Studies on human brain-thymus cross-reactive antigens.

Antigens shared by human brain and thymocytes and by human and mouse tissues were studied with rabbit anti-human thymocyte antiserum (RAHT). It was found that cytotoxicity of RAHT serum against mouse thymus cells was not absorbed by mouse liver or bone marrow cells. Human brain and thymus cells completely absorbed the anti-thymocyte activity from this antiserum. It was suggested that human brain had antigenic determinants identical or very similar to those found on human thymocytes. Activity of RAHT antiserum against mouse thymus cells was completely removed by an absorption of mouse brain and thymocytes. These results demonstrated that there were shared antigenic determinants between human and mouse tissues.     

Comparison of chromosomal proteins of mouse primitive teratocarcinoma, liver and L cells

Histone and non-histone proteins of mouse primitive teratocarcinoma cells have been studied by polyacrylamide gel electrophoresis and compared with those of mouse liver and with those of mouse L cells. The electrophoretic patterns and the ratio of histones/ DNA were identical in all cells studied. The electrophoretic patterns of non-histone proteins of teratocarcinoma cells were significantly different from those of the liver and also of the L cells, although more similarities were encountered between the two types of cultured cells.     

Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven

N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl(4) were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl(4) models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl(4) model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.     

Elimination of the atherogenic effect of beta blocker propranolol by papaverine]

Recently, the ability of beta-blockers to stimulate proliferative activity and induce lipid accumulation in cultured human aortic intimal cells has been demonstrated. Moreover, the addition of calcium antagonists completely blocked the increase in proliferative activity and abolished cholesterol accumulation caused by propranolol. In this study blood serum of rabbits treated with 20 mg of propranolol induced 2-fold cholesterol accumulation in mouse peritoneal macrophages. Papaverin did not influence this effect. In case of simultaneous administration of propranolol and papaverin rabbit serum did not exhibit the ability to accumulate intracellular lipids. Propranolol substantially stimulated the formation of myointimal thickening and neutral lipid accumulation in denuded rabbit aorta. Papaverin completely blocked the propranolol-produced atherogenic changes. The data suggest that in vitro and in vivo atherogenic effects of beta-blockers may be prevented by papaverin.     

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).     

Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.     

The sites of catabolism of murine monomeric IgA

The tissue sites of monomeric IgA (mIgA) catabolism were determined in a BALB/c mouse model. Mouse mIgA myeloma proteins were labeled either by direct iodination or by coupling the residualizing label, dilactitol-125I-tyramine (125I-DLT) to the proteins; catabolites from protein labeled with 125I-DLT accumulate at the site of protein degradation, allowing identification of the tissue and cellular sites involved in catabolism of the protein. The circulating half-lives of 125I- and 125I-DLT-mIgA were the same. The distribution of radioactivity in tissues was measured at 1, 3, 24, and 96 h after iv. injection of 125I-DLT-labeled mIgA, dimeric IgA (dIgA), IgG, or mouse serum albumin. The greatest uptake of 125I-DLT-mIgA was attributable to the liver. This organ accounted for more internal catabolism of mIgA than all other tissues combined. In contrast, 125I-DLT-IgG was catabolized equally in skin, muscle, and liver. These data indicate that, in mice, the liver is the major site of mIgA catabolism. To determine the cell types involved, collagenase digestion was used to isolate parenchymal and non-parenchymal cells from perfused liver of animals injected with 125-DLT-mIgA. Most of the radioactivity was associated with the hepatocyte fraction, even though both cell types showed uptake of 125I-DLT-mIgA. Inhibition studies, with asialofetuin and mouse IgA demonstrated that the uptake of mIgA by liver cells was mediated primarily by the asialoglycoprotein receptor.     

An 18000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [14C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under such incubation conditions, a single protein band with an apparent molecular weight of 18000 was radioactively labeled. [14C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of gamma-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [14C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.     

Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.     

Lysosomes and protein degradation

Evidence from studies on mouse peritoneal macrophages using the inhibitor pepstatin confirms lysosomal involvement in basal protein degradation, and extends its relevance to degradation of long half-life and analogue containing proteins. Studies on the ability of MRC-5 (a limited life-span fibroblast line) cells to selectively degrade analogue-containing proteins are described. These indicate that this capacity is retained even in very old cells; indeed such cells show an increased proportion of rapidly-degradable proteins. Analogue containing proteins bind preferentially to lysosomal membranes, and like liver cytosol proteins of short half-life, are selectively endocytosed and degraded by certain cells in culture. Thus membrane binding allowing selective entry to the lysosomal system may be important in controlling rate of degradation of both intracellular and extracellular protein. A method potentially allowing for determination of the rate of autophagy in cells, is described. This should enable further assessment of the quantitative involvement of lysosomes in protein degradation.     

