Transcriptional activity of the phosphoenolpyruvate carboxykinase gene decreases in regenerating rat liver.

Rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene expression and enzyme activity in liver were studied in rats fasted for 12 hours before and after partial hepatectomy, sham operation or no operation. Transcriptional activity and mRNA levels decreased in regenerating liver compared to sham-operated and unoperated controls. In contrast, PEPCK enzyme activity in regenerating liver was similar to that in the livers of sham-operated and unoperated controls. Since all the animals were fasted the decrease in transcription is probably caused by some factor other than insulin, the known repressor of PEPCK gene expression.

The acute phase response in the rodent.

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

mRNA for kininogens and thiostatins (major acute phase alpha 1-proteins) in the liver of kininogen-deficient rats.

mRNAs for low and high molecular weight kininogens (1.6 and 3.0 kb in size, respectively) and for two thiostatins (1.6 kb in size) were found in the liver of kininogen-deficient Brown-Norway (BN/Mai Pfd) rats. The levels of mRNAs for thiostatins, but not those for low and high molecular weight kininogens (arising from a single kininogen gene), increased strongly during acute inflammation. The pattern of DNA restriction sites for the kininogen gene and the thiostatin genes in the mutant rat strain was identical to that in at least four other rat strains.

ABO blood group and related antigens, natural antibodies and transplantation.

The current success rate of transplant surgery and immunosuppression has led to a demand for organs that has outstripped the supply. This has required investigation of alternate strategies. Therefore, allotransplantation across the ABO blood group barrier has commenced, and pig-to-human xenotransplantation is under consideration. The first immunological barrier to both these types of transplantation is the prevention of the antibody-mediated rejection. This rejection is a result of natural preformed antibodies circulating in the serum of the recipient binding to either ABO (for allo) or alpha-galactose (alpha-Gal) (for xeno) antigens expressed on the donor tissue. These antibodies recognise antigens that are, in both cases, carbohydrate molecules with the characteristic feature that the nonreducing terminal carbohydrate is either a Gal or N-acetlygalactosamine residue in an alpha1,3 linkage. These epitopes are synthesised by closely related members of a single family of glycosyltransferases. This review discusses the carbohydrate antigens, the enzymes involved in their synthesis and the consequences of natural antibodies binding these antigens.

Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry.

Recombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells. Binding of sCD46 to cell surface H was increased about twofold when measles virus fusion protein was coexpressed with H. sH bound to wild-type cell surface CD46 and inhibited measles virus binding onto CD46-expressing cells. sCD46 also inhibited virus infection. Thus, the direct interaction between the ectodomains of H and CD46 is likely to be the primary event in measles virus infection.

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro.

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35 . More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsD55 and rsCD35 to regulate human complement activation mediated by the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46+ rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCd55 and rsCD35, may be useful a therapeutic agent.

Translation is enhanced after silent nucleotide substitutions in A+T- rich sequences of the coding region of CD46 cDNA.

Specific sequences in the coding region of CD46 (membrane cofactor protein) transcripts have been shown to have a marked effect on translation. Two A+T-rich regions of CD46 cDNA were altered by mutation without changing the CD46 amino acid sequence (silent nucleotide substitution). In one region, the A+T content was reduced from 78% to 55% and in the other a putative polyadenylation addition sequence was disrupted. In each example, mutated sequences transfected into COS-7 cells produced significantly more soluble or cell surface protein (up to a 20-fold increase) than wild-type sequences. The amount of cellular plasmid DNA and CD46 mRNA was not increased, suggesting that the effect was not due to increased transfection efficiency, or transcript synthesis or stability. Biosynthetically labelled transfected cells showed an increase in translation rate but cell-free in vitro translation studies demonstrated that wild-type and mutated transcripts were translated with similar efficiency. The data show that translation of CD46 is affected by specific mRNA coding sequences, 400-540 bases from the initiation codon, and suggest that these sequences require the structural integrity of the cell to exert their effect.

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization.

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.

Gene expression in regenerating and acute-phase rat liver.

The integration of growth and the acute-phase response is investigated by comparing the mRNA levels in rat liver during acute inflammation with those after partial hepatectomy. Northern analysis is carried out for the mRNAs for thiostatin, alpha 2-macroglobulin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor subunit 1, haptoglobin, ceruloplasmin, transferrin, vitamin D-binding protein, alpha 1-acid glycoprotein, beta-fibrinogen, apolipoproteins A-IV and E, albumin, transthyretin, alpha 2-HS-glycoprotein, retinol-binding protein, beta-tubulin, c-myc protooncogene, glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, ornithine transcarbamylase, and alcohol dehydrogenase. The acute-phase response dominates during the first 18 h. Changes in mRNA levels related to growth of the liver become important thereafter, and the capacity for an acute-phase response of plasma protein synthesis becomes greatly reduced. The early increase in the level of ceruloplasmin mRNA observed during inflammation is abolished during regeneration, and that of vitamin D-binding protein mRNA is converted into a decrease. The mRNAs levels of glyceraldehyde-3-phosphate dehydrogenase increase, and those for phosphoenolpyruvate carboxykinase decrease during regeneration. Ornithine transcarbamylase mRNA levels are found to exhibit negative acute-phase regulation. The pattern of transcriptional regulation is similar during inflammation and regeneration.

Structure and expression of the rat transthyretin (prealbumin) gene.

The rat transthyretin gene, 7.3 kilobase pairs (kb) long, with 14.5 kb of 5' flanking and 12.2 kb of 3' flanking region was cloned and characterized. The gene contained four exons. A "TATA box" sequence (5'-TATATAA-3') and a "CAAT box" sequence (5'-GTCAAT-3') were located 23 and 95 nucleotides upstream, respectively, from the major transcription start site. Nucleotides -51 to -189 were highly conserved (93% homology between rats and humans, 97% homology between rats and mice). Tandem repeats of sequences of 5'-AC-3' and 5'-ACACATGC-3' in the 5' flanking region, of 5'-GAAA-3' in the first intron, and of 5'-GT-3' in the third intron of the gene were observed. Using specific cDNA probes, tissue specificity and regulation of transthyretin mRNA biosynthesis during embryogenesis were analyzed. Transthyretin expression occurred first in the yolk sac, then decreased when expression increased in fetal liver. Presumptive choroid plexus cells in the inner lining of the neural tube expressed transthyretin early in gestation (11 days before birth) with a maximum immediately preceding the spurt of brain growth around birth. Partial hepatectomy of adult rats induced both an acute phase response and regenerative growth in liver. The decrease in transcription of the transthyretin gene in liver, which is characteristic for the acute phase response, was overridden by stimulation of gene expression after partial hepatectomy. This stimulation also affected transthyretin expression in choroid plexus.

Engineering of recombinant soluble CD46: an inhibitor of complement activation.

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies. Following a single-step immunoaffinity purification, the protein resolved as a single band of approximately 56,000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rsCD46 (51 micrograms/ml) protected Chinese hamster ovary (CHO) cells from lysis initiated by a high titre rabbit anti-CHO antibody and complement from rabbit or human. The protection was specifically mediated by rsCD46 because the monoclonal antibody M177, which blocks interaction between CD46 and C3b/C4b, abrogated the protection. The results demonstrate that rsCD46 is effective as a fluid-phase regulator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Administration of a bolus injection of rsCD46 was effective at delaying hyperacute graft rejection. These data suggest that rsCD46 may have a role as a therapeutic agent.

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.