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The effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the glycosylation and secretion of alpha 1-antitrypsin (three complex type oligosaccharide chains) and of alpha 1-acid glycoprotein (six complex type oligosaccharide chains) was studied in rat hepatocyte primary cultures. In the presence of 4 mM Bay m 1099 the processing of high-mannose to complex type oligosaccharides was partially inhibited leading to the secretion of alpha 1-antitrypsin and alpha 1-acid glycoprotein carrying a mixture of both high-mannose and complex type oligosaccharides. The major part of alpha 1-antitrypsin secreted by Bay m 1099 treated cells still carried two complex type oligosaccharide chains, the majority of alpha 1-acid glycoprotein carried three to five. Despite its effects on protein glycosylation Bay m 1099 did not lead to pronounced changes in the synthesis or secretion of alpha 1-antitrypsin, alpha 1-acid glycoprotein or albumin. At concentrations of Bay m 1099 lower than 0.5 mM no inhibitory effect on oligosaccharide trimming could be observed. After removal of Bay m 1099 from hepatocytes its inhibitory effect on protein glycosylation was immediately reversible.

The effect of age on several messenger RNAs coding for non-specific immune factors were determined in liver RNA isolated from 6-, 12-, 24-, 29- and 37-month-old male Fischer Rats. The levels of complement factors C3 and C4, complement protein factor B, and alpha 1-acid glycoprotein were determined by dot blot hybridization using cDNA probes. All four mRNAs increased slightly between 6 and 29 months of age. Only the mRNAs coding for complement factors C3 and C4 decreased significantly after 29 months of age. In addition, Northern blot analysis of the RNA preparations showed that the size of the four mRNA species did not change with increasing age. There was no evidence for age-related changes in the post-transcriptional processing or degradation of the mRNA species coding for complement factors C3 and C4, complement protein factor B, and alpha 1-acid glycoprotein.

Sera from transgenic mice (TM) carrying human genes of alpha 1-acid glycoprotein (orosomucoid or ORM) have been analyzed by isoelectrofocusing and subsequent immunoblotting with antihuman ORM antibodies. With this technique it is possible to reveal selectively the human protein secreted in the TM sera. Orosomucoid bands present in TM sera have been compared with those of the most common human ORM phenotypes to correlate the products of specific genes to previously identified genetic variants. In this paper, we report the identification of the genes encoding for variants ORM1 F1 and ORM2 A, which are genes AGP-A and AGP-B/B' respectively. The nucleotide sequences of these genes are known; therefore a direct correlation between variants and specific amino acid sequences can be established.

We used a system of co-culture of adult rat hepatocytes with another epithelial cell type from rat liver to study the synthesis of two acute-phase reactants, alpha-1 acid glycoprotein (alpha 1AGP) and the third component of complement (C3), and we have obtained long-term secretion of these two proteins. After a period of adaptation corresponding to the first 2-4 days of the co-culture, hepatocytes secreted C3 and alpha 1AGP for at least 2 weeks at a mean level higher than that observed in the first days of a pure culture of hepatocytes. When pulse-chase analysis was performed on day 6 of co-culture, kinetics of synthesis of alpha 1AGP and C3 were the same as those observed on day 1 of a conventional culture of pure hepatocytes. Furthermore, intracellular and extracellular alpha 1AGP had Mr values respectively of 39,000 and of 42,000-52,000, identical with those observed in pure cultures of hepatocytes. Similarly, the molecular size and subunit structures of C3 were the same in co-culture and in cultures, indicating an identical processing of this protein. C3 produced in co-culture was also haemolytically active. Therefore, the system of adult hepatocytes co-cultured with this liver epithelial cell provides a physiological system in vitro which permits long-term synthesis of the two acute-phase reactants C3 and alpha 1AGP. This model opens the possibility to study the modulation of the synthesis of these two proteins during a long period by inflammatory agents or by hormones.

