Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.     

Interaction of allylisothiocyanate with bovine serum albumin

The interaction of allylisothiocyanate with bovine serum albumin was monitored by fluorescence titration. The interaction was weak with an apparent association constant of 2 × 10². The interaction was unaffected in the pH range of 5.0 to 8.3 and by NaCl. However, the addition of dioxane upto 4% increased the value of the association constant. N-Methyl bovine serum albumin and bovine serum albumin with sulphydryl groups blocked had the same affinity for allylisothiocyanate suggesting that amino and sulphydryl groups may not be involved in the interaction. Polyacrylamide gel electrophoresis and estimation of available lysine suggested that there were perhaps two types of groups involved in the interaction of allylisothiocyanate with bovine serum albumin. An erratum to this article is available at .     

Reduction of immunoreactivity of bovine serum albumin conjugated with polyethylene glycol(PEG) in relation to its esterase activity.

Bovine serum albumin was modified with activated PEG2, 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine. The PEG-modified albumin, in which 15 out of the total 60 amino groups in the albumin molecule were coupled with activated PEG2, lost immunoreactivity towards anti-albumin serum and retained 63% of the esterase activity of native albumin.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

Macrophage recognition of periodate-treated erythrocytes: involvement of disulfide formation of the erythrocyte membrane proteins

Upon exposure to 2 mM periodate at 0 degrees C for 15 min, mouse erythrocytes underwent membrane lipid oxidation, oxidation of cell surface sialyl residues into aldehyde-bearing derivatives, and oxidation of SH groups of the membrane proteins into disulfides. The periodate-treated erythrocytes exhibited a remarkable increase in rosette attachment to resident mouse peritoneal macrophages in the absence of serum. The relationship between the oxidation of the membrane constituents and the macrophage recognition of these cells was investigated. Periodate treatment of erythrocytes in the presence of butylated hydroxytoluene, an inhibitor of lipid oxidation, did not affect the subsequent attachment of the erythrocytes to the macrophages. Reduction of the periodate-treated erythrocytes with borohydride or cyanoborohydride did not affect the erythrocyte attachment. Neuraminidase treatment of erythrocytes before periodate did not affect the attachment either. On reduction of the disulfides of the membrane proteins with dithiothreitol, the periodate-treated erythrocytes lost their ability to attach to the macrophages. Erythrocytes treated with an SH-oxidizing agent, diamide, were then examined for the macrophage recognition. The diamide-treated cells also showed rosette attachment to the macrophages in the absence of serum, but did not when reduced with dithiothreitol. These results indicate that oxidation of the SH groups of the membrane proteins to disulfides causes reversible membrane changes that macrophages recognize, and it is this mechanism that is responsible for the macrophage recognition of the periodate-treated erythrocytes.     

Nature of albumin exported from the frog oocyte following microinjection of mouse liver mRNA

Microinjections of mouse liver mRNA into Xenopus laevis oocytes induced efficient export of a polypeptide with an apparent Mr or 68,000 which was immunoprecipitable with anti-mouse albumin antibody. Analysis of the anti-albumin precipitate of the exported protein by two-dimensional gel electrophoresis showed that the electrophoretic behavior precisely coincided with that of authentic mouse serum albumin. This result indicates that the Xenopus oocyte may perform secretory processes similar or identical to those occurring in liver cells with respect to the processing of albumin.     

Biosynthesis of serum albumin in rat liver. Evidence for the existence of `proalbumin''

1. A protein(s) of rat liver (precipitated from soluble extracts of the microsomal fraction by anti-albumin) yields albumin after limited hydrolysis by trypsin. 2. Evidence that the product of limited tryptic hydrolysis is albumin, is based upon ion-exchange chromatography, electrofocusing and peptide `mapping'. 3. The albumin `precursor' is recognized by anti-albumin and is apparently not distinguished from albumin by anti-albumin. 4. A small peptide is liberated from the presumptive albumin precursor during limited tryptic hydrolysis. This peptide is labelled by arginine, but not by leucine, lysine or methionine. 5. These results support our previous suggestion based on kinetic evidence that the albumin-like protein(s), in the anti-albumin precipitate from rat liver, is an albumin precursor.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

A protein kinase inhibitor in brown adipose tissue of developing rats

A heat-stable protein was extracted from brown adipose tissue of infant rats that inhibited both purified bovine heart protein kinase and a crude preparation of protein kinase from brown fat. It did not act as an adenosine triphosphatase nor did it exert its effect by proteolytic action. Inhibition of protein kinase was affected in both the presence and the absence of cyclic AMP. Most of the inhibitory activity was present in the 100000g supernatant fraction of tissue homogenates. Inhibitory activity was highest perinatally and it declined 10 days after birth. It is suggested that the protein kinase inhibitor may play a role in regulating cyclic AMP actions.     