An 18 000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [¹⁴C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [¹⁴C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [¹⁴C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Partial characterization of the PAF-induced soluble factors which mimic the activity of 'early pregnancy factor'

Platelet-activating factor (PAF) stimulated mouse spleen cells to release soluble factors (termed S2 factors) which were capable of inducing increased rosette inhibition titres when applied to fresh mouse spleen cells in the rosette inhibition assay. In this ability the S2 factors mimic that of pregnancy serum, an action previously ascribed to 'early pregnancy factor'. The PAF-stimulated production of these S2 factors was not influenced by inhibitors of cyclooxygenase metabolism, but was completely inhibited by the lipoxygenase inhibitors, diethyl carbamazine and nordihydroguaiaretic acid. The S2 factors had a lipid-like character in that they were extractable in organic solvents. The calcium ionophore A23187 also stimulated the production of these factors which may well be products of the lipoxygenase pathway of arachidonic acid metabolism.     

Major component of Ra-reactive factor, a complement-activating bactericidal protein, in mouse serum

A complement-activating bactericidal protein, Ra-reactive factor was isolated from mouse serum by an affinity method. The m.w. of the isolated RaRF estimated by glycerol density gradient sedimentation (around 300,000) was the same as that of the active material in mouse serum. As evidenced by gel filtration, the intact RaRF was decomposed into high (higher than 200,000) and low (50,000 to 200,000) m.w. components by treatment with 10% acetonitrile. SDS- and acid/urea-PAGE demonstrated that the high m.w. component was completely dissociated into equimolar quantities of two kinds of 28 kDa polypeptides, P28a and P28b, under reducing conditions, indicating that the association of these polypeptides was stabilized by disulfide bonds. The ability to bind specifically to the Ra determinant was retained in the high m.w. component, although the complement-activating potency was lost. The amino acid compositions of P28a and P28b polypeptides were compared with those of related serum proteins. The P28a and P28b polypeptides were found to have the highest homology to rat mannan-binding protein and mouse and human C1q subcomponent of complement.     

Towards a biomarker of mammalian senescence: carbonic anhydrase III

Three proteins (D2, D3, D4) have been identified in the male Fischer 344 rat liver that decrease their concentration dramatically to virtually zero during the transition from physiological maturity to senescence. D3 (Mr 28 kDa), absent (or at a very low concentration) from the livers of newborns and females of all ages, reaches at 60 days (sexual maturity) its maximum concentration, which declines almost linearly thereafter. A homologous protein (CNBr peptide map) occurs in the BALB/c mouse under similar conditions. D3 was purified and since its N-terminal is blocked, digested with CNBr. SDS-PAGE-separated peptides were blotted upon Immobilon and sequenced. The partial sequence matches that of rat carbonic anhydrase III. Treatment of senescent rats with 5 alpha-dihydrotestosterone restores D4 completely, yet D2 and D3 only partially, towards their maximum life-time concentration. Thus senescence-related factors (e.g. hepatic androgen receptor) aside from serum testosterone are responsible for the disappearance of the three proteins from the senescent liver.     

Serum mediated changes in cell attachment and cytoplasmic organization of B16 melanoma correlates with selective alterations in secreted proteins

B16 melanoma cells exhibited decreased adhesion to substration and the presence of cytoplasmic granules, resembling lipid inclusions, when cultured in vitro in the presence of syngenic serum from C57/BL6 mice. This constrasted with the rapid cell attachment and absence of cytoplasmic granules in cultures seeded in medium supplemented with an identical concentration of fetal bovine serum. Electrophoretic comparison of intracellular proteins revealed similar patterns in detergent-soluble and matrix-associated proteins from cells grown with bovine or mouse serum. However, a similar analysis of the conditioned media showed clear differences in methionine-rich species which migrated in the 100 kd region in cells grown with bovine serum, and as 110 kd component in cells grown with mouse serum. Our data indicate that the poor attachment to substratum and changes in cytoplasmic organization of B16 melanoma cells, is primarily associated with specific changes in secreted proteins.     

Synthesis of alpha-fetoprotein and albumin by fetal mouse liver cultured in chemically defined medium

Fetal mouse liver synthesizes two major secretory proteins: α-fetoprotein and albumin. The relative proportions of these proteins change during development. Fetal mouse liver secretes predominantly α-fetoprotein, and the neonatal mouse liver secretes predominantly albumin. α-Fetoprotein and albumin synthesis were studied in vitro using an organ culture system and a chemically defined medium (BGJ_b). This medium does not support hepatocellular replication but maintains protein synthesis at high levels for prolonged periods of culture. Patterns of protein synthesis were analyzed as functions of gestational age and time in culture. The ratio of α-fetoprotein/albumin decreases from 1.4 on gestational Day 14 to 0.4 on gestational Day 18. However, when liver cultures were begun on any given gestational day, the ratio of α-fetoprotein/albumin remains constant for as long as 8 days in culture. Thus, developmental changes in α-fetoprotein and albumin synthesis are arrested under the conditions of this culture system. Fetal mouse liver secretes two electrophoretically distinguishable forms of albumin. One form is similar in mobility to albumin from adult mouse serum; the other more electropositive species is similar in mobility to proalbumin isolated from adult mouse liver microsomes and can be converted to albumin by mild trypsin treatment.     

Immunological characterization of basement membrane types of heparan sulfate proteoglycan

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extracellular matrix proteins.