To study the effect of intraabdominal sepsis on hepatic protein synthesis, male Sprague-Dawley rats underwent celiotomy with either cecal ligation and puncture (CLP) or sham operation. Eight and sixteen hours later total hepatic protein synthesis was measured by flooding dose technique. Specific synthetic rates of structural or secreted hepatic proteins were further studied 16 hr after CLP in an isolated perfused liver model. Total hepatic protein synthesis was significantly elevated at 16 hr (59 +/- 6%/day vs 37 +/- 6%/day, P less than 0.05), but not 8 hr post-CLP. Structural hepatic protein synthesis was unchanged after CLP; however, the synthetic rates of the acute-phase secretory proteins alpha 1-acid glycoprotein, transferrin and complement component C3 were significantly increased 16 hr after CLP. However, the albumin synthetic rate was not increased during sepsis. We conclude that sepsis causes augmentation of hepatic protein synthesis primarily to increase acute-phase proteins for host defense.

In vivo incorporations of (14)C-leucine and (3)H-galactose into the seromucoid proteins are decreased in streptozotocin diabetic rats. The decrease in galactose incorporation is greater than that of leucine, and suggests that streptozotocin diabetes influences the metabolism of both the protein and sugar moieties of seromucoid glycoproteins.

The effect of dexamethasone on the synthesis of acute phase proteins has been studied in primary cultures of rat hepatocytes. In the absence of dexamethasone no detectable amounts of alpha 2-macroglobulin were synthesized by hepatocytes cultured for 1 day. alpha 2-Macroglobulin synthesis was induced by dexamethasone concentrations of 10(-8) M or higher with a maximum at a concentration of 10(-7) M. alpha 1-Acid glycoprotein was synthesized in the absence of dexamethasone; however, its synthesis was also greatly stimulated by dexamethasone concentrations of 10(-8)-10(-6) M. Synthesis of alpha 1-proteinase inhibitor was stimulated only 1.4-fold at a dexamethasone concentration of 10(-7) M. The kinetics of induction of alpha 2-macroglobulin and alpha 1-acid glycoprotein were studied at a dexamethasone concentration of 10(-7) M. After an initial lag phase of 3 h the synthesis of both proteins showed a steady increase during 2 days. Synthesis of albumin remained unchanged under these experimental conditions. Unlike alpha 2-macroglobulin and alpha 1-acid glycoprotein tyrosine aminotransferase activity increased already during the first 3 h of induction by dexamethasone with a maximum at 12 h followed by a slight decrease.

Automated Edman degradation of alpha 1-acid glycoprotein from serum (serum AGP) was successful only after the desialylated, reduced, and alkylated protein had been treated with pyroglutamate aminopeptidase. The sequence is (less than Glu)-Asn-Pro-Glu-Pro-Ala-X-Ile-Thr-Leu-Gly-Ile-Pro-Ile-. For radioactive sequencing of the NH2 terminus of the intracellular precursor form (liver AGP) of serum AGP, protein was labeled by incubating liver cells in suspension with either [3H]proline or [3H]isoleucine for a 15-min period. Liver AGP was separated from serum AGP by ion exchange chromatography. Between 78 and 90% of the radioactivity incorporated into total AGP was associated with liver AGP, indicating that little conversion of newly synthesized liver AGP to serum AGP had occurred during the 15-min incubation period. Liver AGP was then isolated by immunoprecipitation. The antigen-antibody complex was exposed to 15 Edman degradation cycles; however, no radioactivity was released. Automated Edman degradation of liver AGP which had been extracted from the immunoprecipitate with dilute acid and subsequently treated with pyroglutamate aminopeptidase yielded a sequence of proline and isoleucine identical with that in serum AGP. We conclude that processing of the intracellular precursor form of AGP does not involve a proteolytic trimming near the NH2 terminus.

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.

The administration of phenobarbital (80 mg/kg body weight for 3 days) increased the plasma orosomucoid level with dramatic alterations in the relative proportions of heteroglycans. A crossed immunoaffinoelectrophoresis with Con A revealed an increase of strongly reactive fractions with a concomitant decrease of non and weakly reactive fractions.

