The inter-alpha-inhibitor (I alpha I) family encompasses four plasma proteins, namely free bikunin as well as I alpha I, pre-alpha-inhibitor (P alpha I) and inter-alpha-like inhibitor (I alpha LI). Each of the last three proteins is a distinct assembly of one bikunin chain with one or more unique heavy (H) chains designated H1, H2 and H3. The three H chains and the bikunin chain are encoded by four distinct mRNAs. These molecules and chains, as well as the corresponding mRNAs, were quantified in sera and liver biopsies from a series of patients with or without mild or severe acute infection. The decrease or increase observed for a given molecule or chain in the serum was in agreement with a similar change in the corresponding liver mRNA. In acute inflammation the H2 and bikunin chains are down-regulated and the relevant molecules (I alpha I, I alpha LI) behave as negative acute-phase proteins, whereas the H3 chain is up-regulated and the corresponding P alpha I molecule is a positive acute-phase protein. Also, P alpha I displays a higher-than-usual M(r); this is probably due to ligand binding. The H1 gene does not seem to be affected by the inflammatory condition. The quantitative changes in RNA levels seen in vivo were confirmed in vitro in the human hepatoma Hep3B cell line prior to or after induction with the acute-phase mediators interleukin-1 and/or -6. These results provide the first example in humans of positive and negative acute-phase proteins that are encoded by evolutionary related genes.

The acute-phase protein (APP) response is regulated by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor (TNF), but may also be influenced by malnutrition. The aims of this study were as follows: 1) to determine in rats the effect of a protein-deficient diet on IL-6 mRNA expression in intestine, liver and peripheral blood mononuclear cells (PBMC), and on alpha-1 acid glycoprotein (AGP) and alpha-2 macroglobulin (A2M) serum levels and hepatic mRNA expression; 2) to compare, in protein-deficient rats, the IL-6 and APP responses after a turpentine (TO)- or a lipopolysaccharide (LPS)-induced inflammation; and 3) to determine the effect of a protein malnutrition on IL-6 mRNA expression in rat PBMC treated ex vivo with LPS. Interleukin-6 mRNA was present in intestine and PBMC but not in the liver of malnourished rats, and was absent in any tissue or cells of controls. A2M was present in the serum from malnourished rats but not after refeeding. AGP mRNA expression was not influenced by protein malnutrition. In malnourished rats, IL-6 serum level peaked later than in controls after TO and LPS treatment. In malnourished TO-treated rats, A2M mRNA increased earlier than in controls and remained detectable later than in controls. AGP mRNA expression after TO was not influenced by protein malnutrition. In PBMC of malnourished rats, LPS-induced IL-6 mRNA expression occurred earlier and lasted longer than in controls. Our results indicate that protein malnutrition by itself induces IL-6 and A2M expression, and that it modulates the APP response to inflammation.

Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.

Complementary DNA clones for human Cls were isolated from cDNA libraries that were prepared with poly(A)+ RNAs of human liver and HepG2 cells. A clone with the largest cDNA insert of 2664 base pairs (bp) was analyzed for its complete nucleotide sequence. It contained 202 bp of a 5' untranslated region, 45 bp of coding for a signal peptide (15 amino acid residues), 2019 bp for complement component Cls zymogen (673 amino acid residues), 378 bp for a 3' untranslated region, a stop codon, and 17 bp of a poly(A) tail. The amino acid sequence of Cis was 40.5% identical to that of COr, with excellent matches of tentative disulfide bond locations conserving the overall domain structure of COr. DNA blotting and sequencing analyses of genomic DNA and of an isolated genomic DNA clone clearly showed that the human genes for COr and Cls are closely located in a "tail-to-tail" arrangement at a distance of about 9.5 kilobases. Furthermore, RNA blot analyses showed that both COr and Cis genes are primarily expressed in liver, whereas most other tissues expressed both COr and Cis genes at much lower levels (less than 10% of that in liver). Multiple molecular sizes of specific mRNAs were observed in the RNA blot analyses for both COr and Cis, indicating that alternative RNA processing(s), likely an alternative polyadenylylation, might take place for bith genes.The human complement system, which is composed of the classical and alternative pathways, involves about two dozen plasma proteins, including proteases and cofactors (1-3). These proteins are sequentially activated to form a lytic complex that attacks the foreign cell. Anaphylatoxic and vasoactive peptides are also generated during the course of this reaction (4). Clr and Cls are single-chain plasma glycoproteins of about 85 kDa. These proteins are highly homologous to each other, and both are subcomponents of the complement C1 complex in which two molecules of each Cir and Cls form a complex with one molecule of Clq, another subcomponent of C1, in the presence of calcium ions (2,3). Clq in the C1 complex binds to the antigen-antibody immune complex through the constant region of immunoglobulin heavy chains, resulting in autoactivation of Clr (5). The activated Clr then proteolytically activates Cls, which in turn activates C2 and C4 in the complement cascade reactions. Upon proteolytic activation, both Clr and Cls are converted to two-chain-form proteases comprised of a heavy chain (58 kDa) and a light chain (27 kDa) and are readily inhibited by C1 inhibitor, forming a stoichiometric complex.Recently, an isolation and characterization of Clr and its unique domain structures have been reported (6). In addition to the serine protease module contained in the light chain of Cir, the heavy chain of Clr contains five distinct structural domains. Domains I and III are homologous repeats, and domain II is an epidermal growth factor precursor-like sequence that is also found in many other proteins such as blood coagulation factors and low density lipoprotein receptors. Doma...