproduct, dehydroascorbate (DHAA), is necessary to maintain sufficient tissue levels of the reduced form of the vitamin. Additionally, the recycling may be more significant in mammals, such as guinea pigs and humans, who have lost the ability to synthesize ascorbate de novo, than it is in most other mammals who have retained the ability to synthesize the vitamin from glucose. Both a chemical and an enzymatic reduction of DHAA to ascorbate have been proposed. Several reports have appeared in which proteins, including thioltransferase, protein disulfide isomerase, and 3-alpha-hydroxysteroid dehydrogenase, characterized for other activities have been identified as having DHAA reductase activity in vitro. Whether these previously characterized proteins catalyze the reduction of DHAA in vivo is unclear. In the present study, a 66 kD protein was purified strictly on the basis of its DHAA-reductase activity and was identified as rat serum albumin. The protein was further characterized and results support the suggestion that serum albumin acts as an antioxidant and exerts a significant glutathione-dependent DHAA-reductase activity that may be important in the physiologic recycling of ascorbic acid.     

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