The isolated rat liver perfused for 12 hours at pH 7.10 with a suspension of bovine erythrocytes in Krebs-Ringer bicarbonate buffer containing 3 per cent bovine serum albumin has been used as a test system to study effects of glucagon and of dexamethasone in the presence and absence of insulin on net biosynthesis of rat serum albumin, fibrinogen, alpah1-acid glycoprotein, alpha2-(acute phase) globulin, and haptoglobin. Quantitative measurement of perfusate glucose, amino acid nitrogen, and urea affords a basis for determining net glucose and nitrogen balance in the perfusion system. Although the dose of dexamethasone (total 1.0 mug.) used was insufficient to induce synthesis of alpha2-acute phase globulin, net syntheses of albumin, fibrogen, alpha1-acid glycoprotein, and haptoglobin were increased. Glucagon given with dexamethasone depressed albumin and haptoglobin synthesis markedly, but not that of fibrinogen and alpha1-acid glycoprotein. Glucagon with dexamethasone markedly enhanced ureogenesis and glycogenolysis and elicited an exaggerated negative nitrogen balance. The unfavorable effects of glucagon on albumin and haptoglobin synthesis and on nitrogen balance were reversed by giving insulin simultaneously. It is emphasized that insulin is essential for positive nitrogen balance.

Inflammation results in an increase in the levels of a variety of glycoproteins in serum. The glycoproteins that respond in this way are usually referred to as acute-phase reactants. Studies on the acute-phase response of rat alpha 1-acid glycoprotein showed that there was an increase in the liver levels of this glycoprotein at 12 h after turpentine inflammation. This was followed by increased serum levels at 48-72 h after inflammation, suggesting a precursor-product relationship between liver and serum alpha 1-acid glycoprotein. Incorporation studies coupled with measurements of synthesis rates of alpha 1-acid glycoprotein showed that increased synthesis was responsible for the acute-phase response of this protein to inflammation. These studies also showed that albumin was a negative acute-phase reactant. The acute-phase response of alpha 1-acid glycoprotein was accompanied by increased liver pools of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) and increased liver activities of glucosamine-6-phosphate synthase and UDP-GlcNAc 2-epimerase. Activities of galactosyl and sialyl transferases in liver were also elevated and serum sialyl transferase was increased substantially in inflammation, suggesting that it may also be an acute-phase reactant. Liver activities of beta-N-acetylhexosaminidase and beta-galactosidase declined by about 50% at 24 h after inflammation; there was evidence that serum levels of these enzymes increased at 24-72 h after inflammation, suggesting that the lysosomal glycosidases may be released from liver during inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant and uninvolved human breast tissues were maintained in organ culture for 3 to 6 days. Under these conditions, the three-dimensional glandular architecture is maintained with the least disruption of tissue integrity. The biosynthesis and release of glycoproteins were studied by using the incorporation of [14C]glucosamine and [14C]leucine by the breast surgical specimens. Five major families of labeled glycoproteins were identified from culture supernatants using two-dimensional gel electrophoresis. Quantitative immunoprecipitation established that 16 to 30% of the total of labeled glycoproteins were recognized as normal serum components. Two of these glycoproteins were antigenically related to normal human serum components as demonstrated with crossed immunoelectrophoresis. Evidence was obtained for the active synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. alpha 1-Antichymotrypsin accounted for 0.9 to 7.8% of the biosynthetically labeled glycoproteins from organ culture supernatants. This component was 11.9% of the glycoproteins released by a monolayer culture of the established breast carcinoma cell line, MCF-7. alpha 1-Acid glycoprotein made up 0.7 to 3.1% of the labeled glycoproteins. alpha 1-Antichymotrypsin is a known neutral serine proteinase inhibitor with a particularly strong affinity for cathepsin G. alpha 1-Acid glycoprotein may function primarily as a potent immunomodulator by suppressing lymphoblastogenesis. These glycoproteins may thus have regulatory roles in the proteolytic modification of breast tissue and represent the tissue's own protecting shield against invading leukocytes.