The effect of serum albumin and the effect of cell concentration on the in vitro growth of mouse and rat lymphocytes.

The growth of mouse and rat T and B lymphocytes, activated by concanavalin A or lipopolysaccharide, is increased over growth in protein-free medium 5–20-fold by human or bovine serum albumin. The growth is dependent on the concentration of albumin. At a concentration of 2–4 mg/ml the growth rate is the same as in the presence of 10% fetal bovine serum. Of the other serum proteins (Cohn fractions) only human fraction VI supports growth somewhat while human fractions II–IV and bovine fraction VI do not support growth. The growth of mouse and rat lymphocytes is greatly suppressed if lymphocytes are cultured at high cell concentrations, and the growth-promoting ability of serum albumin cannot be detected under such conditions. The growth rate can be improved by daily adjustment of the pH, by daily refeeding, and by daily change of medium. The growth inhibitory activity can be removed largely by dialysis. It is concluded that the suppression of growth at high cell concentrations is caused by a combination of effects, i.e., a shift of pH, lack of nutrients, and accumulation of cellular metabolites.     

Characterization of inhibitor to leptospiral hemolysin present in bovine serum

Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform-methanol-soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin-layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective. From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.     

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.     

Further characterization studies of the alpha-amylase protein inhibitor of gel electrophoretic mobility 0.19 from the wheat kernel.

A highly purified amylase protein inhibitor from the kernels of hexaplois wheat, designated 0.19 according to its gel electrophoretic mobility, has been characterized according to its circular dichroism spectra determined at different pH values and in the presence or absence of dissociating and reducing agents. The 0.19 albumin has also been characterized according to the specificity with which it inhibits 21 alpha-amylases from different origins and according to its sensitivity to a number of chemical and enzymatic treatments of its inhibitory action on human saliva and Tenebrio molitor L. larval midgut alpha-amylases. Inhibitory activity of 0.19 toward human saliva amylase significantly increased when the inhibitor was incubated with the enzyme before the addition of starch, but it was not affected by the preincubation of 0.19 with starch. Maltose reversed the inhibition of human saliva by 0.19 and showed some inhibitory activity toward the enzyme. However, maltose concentrations that only slightly affected amylase activity were very effective in restoring the amylase activity inhibited by 0.19. The inhibitory action of 0.19 on human saliva and T. molitor L. amylases were equally resistant to trypsin and thermal treatments, but 0.19 was readily inactivated by incubation with pepsin or by reduction of disulfide bonds. The inhibition of the mammalian amylase by 0.19 was adversely affected by a treatment with CNBr (1:100 ratio of methionine residues to CNBr) whereas the inhibition of the insect amylase was not. As shown by circular dichroism measurements in the far ultraviolet, 0.19 is a protein with about 50% of ordered structure. Significant and largely reversible changes have been observed in the aromatic CD spectrum of 0.19 at alkaline pH values or in the presence of sodium dodecyl sulfate. These changes, which were associated with a partial loss of inhibitory activity, indicate that ionizable tyrosine groups contribute significantly to the ellipticity bands of 0.19 in the near ultraviolet.     

Binding of low-density lipoprotein to heparin-Sepharose: characterization and inhibition by serum high-molecular-weight components

In this study, the interaction of human serum low-density lipoprotein (LDL) with heparin immobilized on Sepharose was reinvestigated. Binding of isolated LDL (stabilized with human serum albumin (HSA] was compared with that of LDL in full serum. (1) Binding of isolated LDL was slightly decreased by CaCl2 and was not affected by MgCl2. In contrast, with full serum LDL binding was increased by these divalent cations. (2) In both situations, binding of LDL was saturable, but the maximum degree of binding that could be reached was much higher with isolated LDL than with LDL in full serum. This could be ascribed to an inhibitory action of a factor found in the d greater than 1.24 fraction of serum. (3) The effect of this factor was diminished in the presence of CaCl2 or MgCl2, which suggests that the stimulation of LDL binding by these cations in full serum is due to suppression of the inhibitory activity of this factor. (4) The inhibitory factor in the d greater than 1.24 fraction can be partially purified by absorption to heparin-Sepharose, followed by elution with 6 M guanidine chloride. The resulting preparation had a 30- to 50-fold higher specific activity. Attempts to purify the factor further resulted in loss of activity. (5) The activity is decreased upon treatment with trypsin and also upon acetylation or reduction with dithiothreitol, indicating that free amino groups and S-S bridges are essential.     

