Differential changes in alpha2-macroglobulin and hemopexin in brain and liver in response to acute inflammation.

Changes in serum and cerebrospinal fluid (CSF) proteins following generalized acute inflammation induced by fermented yeast in the rat was examined by concanavalin A-blotting, immunoblotting, and radioimmunoassay. Using alpha2-macroglobulin (alpha2-M) and hemopexin (HPX) as marker proteins, the concentration alpha2-M was found to increase in serum and CSF by 150- and 5-fold, respectively, whereas the concentration of HPX increased by about 4-fold in both fluids following yeast-induced inflammation. The lesser increase in alpha2-M in the CSF versus the systemic circulation is not likely to be the result of changes in the permeability of the blood--brain barrier, since no change in the total protein content of CSF was detected in inflamed rats when compared to control animals. These results, however, illustrate the regulation of the same protein, such as alpha2-M, in two separate organs within the same animal can be drastically different. These results also suggest a possible protective role of alpha2-M in the brain during acute inflammation. Moreover, these observations are consistent with the previous observation that there is a differential response in the level of alpha2-M between the testis and the systemic circulation during inflammation.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas

Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.     

Role of the Barhl2 homeobox gene in the specification of glycinergic amacrine cells

The mammalian retina contains numerous morphological and physiological subtypes of amacrine cells necessary for integrating and modulating visual signals presented to the output neurons. Among subtypes of amacrine cells grouped by neurotransmitter phenotypes, the glycinergic and gamma-aminobutyric acid (GABA)ergic amacrine cells constitute two major subpopulations. To date, the molecular mechanisms governing the specification of subtype identity of amacrine cells remain elusive. We report here that during mouse development, the Barhl2 homeobox gene displays an expression pattern in the nervous system that is distinct from that of its homologue Barhl1. In the developing retina, Barhl2 expression is found in postmitotic amacrine, horizontal and ganglion cells, while Barhl1 expression is absent. Forced expression of Barhl2 in retinal progenitors promotes the differentiation of glycinergic amacrine cells, whereas a dominant-negative form of Barhl2 has the opposite effect. By contrast, they exert no effect on the formation of GABAergic neurons. Moreover, misexpressed Barhl2 inhibits the formation of bipolar and Müller glial cells, indicating that Barhl2 is able to function both as a positive and negative regulator, depending on different types of cells. Taken together, our data suggest that Barhl2 may function to specify the identity of glycinergic amacrine cells from competent progenitors during retinogenesis.     

Comparing the performance of one-column process and four-zone simulated moving bed by computer simulation

A new one-column chromatography process, analogous to a four-zone simulated moving bed (SMB), was presented. The basic principle of the process was identical to that of a four-zone SMB. The process consisted of one chromatographic column and four tanks, instead of the four columns in the four-zone SMB (1-1-1-1), and has been used for the separation of two amino acids, phenylalanine and tryptophan, using an ion exchange resin. The operating parameters for the one-column process and four-zone SMB were obtained from equilibrium theory. Computer simulations were used to compare the performances of the new one column process to that of the general four-zone SMB, using Aspen Chromatography^™ v 11.1. The differences between the one-column and SMB processes in terms of the purities and yields of phenylalanine and tryptophan were less than 4 and about 6%, respectively. The lower purities of the one-column process were due to the loss of the developed concentration profiles in the column when the liquid was stored in tanks. The one-column process gave great flexibility, and would be useful for reconstructing an existing conventional chromatography process to one of a SMB.     

Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.