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

We have studied the glucocorticoid-mediated accumulation of alpha 1-acid glycoprotein (AGP) in mRNA in HTC rat hepatoma cells. In contrast to the well-characterized primary response of mouse mammary tumor virus, in vitro transcription assays in isolated nuclei show that the rate of transcription of the AGP gene is high even in the absence of hormone. Despite the constitutive transcription of the AGP gene, no detectable AGP RNA can be found in either the cytoplasm or the nuclei of untreated cells. Previous experiments have shown that the glucocorticoid induction of AGP RNA requires ongoing protein synthesis. In conjunction with the present study, our data suggest that glucocorticoids stimulate accumulation of AGP RNA by inducing an RNA processing factor that allows production of stable transcripts.

Blood samples were obtained on four different occasions from 18 cigarette smoking and 34 non-smoking healthy men (age 40-69) and analyzed to assess age- and smoking-associated changes in plasma proteins, blood coagulation and platelet functions. Collagen-induced platelet aggregation was significantly increased with aging in non-smokers. Significant changes in chronic smokers were increases in platelet count and fibrinogen in plasma; elevation of platelet factor-3 (PF-3) activity in platelet-poor plasma (PPP); increase in serum levels of alpha 1-antitrypsin, orosomucoid, haptoglobin and properdin factor B; and shortening of the lag period of collagen-induced platelet aggregation. Filtration of PPP through Millipore filters removed PF-3 membranes. The differences in PF-3 activities in filtered plasma were no longer significant between smokers and non-smokers. Results suggest that chronic smokers have higher levels of acute phase proteins reflecting underlying inflammatory processes, and higher levels of PF-3 activity in plasma due to liberation of PF-3 membranes from platelets.

The plasma half life of recombinant human interleukin 1 beta (rhIL 1 beta) was determined in rats by measuring the disappearance of the radioactivity of 125I-labeled rhIL 1 beta from the circulation. The plasma clearance showed a biphasic behavior: an initial fast disappearance (half life of about 3 min) was followed by a second slower one (half life of about 4 h). Twenty minutes after a single-dose injection of 125I-labeled rhIL 1 beta most of the radioactivity was concentrated in kidneys, liver and intestine. rhIL 1 beta induced the synthesis of alpha 1-acid glycoprotein (AGP), alpha 1-cysteine proteinase inhibitor (CPI) and beta-fibrinogen mRNA in liver. Half maximal stimulation was elicited by approximately 3000 U of rhIL 1 beta per animal. The mRNA changes for AGP and CPI were followed by corresponding protein increases in serum. Twenty hours after rhIL 1 beta injection, serum AGP rose from 0.7 to 2.5 mg/ml. CPI increased from 0.3 to 1.9 mg/ml 25 h after administration of rhIL 1 beta. Within 20 h after rhIL 1 beta injection, albumin serum concentration showed a strong decrease, preceded by a reduction in hepatic albumin mRNA levels. Neither changes in albumin synthesis nor degradation can explain this decrease suggesting that other mechanisms such as increased transvascular permeability are involved.

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.

Rat alpha 1-acid glycoprotein is an acute phase reactant which shows a marked elevation in mRNA level following inflammatory induction. It has been proposed that both transcriptional and post-transcriptional mechanisms regulate the induction of the gene. We have studied the processing of the primary transcript of alpha 1-acid glycoprotein. The preferred pathway of intron removal was determined by Northern blot analysis and was found to be unaltered after inflammatory stimulation. The final nuclear precursor did exhibit size alterations, manifest as a quantitative shift from the final precursor at 6 h to a second progressively shorter form at 18 and 24 h. Deadenylation of nuclear RNA showed that the difference in size of the precursor is due to a change in poly(A) tail length, which occurs after the splicing out of the last intron. Nuclear run-on transcription assays measured a 2.5-fold increase in transcriptional activity, with a peak at 12 h. The highest level of cytoplasmic RNA with a long poly(A) tail, however, occurs before 12 h. Our data suggest that the reduction in poly(A) tail size is due to a rapid trimming of the tail in the nucleus and that this process is modified upon inflammatory induction.