Effects of subacute treatment of ethylene glycol on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats

The present study was aimed to investigate the effects of ethylene glycol (EG) on serum marker enzymes, antioxidant defense systems and lipid peroxidation concentration (malondialdehyde=MDA) in various tissues of rats exposed to ethylene glycol. EG (1.25% or 2.5%) in drinking water was administered orally to rats (Sprague-Dawley albino) ad libitum for 21 days continuously. EG treatments caused different effects on the serum marker enzymes, antioxidant defense system and MDA content in various tissues of the treatment groups as compared with the controls. EG also caused a significant increase in the serum marker enzyme activities with 2.5% dosage whereas, no changes were not observed with 1.25% dosage of EG treatment. Lipid peroxidation significantly increased in all the tissues except for in the heart and stomach of rats treated with both dosages of EG. Also, the antioxidative systems were also seriously affected by EG. For example, SOD significantly decreased in the liver treated with both dosages whereas, SOD activity in the erythrocytes, kidney, heart and stomach were significantly increased and not changed in the brain with two dosages of EG. Also, while CAT activity significantly decreased in the erythrocytes, liver and kidney, the activity in the stomach significantly increased, but did not change in the brain and heart with two doses of EG. GR activity significantly decreased in the erythrocytes treated with both dosages of EG whereas GR was not affected in other tissues by EG treatment. GST activity significantly elevated in the heart and brain but did not change in the other tissues of rats treated with both dosages of EG. Meanwhile, GSH depletion in the erythrocytes of rats treated with 2.5% dosage of EG was found to be significant whereas, the level of GSH in the brain was significantly increased treated with both the dosages of EG. The observations presented led us to conclude that the administration of subacute EG promotes lipid peroxidatin content, elevates tissue damage serum marker enzymes and changes in the antioxidative systems in rats. These data, along with the determined changes suggest that EG produced substantial systemic organ toxicity in the erythrocyte, liver, brain, heart kidney and stomach during the period of a 21-day subacute exposure.     

Investigation of the haemolytic activity of Proteus mirabilis strains

Young broth cultures of all P. mirabilis strains tested exhibited haemolytic activity. This activity seemed to be strongly cell-associated as only a very small fraction of this activity was found in the cell-free supernatant. The haemolysin was only produced by actively growing cells. Inhibition studies with trypsin and chloramphenicol suggested that the haemolysin is of protein nature. Lecithin and serum of several species had an inhibitory effect on the haemolysin. Besides erythrocytes of various species also VERO cells were affected by the haemolysin. A correlation was found between the haemolytic activity of a strain and its virulence in an experimental mouse model.     

The reaction of S-mercuric-N-dansylcysteine with acetylcholinesterase and butyrylcholinesterase

S-mercuric-N-dansylcysteine was investigated as a potential probe of protein sulphydryl groups using bovine serum albumin, S-carboxymethyl-bovine serum albumin, lysozyme, and partially reduced lysozyme as test proteins. Criteria used to assess covalent binding through mercury-bridged mercaptide linkages include a finite reaction time (minutes to hours), abolition of the characteristic fluorescence spectrum following addition of a reducing agent, and failure to separate probe and protein after chromatography or electrophoresis. By these criteria, both Torpedo californica acetylcholinesterase and human serum cholinesterase (butyrylcholinesterase) contain four free sulphydryl groups per tetrameric enzyme molecule whereas Electrophorus electricus acetylcholinesterase has none. Labeled acetylcholinesterase and butyrylcholinesterase remain active and responsive to the inactivator Zn2+. Zn2+ promotes an increase in the fluorescence of bound S-mercuric-N-dansylcysteine, whereas activators such as Mg2+ or gallamine promote a decrease, suggesting that the label may be a useful probe of ligand-induced conformational changes. With T. californica acetylcholinesterase, but not with human serum cholinesterase, Zn2+ also promotes access to two additional groups that are reactive towards the sulphydryl reagent.     

Complement inhibitor(s) released by leukocytes. I. Pretreatment of sheep erythrocytes with supernatants of mouse spleen and thymus cells inhibit whole complement activity and C2 utilization.

Sheep erythrocytes pretreated with supernatants of mouse spleen or thymus cells become resistant to lysis by guinea pig complement. The inhibitory activity (IA) reduces the utilization of C2 by EAC14. Because IA binds to the surface of sheep erythrocytes and does not inhibit C1 irreversibly, it is probably a hitherto undescribed inhibitor of complement.