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.

Hybridization studies using specific cDNA probes have been used to determine the specific mRNA levels for apolipoproteins B and E, alpha 1 acid glycoprotein and beta actin in extracts of rat liver. Injection of rats with recombinant mouse tumor necrosis factor had led to a rapid increase in liver mRNA levels for alpha 1 acid glycoprotein (x 12) and for beta actin (x 2.5) whereas mRNA levels for Apolipoprotein B and E remained stable over the same period.

alpha 1-acid glycoprotein (alpha 1-AGP), or orosomucoid, is shown to be inducible by glucocorticoids in HTC rat hepatoma cells. Immunoprecipitation of [35S]methionine pulse-labeled proteins from these cells reveals secreted proteins of Mr = 35,000-48,000 (alpha 1-AGP) and Mr greater than 180,000, both of which are greatly enhanced by glucocorticoid treatment. The amount of alpha 1-AGP-specific mRNA in HTC cells is greatly increased (at least 100-fold) in response to glucocorticoids. The new steady-state level of RNA is approached with a t 1/2 of about 8 hr and the RNA consists of a single species of approximately 850 bases. The response is specific for glucocorticoids since: (i) the EC50 for dexamethasone is 30 nM; (ii) the glucocorticoid antagonist, progesterone, inhibits the induction by dexamethasone; and (iii) a glucocorticoid receptor-deficient cell line is incapable of alpha 1-AGP mRNA induction. This is a secondary hormonal response since inhibition of protein synthesis blocks the induction of alpha 1-AGP mRNA by dexamethasone whereas the induction of mouse mammary tumor virus (MMTV) RNA is unaffected.

In order better to define the extent of protein synthesis capacity of the human prostate, we have studied the translation of selected serum proteins using isolated poly(A)+ RNA preparations and the rabbit reticulocyte lysate system. The translation of alpha 1-acid glycoprotein could be conclusively demonstrated but there was no apparent translation of albumin and plasmatic transferrin. Labeled alpha 1-acid glycoprotein was identified by specific immunoprecipitation with a commercial anti alpha 1-acid glycoprotein antiserum and correct processing by canine pancreatic microsomal membranes. Furthermore, we have shown by the immunoperoxidase technique that alpha 1-acid glycoprotein was indeed localized mainly in prostatic epithelial cells in 2 out of 2 patients with benign prostatic hypertrophy and in 3 out of 11 patients with prostatic adenocarcinoma. The significance of the synthesis and secretion of alpha 1-acid glycoprotein by prostatic cell themselves is presently unknown. However, we think that it could represent an interesting subject to explore further in relation with prostatic inflammation.

A new drug interaction is described in which the plasma binding of propranolol is markedly stimulated by chronic phenobarbital administration in dogs. After 3 weeks of phenobarbital therapy, the percentages of unbound propranolol fell from 15.2 +/- 1.2 (mean +/- S.E.) to 2.4 +/- 0.3 (P less than .001). The time course of the induction and recovery of the binding of propranolol was also investigated. A half-maximal stimulation of binding was seen by day 3 of phenobarbital therapy and a plateau was reached after 2 weeks. After discontinuing phenobarbital, the recovery was delayed with the midpoint of the return to a normal binding value occurring on day 17. A new model of the time course for induction which includes the pharmacokinetics of the inducing agent has been developed. The stimulation of protein production by phenobarbital is assumed to follow Michaelis-Menten kinetics, but with a lag phase for the expression of the induction. Using this model, the Km for phenobarbital-stimulated production of the binding protein was 1200 ng/ml, the turnover T1/2 of the protein was 3.4 days and the T1/2 for the lag phase was also 3.4 days. The increased plasma protein binding was highly correlated (r = 0.97) with the elevated concentration of nonprecipitable glycoproteins. This most likely represents induction of alpha-1 acid glycoprotein which is the principal binding protein of propranolol in man. The plasma binding of propranolol was also stimulated with chronic phenytoin in these dogs and with a single dose of Arochlor 1254 in rats.

Expression of hepatocyte protein gene-products changes as cells adapt to the environment of tissue culture. We examined such changes, using "giant" two-dimensional (2-D) gel electrophoresis. Over 80 cellular and secreted proteins, about 3% of the 2500 we examined, were found to change spontaneously during the 20-h culture period. The magnitude of these changes was estimated to range from 1.5- to nearly 100-fold. If dexamethasone is included in the culture medium, the overall production of secreted proteins is maintained, but in addition it exerts major influences on the expression of individual protein gene-products. Ten of the spontaneous changes are enhanced and eight are unaffected, but most are substantially retarded (32) or even reversed (27). There are also large inductions or repressions in 12 proteins that do not change spontaneously in culture. We have tentatively identified tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, gamma-fibrinogen, and alpha 1-acid glycoprotein to be among those induced by dexamethasone. Evidently, many more changes occur as these cells adapt to tissue culture than was previously appreciated. These results emphasize the power of 2-D gel analysis in monitoring the protein phenotype of cells. We have also shown that glucocorticoids are important in maintaining the overall synthesis of secreted proteins and the protein phenotype of isolated hepatocytes in primary culture.

Soluble alpha 1 acid-glycoprotein is considered an "acute phase protein" with an inhibitory effect on lymphocyte activity; it has recently been shown that a lymphocyte modulatory variant of alpha 1 acid-glycoprotein has a positive role on T cell activation. It is not clear whether the presence of this glycoprotein on lymphocyte membranes is due to an endogenous production or to a passive uptake of soluble alpha 1 acid-glycoprotein by its carbohydrate moiety. Our data show an increase of membrane alpha 1 acid-glycoprotein both in peripheral blood lymphocyte and T-enriched lymphocytes after phytohemagglutinin stimulation. Peripheral blood lymphocyte enzymatic treatment by neuraminidase does not affect alpha 1 acid-glycoprotein expression while pronase digestion induces a strong decrease of alpha 1 acid-glycoprotein positive lymphocytes and a resynthesis after phytohemagglutinin stimulation. Furthermore, the presence of alpha 1 acid-glycoprotein was prevalently, found on helper/inducer lymphocytes. These data support the hypothesis of a synthesis of alpha 1 acid-glycoprotein by T lymphocytes during their activation process.

The incorporation of [14C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt). The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of alpha 1-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.

The hepatic synthesis of four "acute phase reactants" (APR), i.e., fibrinogen, alpha 1-acid glycoprotein, alpha 2-macroglobulin, and haptoglobin has been investigated in rats suffering from turpentine-induced inflammation. To follow the change in the rates of synthesis of the four APR, their concentrations were measured by immunonephelemetry in both the plasma and the hepatic microsomal fraction at various times after injury. A synchronous increase in the concentrations of these four proteins was observed in the liver (maximum 24 hours) as well as in the plasma (maximum 40 hours) of the same animals. In parallel, the site of their synthesis was localized in liver sections by light and electron microscopy using direct immunoperoxidase labeling. Of the liver cells, only the hepatocytes were labeled. In the early period of the inflammatory reaction (10 to 16 hours), synthesizing cells were detected principally in the periportal zone, but later (24 hours), the labeled area was extended to nearly the entire hepatic lobule. When serial sections of liver were examined at that time, the same cells were found to contain simultaneously the four APR. Within the cells examined by electron microscopy, the four proteins were localized in the secretory pathway, i.e., rough and smooth endoplasmic reticulum, Golgi apparatus, and secretory vacuoles. Therefore, we conclude that: (1) the experimental inflammatory reaction induces a synchronous increase in the synthesis of four APR by the liver; (2) this increased synthesis apparently results from an increased number of synthesizing hepatocytes; (3) these cells have a preferential periportal localization; and (4) individual hepatocytes are not specialized in the synthesis of a single plasma protein.

Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins.